International Journal of Molecular Sciences

Article Expression Silencing of Glutathione 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis

Marlena Rademacher, Hartmut Kuhn and Astrid Borchert *

Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Biochemistry, Charitéplatz 1, 10117 Berlin, Germany; [email protected] (M.R.); [email protected] (H.K.) * Correspondence: [email protected]; Tel.: +49-30-450-528-034

Abstract: Among the eight human isoforms, glutathione peroxidase 4 (GPX4) is the only capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebroge- nesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythro- poiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective ery-  thropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a  role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a Citation: Rademacher, M.; Kuhn, H.; catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis. Borchert, A. Expression Silencing of Glutathione Peroxidase 4 in Mouse Keywords: cell differentiation; lipid peroxidation; oxidative stress; eicosanoids Erythroleukemia Cells Delays In Vitro Erythropoiesis. Int. J. Mol. Sci. 2021, 22, 7795. https://doi.org/ 10.3390/ijms22157795 1. Introduction Erythropoiesis is a multi-step process converting multipotent hematopoietic stem Academic Editor: Dimitrios Kouretas cells (HSC) to the mature peripheral erythrocytes circulating in the peripheral blood [1]. During the early steps of erythropoiesis HSC differentiate from the common myeloid Received: 18 March 2021 progenitor cell of the megakaryocytic-erythroid linage to the burst-forming unit- erythroid Accepted: 20 July 2021 Published: 21 July 2021 (BFU-E). BFU-E cells are the first cellular progenitors committed solely to the erythroid lineage [2]. These BFU-Es further differentiate into the colony-forming unit erythroid

Publisher’s Note: MDPI stays neutral (CFU-E) and this process concludes the first phase of erythroid differentiation. The second with regard to jurisdictional claims in phase of erythroid differentiation involves the maturation of the nucleated precursors published maps and institutional affil- from the proerythroblasts to basophilic, polychromatic and orthochromatic erythroblasts. iations. This phase is characterized by the gradual accumulation of hemoglobin, progressive decrease in cell size and nuclear condensation ultimately resulting in enucleation [1,2]. The final phase of erythropoiesis involves the development of the reticulocyte into mature erythrocytes. During this developmental stage erythrocytes acquire their characteristic biconcave shape and will be released into the bloodstream. Mature human red blood Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. cells remain in circulation for about 120 days and during this time period residential This article is an open access article macrophages in liver and spleen continuously survey them for structural and functional distributed under the terms and integrity [3]. Aged or damaged erythrocytes are phagocytosed by these macrophages and conditions of the Creative Commons this concludes the life cycle of the red cells. In mammals terminal erythroid differentiation Attribution (CC BY) license (https:// proceeds in erythroblastic islands within the bone marrow. These islands consist of a creativecommons.org/licenses/by/ central macrophage, which is surrounded by up to 30 erythroid cells of variable degrees 4.0/). of maturation [4]. The central macrophage fulfills many functions [5–7]: (i) It anchors the

Int. J. Mol. Sci. 2021, 22, 7795. https://doi.org/10.3390/ijms22157795 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 7795 2 of 20

erythroid cells within an erythroblastic island. (ii) It regulates the cellular interactions required for effective erythroid differentiation. (iii) It phagocytoses the nuclei extruded from erythroid precursors during terminal erythroid differentiation. (iv) It supplies the erythroid precursors with iron for effective heme synthesis. (v) It secretes cytokines and other signaling molecules regulating erythroid differentiation and cell proliferation in the immediate surrounding. Glutathione (Gpx-isoforms) form a family of capable of reducing organic and inorganic peroxides at the expense of reduced glutathione or alternative electron donors [8]. The involves eight different functional GPX (GPX1-8), which encode for eight functionally distinct GPX-isoforms. In humans GPX1, GPX2, GPX3, GPX4 and GPX6 are carrying selenocysteine as catalytically active amino acid at the . The other human GPX-isoforms contain a cysteine at this position. The mouse genome involves an ortholog for each human gene but in this species the Gpx6 gene encodes for a cysteine-containing enzyme. Among glutathione peroxidase isoforms GPX4 is somewhat unique since these enzyme orthologs are capable of reducing not only simple inorganic hydroperoxides such as H2O2 but also complex hydroperoxy ester lipids [9,10]. In humans and mice, GPX4 is expressed in three isoforms, which differ from each other with respect to their subcellular localization. cGPX4 is mainly localized in the cytosol, mGPX4 occurs in the mitochondria and nGPX4 prevails in the nucleus [11–13]. Although these isoforms are encoded for by the same gene (Gpx4) the primary translation products are protein-chemically distinct. Gpx4−/− mice are not viable but die at early stages (E6.5) of embryogenesis [14,15] and this is also the case for mice expressing catalytically silent mutants (Sec46Ala, Sec46Ser) of the enzyme [16,17]. However, mice expressing the Sec46Ala mutant heterozygously are viable and do not show obvious phenotypic alterations [16]. More detailed studies of the reproduction behavior of these animals indicated that males are subfertile, which has been related to structural and functional defects of their sperm [18]. The molecular basis for the embryonic lethality of Gpx4−/− mice is still a matter of discussion but siRNA induced Gpx4 expression silencing during in vitro embryogenesis [19] and neuron specific disruption of the Gpx4 gene [20] suggested a role of the enzyme in early cerebral embryogenesis. More recently, it was reported that conditional knockout of the Gpx4 gene in hematopoietic cells compromised erythropoiesis of adult mice [21]. Functional inactivation of Gpx4 gene induces necroptotic death of erythroid precursors, which leads to anemia that is partly compensated by increased extramedullary erythropoiesis [21]. If similar mechanisms pro- ceed during early embryogenesis malfunction of the erythropoietic system may contribute to embryonic lethality of Gpx4−/− mice. To contribute to the discussion on the potential role of Gpx4 in erythropoiesis we employed mouse erythroleukemia cells (MEL), which differentiate along the erythroid linage [22,23] when stimulated with dimethylsulfoxide (DMSO). This cellular erythro- poiesis model is frequently used and is considered a suitable cellular model for later stages of erythropoiesis [24,25]. Stimulated MEL cells reach the differentiation levels of nondividing normoblasts, synthesize hemoglobin but do not proceed into final erythroid differentiation [26–28]. For our studies, we first tested whether resting and DMSO-treated MEL cells express different Gpx4-isoforms. Next, we silenced Gpx4 expression in these cells using the RNAi technology (stable transfection with an anti-Gpx4 siRNA plasmid) and tested the impact of this genetic manipulation on DMSO-induced erythropoiesis. We found that Gpx4 isoforms are expressed in MEL cells and that expression silencing delays in vitro erythropoiesis. This data is consistent with the above referenced in vivo studies [21,29]. However, when we tested Gpx4+/Sec46Ala mice, which heterozygously express a catalytically silent Gpx4 mutant, we did not observe a defective hematopoietic phenotype. Moreover, ex vivo erythropoiesis of embryonic liver cells did not reveal significant differences between wildtype and Gpx4+/Sec46Ala hepatocytes. Taken together these data suggest that heterozy- gous expression of a catalytically silent Gpx4 mutant may not be sufficient to compromise in vivo erythropoiesis. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 20

Moreover, ex vivo erythropoiesis of embryonic liver cells did not reveal significant differ- ences between wildtype and Gpx4+/Sec46Ala hepatocytes. Taken together these data suggest Int. J. Mol. Sci. 2021, 22, 7795 3 of 20 that heterozygous expression of a catalytically silent Gpx4 mutant may not be sufficient to compromise in vivo erythropoiesis.

2. Results Results 2.1. Expression Expression of Gpx4 Isoforms in Mouse Erythroleukemia Cells Mouse erythroleukemiaerythroleukemia (MEL) (MEL) cells cells are are frequently frequently employed employed as cellular as cellularin vitro inmodel vitro modelfor studies for studies on mechanistic on mechanistic details of details erythroid of erythroid differentiation. differentiation. Resting MEL Resting cells doMEL neither cells doexpress neither hemoglobin express hemoglobin nor other red nor cell other specific red proteins, cell specific but stimulation proteins, but with stimulation DMSO induces with DMSOerythroid induces differentiation erythroid differentiation reaching the level reaching of orthochromatic the level of orthochromatic normoblasts normoblasts [28,30]. To [28,30].explore whetherTo explore these whether cells express these cells Gpx4 expr isoformsess Gpx4 we first isoforms carried we out first qRT-PCR carried to out quantify qRT- PCRthe steady to quantify state concentrationsthe steady state of concentrations Gpx4 mRNA in of resting Gpx4 mRNA MEL cells in resting (Figure 1MELa). With cells more (Fig- urethan 1a). 150 With copies more mRNA than per150 1000copies copies mRNA of Gapdhper 1000 mRNA, copies theof Gapdh cytosolic mRNA, isoform the (cGpx4) cytosolic is isoformexpressed (cGpx4) at high is levels. expressed The mGpx4at high mRNA,levels. The which mGpx4 encodes mRNA, for the which mitochondrial encodes for Gpx4 the mitochondrialisoform, was also Gpx4 present, isoform, but itswas expression also present, level but was its 4-fold expression lower thanlevel that was of 4-fold cGpx4. lower The thannuclear that Gpx4 of cGpx4. mRNA The was nuclear only Gpx4 present mRNA in small was amounts only present (less in than small 1% amounts of cGpx4 (less mRNA). than 1%A similar of cGpx4 Gpx4 mRNA). expression A similar pattern Gpx4 was expressi previouslyon pattern observed was inpreviously different observed mouse somatic in dif- ferenttissues mouse [16]. somatic tissues [16].

Figure 1. Expression of Gpx4 isoforms in native and transfected MEL cells. Total RNA was extractedextracted from MEL cells, and the steady-state concentrations of mRNAs encoding for diff differenterent Gpx4-isoforms were quantified quantified by qRT-PCR using the specifiedspecified amplificationamplification primers as describeddescribed in the Material and Method section. (a) Expression of Gpx4-mRNA isoforms in native native MEL MEL cells. cells. Each Each sample sample was was quantified quantified in triplicates. in triplicates. (b) Expression (b) Expression of Gpx4-isoforms of Gpx4-isoforms in stably in stably transfected transfected RNAi RNAiMEL cells. MEL Native cells. NativeMEL cells MEL were cells transfected were transfected with a Gpx4-s with a Gpx4-siRNAiRNA plasmid plasmid (Gpx4 RNAi) (Gpx4 or RNAi) with ora corresponding with a corresponding control controlvector (MOCK vector (MOCK RNAi). Different RNAi). Different isolated isolatedgrowing growing cell clones cell we clonesre selected were selectedas described as described in Material in Materialand Methods. and Methods. Expres- Expressionsion of Gpx4-mRNA of Gpx4-mRNA isoforms isoforms was quantifi was quantifieded in three inselected three selectedcell clones cell and clones Gpx4 and mRNA Gpx4 copy mRNA numbers copy were numbers related were to the Gapdh mRNA copy numbers. Inset: Quantification of Gapdh mRNA in MOCK RNAi and Gpx4 RNAi MEL cells related to the Gapdh mRNA copy numbers. Inset: Quantification of Gapdh mRNA in MOCK RNAi and Gpx4 RNAi MEL related to the amount of total RNA. RNA preparations of three different samples were analyzed and each sample was cells related to the amount of total RNA. RNA preparations of three different samples were analyzed and each sample quantified in triplicates. For statistical comparison, the Student’s t-test was performed. * p < 0.05, ** p < 0.005; mGpx4: wasmitochondrial, quantified incGpx4: triplicates. cytosolic, For statisticalnGpx4: nuclear comparison, isoform. the Student’s t-test was performed. * p < 0.05, ** p < 0.005; mGpx4: mitochondrial, cGpx4: cytosolic, nGpx4: nuclear isoform. 2.2. Stable Transfection of MEL Cells with Anti-Gpx4 Antisense Construct Reduced Gpx4 2.2. Stable Transfection of MEL Cells with Anti-Gpx4 Antisense Construct Reduced ExpressionGpx4 Expression To explore the impact of expression silencing of Gpx4 we firstfirst established a cellular model, in which which expression expression of of the the enzyme enzyme was was compromised compromised using using the the siRNA siRNA technology. technology. For this purpose, we stably transfected resting MEL cells with an anti-Gpx4anti-Gpx4 anti-senseanti-sense plasmid and analyzed a number of cell clones that survive the selection procedure. During the initial initial screen screen three three well well separated separated cell cell clones clones were were selected selected and and expression expression of the of thec + mGpx4c + mGpx4 was wasquantified quantified by qRT-PCR by qRT-PCR (Figure (Figure 1b).1 b).Here Here we wefound found that that in all in selected all selected cell cell clones Gpx4 expression was silenced by about 50%. To exclude that our transfection strategy impacted the expression of the reference gene we also quantified Gapdh expression in the three cell clones but did not observe significant differences when the Gapdh mRNA copy numbers were related to the amount of total cellular RNA (inset to Figure1b). For Int. J. Mol. Sci. 2021, 22, 7795 4 of 20

detailed erythropoiesis studies we selected clone 2 since in these cells Gpx4 expression was most strongly (51 ± 2%) silenced.

2.3. Basic Characterization of Gpx4 Compromised MEL Cells To characterize the Gpx4 deficient MEL cells in more detail we first quantified expres- sion silencing of the two major Gpx4 isoforms on the mRNA level. Here we found that the Gpx4 expression levels (cGpx4 + mGpx4) in wildtype MEL cells (about 200 copies of m + cGpx4 mRNA per 1000 Gapdh mRNA copies, Figure1a) were somewhat higher than those in the mock-transfected cells (about 120 copies of m + cGpx4 mRNA per 1000 Gapdh mRNA copies, Figure2a). These data suggested that the Gpx4 expression level was somewhat reduced by the transfection procedure. Next, we quantified the different Gpx4 mRNA isoforms (cGpx4, mGpx4, nGpx4) in wild type MEL cells (Figure1a), in MEL cells transfected with a Gpx4-siRNA plasmid (Gpx4 RNAi) or with a corresponding control vector (MOCK RNAi) (Figure2a) and observed similar expression patterns. In all cell types cGpx4 was dominant followed by mGpx4. The nuclear isoform (nGpx4 mRNA) was always present in small amounts. Most importantly, when we compared the steady state concentrations of the Gpx4 isoforms in mock-transfected cells with those in cells transfected with the anti-Gpx4 antisense plasmid we found significantly reduced mRNA concentrations (>50% reduction) for all isoforms (Figure2a). This was even the case for nGpx4 (Figure2a, inset), which was only present in small amounts. To explore whether Gpx4 expression silencing could also be detected on the level of the Gpx4 protein we carried out immunoblotting. Cell lysis supernatants of mock-transfected MEL cells and corresponding lysates supernatants of MEL cells transfected with the anti- Gpx4 antisense constructs were analyzed by SDS-PAGE and the blots were probed with an anti-human GPX4 antibody, which cross-reacts with the mouse ortholog [31]. As shown in Figure2b an immunoreactive protein with an apparent molecular weight of 18 kDa was detected in the lysis supernatant of mock-transfected cells. This band was not visible anymore when the lysate supernatant of MEL cells was analyzed, which were transfected with the anti-Gpx4 antisense plasmid. Next, we carried out activity assays with the lysate supernatants of mock-transfected and antisense-transfected cells. For specific measurements of the Gpx4 activity we em- ployed HPLC purified hydroperoxy phosphatidylcholine as substrate (see Materials and Methods). Here we found that the Gpx4 activity of MEL cells transfected with the anti-Gpx4 antisense plasmid was about 60% lower than that of the mock-transfected cells (Figure2c). Taken together the data shown in Figure2 indicated that expression of the Gpx4 gene was strongly reduced but not abolished in siRNA transfected cells. To test whether expression silencing of Gpx4 impacts basic cellular functions we compared the cell division kinetics of mock-transfected and Gpx4 siRNA transfected cells (Figure2d). When equal numbers of cells (104 cells/mL) were seeded into the culture dishes we did not find significant differences between the two cell types after 24 h of incubation. After 48 h the cell division rate of Gpx4 RNAi cells was slightly increased compared to the corresponding control transfectant (MOCK RNAi) (p = 0.087). After 72 and 96 h, the division rate of Gpx4 RNAi cells was significantly higher compared to MOCK RNAi cells. Moreover, the cell doubling time of Gpx4-deficient MEL cells (Td = 15.05 ± 0.21) was significantly (p = 0.0006) lower when compared with that of mock-transfected MEL cells (Td = 18.76 ± 0.38). On the other hand, we did not observe obvious signs of premature cell death (Trypan blue staining, data not shown) and these findings were somewhat surprising in the lights of the previous reports on the cell protecting character of Gpx4 in mouse erythroid precursors [21]. Int. J. Mol. Sci. 2021, 22, 7795 5 of 20 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 20

Figure 2. ExpressionFigure silencing 2. Expression of Gpx4 silencing in MEL cells of Gpx4 stably in transfected MEL cells stablywith an transfected anti-Gpx4with siRNA. an anti-Gpx4(a) Expression siRNA. of Gpx4 mRNA isoforms (ina) stably Expression transfected of Gpx4 RNAi mRNA MEL isoforms cells. Tota inl stably RNA transfectedwas extracted RNAi from MEL MEL cells. cells, Total and the RNA steady-state was concentration of extractedmRNAs of from the MELdifferent cells, Gpx4-isoforms and the steady-state was quantified concentration by qRT-PCR of mRNAs using ofthe the specified different amplification Gpx4- primers as described in the Material and Method section. Inset: Quantification of nGpx4 mRNA isoform. (b) Expression isoforms was quantified by qRT-PCR using the specified amplification primers as described in the of Gpx4 protein in stably transfected RNAi MEL cells. MOCK RNAi and GPx4 RNAi MEL cells were homogenized, and Material and Method section. Inset: Quantification of nGpx4 mRNA isoform. (b) Expression of aliquots (100 µg lysate supernatant protein) were applied to Western blotting using the monoclonal anti-human Gpx4 antibody (#MABF1969,Gpx4 protein Merck) in [31] stably as descr transfectedibed in RNAiMaterial MEL and cells. Methods. MOCK The RNAi absence and GPx4 of Gpx4 RNAi protein MEL cellsin GPx4 were RNAi treated MEL cellshomogenized, was indicated and byaliquots the absence (100 µofg lysatethe immunoreactive supernatant protein) band werein the applied molecular to Western weight blotting range of using about 20 kD. The chemicalthe identity monoclonal of the immunoreactive anti-human Gpx4 bands antibody at higher (#MABF1969, molecular Merck)weight [remains31] as described unclear. Since in Material the monoclonal and antibody employedMethods. for these The analyses absence reco of Gpx4gnizes protein a specific in GPx4 amino RNAi acid treated sequence MEL mo cellstif [31], was indicatedwhich is also by the present absence in other mouse proteins. ofThus, the immunoreactivethe most plausibl bande explanation in the molecular for the immunoreactive weight range of aboutbands 20with kD. higher The chemical molecular identity weights is cross-reactivity ofof the the antibody immunoreactive with non-Gpx4 bands related at higher protei molecularns. Expression weight remainsof the Gapdh unclear. protein Since was the quantified monoclonal as load- ing control. (c) Gpx4antibody activity employed in stably for transfected these analyses RNAi recognizes MEL cells. aspecific Cells were amino homogenized, acid sequence and motif the [Gpx431], which activity is was assayed in the 10,000 g cell lysate supernatant using purified phosphatidylcholine hydroperoxide as substrate (see Mate- also present in other mouse proteins. Thus, the most plausible explanation for the immunoreactive rial and Methods). (d) Cell division rate of stably transfected RNAi MEL cells. MEL cells, stably transfected with a Gpx4 bands with higher molecular weights is cross-reactivity of the antibody with non-Gpx4 related siRNA plasmid (Gpx4 RNAi) or with a corresponding control vector (MOCK RNAi), were seeded in culture dishes (104 cells/mL) in triplicatesproteins. and Expression cultured at of37 the °C Gapdhfor 7 days protein as described was quantified in Material as loading and Methods. control. Cells (c) Gpx4 were activity counted in daily. Significances werestably calculated transfected using RNAi the Student’s MEL cells. t-test, Cells n = were 3, * p homogenized, < 0.05, ** p < 0.005. and the Gpx4 activity was assayed in the 10,000 g cell lysate supernatant using purified phosphatidylcholine hydroperoxide as substrate (see Material2.4. and Erythropoiesis Methods). (d Can) Cell Be division Induced rate in Mock-Transfected of stably transfected and RNAi Gpx4 MEL siRNA cells. Transfected MEL cells, MEL stably transfectedCells with a Gpx4 siRNA plasmid (Gpx4 RNAi) or with a corresponding control vector 4 ◦ (MOCK RNAi),Erythropoiesis were seeded in culture can be dishes induced (10 incells/mL) resting MEL in triplicates cells when and culturedthe cells atare 37 stimulatedC for with 7 days as describedDMSO and in Material erythroid and Methods.differentiation Cells were can countedbe followed daily. Significancesby quantification were calculated of the expression using the Student’sof severalt-test, erythroid-specific n = 3, * p < 0.05, ** genesp < 0.005. [26,28,30]. For our experiments we quantified the 2.4. Erythropoiesissteady state Can concentrations Be Induced in Mock-Transfected of the mRNAs andenco Gpx4ding siRNAfor hemoglobin Transfected a-a1 MEL (Hba-a1) Cells and for the 5-aminolevulinic acid synthase 2 (Alas2), the key enzyme of heme biosynthesis, at dif- Erythropoiesis can be induced in resting MEL cells when the cells are stimulated with ferent time-points of the differentiation process. From Figure 3a it can be seen that in DMSO and erythroid differentiation can be followed by quantification of the expression mock-transfected cells the expression of the Hba-a1 mRNA is elevated following DMSO of several erythroid-specific genes [26,28,30]. For our experiments we quantified the stimulation. In contrast, Hba-a1 expression remained minimal in cells cultured in the ab- steady state concentrations of the mRNAs encoding for hemoglobin a-a1 (Hba-a1) and sence of DMSO and there were highly significant differences between DMSO-treated and for the 5-aminolevulinic acid synthase 2 (Alas2), the key enzyme of heme biosynthesis, at

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different time-points of the differentiation process. From Figure3a it can be seen that in Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWmock-transfected cells the expression of the Hba-a1 mRNA is elevated following DMSO6 of 20

stimulation. In contrast, Hba-a1 expression remained minimal in cells cultured in the absence of DMSO and there were highly significant differences between DMSO-treated untreatedand untreated mock-cells mock-cells at all at time all timepoints points of the of thedifferentiation differentiation period. period. Similar Similar Hba-a1 Hba-a1 ex- pressionexpression kinetics kinetics were were previously previously reported reported for forwildtype wildtype Mel Mel cells cells [24,27,32]. [24,27,32 Next,]. Next, we weex- ploredexplored the the expression expression profiles profiles for forAlas2 Alas2 and and observed observed similar similar kinetics kinetics.. Expression Expression of this of mRNAthis mRNA was wasinduced induced by DMSO by DMSO and andsignificant significant differences differences to unstimulated to unstimulated cells cells were were ob- servedobserved (Figure (Figure 3b).3b).

Figure 3. DMSO induces erythroid differentiation in mock-transfected and Gpx4 siRNA transfected MEL cells. Total Total RNA was extracted from untreated and DMSO-treated MEL cell transfectants at different days of the culture period. The steady- was extracted from untreated and DMSO-treated MEL cell transfectants at different days of the culture period. The steady- state concentrations of Hba-a1, Alas2, and Gapdh mRNAs were quantified by qRT-PCR using the specified amplification state concentrations of Hba-a1, Alas2, and Gapdh mRNAs were quantified by qRT-PCR using the specified amplification primers as described in the Material and Methods section. Each sample was quantified in triplicates. (a) Expression of primersHba-a1 mRNA as described in MOCK in the RNAi Material MEL andcells. Methods (b) Expression section. of Each Alas2 sample mRNA was in quantifiedMOCK RNAi in triplicates.MEL cells. ((ac) ExpressionExpression of Hba-a1 mRNA in MOCKGpx4 RNAi RNAi MEL MEL cells. cells. (d (b) )Expression Expression of of Alas2 Alas2 mRNA mRNA in in Gpx4 MOCK RNAi RNAi MEL MEL cells. cells. Significances (c) Expression were of Hba-a1calculated mRNA using in the Gpx4 Student’s RNAi t MEL-test, * cells. p < 0.05, (d) Expression** p < 0.005. of Alas2 mRNA in Gpx4 RNAi MEL cells. Significances were calculated using the Student’s t-test, * p < 0.05, ** p < 0.005. To explore whether erythropoiesis can also be induced in Gpx4-deficient MEL cells similarTo differentiation explore whether studies erythropoiesis were carried can ou alsot with be induced cells transfected in Gpx4-deficient with the anti-Gpx4 MEL cells antisensesimilar differentiation plasmid. Here studies we found were that carried expres outsion with of cells the Hba-a1 transfected mRNA with is the also anti-Gpx4 induced byantisense DMSO plasmid.and significant Here we differences found that to expressionuntreated (- of DMSO) the Hba-a1 cells mRNAwere observed is also induced at all time by pointsDMSO of and the significantdifferentiation differences period. toInterestingl untreatedy, (- at DMSO) day 3 of cells the weredifferentiation observed period at all time the degreepoints of theDMSO-dependent differentiation upregulation period. Interestingly, of Hba-a1 at expression day 3 of the was differentiation less pronounced period for the degree of DMSO-dependent upregulation of Hba-a1 expression was less pronounced Gpx4 deficient MEL cells (Figure 3c) when compared with the corresponding mock-con- for Gpx4 deficient MEL cells (Figure3c) when compared with the corresponding mock- trols (Figure 3a). In fact, for the mock-cells we observed a 7-fold higher (720%) steady state controls (Figure3a). In fact, for the mock-cells we observed a 7-fold higher (720%) steady concentration of the Hba-a1 mRNA in DMSO-treated cells when compared with un- state concentration of the Hba-a1 mRNA in DMSO-treated cells when compared with treated mock-controls (Figure 3a, day 3). In contrast, for Gpx4-deficient MEL cells the untreated mock-controls (Figure3a, day 3). In contrast, for Gpx4-deficient MEL cells the Hba-a1 mRNA concentration was only increased by about 30%. Similarly, differential ex- Hba-a1 mRNA concentration was only increased by about 30%. Similarly, differential pression kinetics were observed when Alas2 mRNA was profiled. Here again, significant expression kinetics were observed when Alas2 mRNA was profiled. Here again, significant differences were detected at day 5 and day 6 of the differentiation period when DMSO- differences were detected at day 5 and day 6 of the differentiation period when DMSO- treated cells were compared with untreated cells (Figure 3d). In contrast, no difference treated cells were compared with untreated cells (Figure3d). In contrast, no difference between DMSO-treated and untreated cells was found on day 3. between DMSO-treated and untreated cells was found on day 3.

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2.5. Stable Knockdown of Gpx4 Expression Delays DMSO-Induced Erythroid Differentiation The data shown in Figure3 suggest that at day 3 of the differentiation period the steady state concentrations of the Hba-a1 and Alas2 mRNA are significantly elevated in DMSO- treated mock-transfected MEL cells when compared with untreated controls (Figure3a,b). In contrast, in Gpx4-deficient cells the degree of elevation is strongly reduced when Hba-a1 mRNA is used as readout parameter (Figure3c) and no significant difference is seen for Alas2 (Figure3d). These data suggest that in Gpx4-deficient MEL cells DMSO induced erythropoiesis may be delayed. To obtain additional evidence for the delay of erythroid differentiation in Gpx4-deficient MEL cells, we next explored the impact of DMSO on cell proliferation of Gpx4-sufficient and Gpx4-deficient MEL cells. In principle, erythropoiesis involves two counterbalanced processes: (i) Upregulation of functional specialization that is paralleled by a loss in functional multiplicity. (ii) Downregulation of the proliferation potency [28]. In other words, cells with a high proliferation capacity exhibit a low degree of differentiation. Vice versa, differentiated cells have a reduced proliferation potential. To explore whether Gpx4 expression silencing impacts the proliferative potential of MEL cells during erythroid differentiation, stably transfected Gpx4 RNAi MEL cells and the corresponding control transfectant (MOCK RNAi) were cultured in suspension for 5 days. When the MEL transfectants were cultured in the absence of DMSO, they divided with a certain rate (Figure4a,b). In the presence of DMSO the cell division rate was significantly reduced (Figure4a, dotted line). This observation is consistent with the higher degree of differentiation of the cells. In contrast, when Gpx4 RNAi MEL cells were taken through the same experimental protocol we did not observe impairment of cell division at day 3 of the culture period. From this data one may conclude that there is no major alteration in the degree of erythroid differentiation between Gpx4-sufficient and Gpx4-deficient cells at this time point. However, at later time points of the culturing period (5 days of DMSO treatment) we observed significant differences in the cell division characteristics (Figure4b, dotted line). The impaired proliferation potential of DMSO-treated Gpx4-deficient cells (compared with untreated Gpx4-deficient cells) suggested a higher degree of erythroid differentiation of the untreated cells. Thus, on the basis of these proliferation data one might conclude that Gpx4-deficiency delays DMSO-induced erythroid differentiation. To obtained more direct evidence for this conclusion we next quantified the DMSO sensitivity of mock-transfected MEL cells with that of Gpx4-deficient cells employing the steady state concentration of the Hba-a1 and Alas2 mRNA as readout parameter. For this purpose, we first subtracted from the mRNA copy numbers of DMSO-treated mock- transfected cells at day 3 of the differentiation period the corresponding value of untreated mock transfectants. The resulting difference appears to be a suitable measure for the DMSO sensitivity of these cells. Then, we repeated this procedure for Gpx4-deficient cells at day 3 of the differentiation period and compared the resulting differences. From Figure4c,d it can be seen that at day 3 of the differentiation period mock-transfected MEL cells were more sensitive for DMSO-induced Hba-a1 and Alas2 expression than Gpx4-deficient cells. In contrast, on day 5 of the differentiation period, there was no significant difference in the DMSO sensitivity of the two cell types anymore. These data are consistent with our previous conclusion that Gpx4-deficiency delays in vitro erythropoiesis of MEL cells by reducing their sensitivity for DMSO. To further support this conclusion we quantified the hemoglobin content of DMSO- stimulated mock-transfected MEL cells with the corresponding Gpx4-deficient counterparts. As indicated in Figure4(eI) unstimulated MEL cells did only express small amounts of hemoglobin regardless of whether the expression of Gpx4 was compromised or not. When mock-transfected cells were stimulated with DMSO a time-dependent increase in hemoglobin expression was observed (Figure4(eII)) and significant differences between DMSO-treated and untreated cells were detected at day 5 and day 6 of the differentiation period. In contrast, when Gpx4-deficient cells were taken through the same experimental protocol we did not see significant differences between DMSO-treated and untreated cells at day 5 of the differentiation period (Figure4(eIII)). Compared with mock-transfected cells Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 20 Int. J. Mol. Sci. 2021, 22, 7795 8 of 20

at day 5 of the differentiation period (Figure 4(eIII)). Compared with mock-transfected cellsthe hemoglobin the hemoglobin content content was significantly was significantly lower afterlower DMSO after DMSO stimulation stimulation (Figure 4(Figure(eIV)). 4(eIV)).These data These support data support our conclusion our conclusion that partial thatGpx4 partial deficiency Gpx4 deficiency delays but delays did but not did abolish not abolishin vitro in erythropoiesis vitro erythropoiesis in this cellular in this cellular in vitro in model. vitro model.

Figure 4. Stable knockdown of Gpx4 expression retard retardss DMSO-induced erythroid differentiation. (a,b) MEL cell division rate of stably transfected RNAi MEL cells. MEL cells, stably transfected with a Gpx4 siRNA plasmid (Gpx4 RNAi) ( b) or with a corresponding control vector (MOCK RNAi) (a) were seeded into culture dishes (104 cells/mL) in triplicates and with a corresponding control vector (MOCK RNAi) (a) were seeded into culture dishes (104 cells/mL) in triplicates and cultured at 37 °C for 5 days in the absence (- DMSO) or in the presence of DMSO (+ DMSO) as described in Material and cultured at 37 ◦C for 5 days in the absence (− DMSO) or in the presence of DMSO (+ DMSO) as described in Material and Methods. Cells were counted on day 3 and day 5. The results are presented as cumulative cell number versus days in Methods.culture. Significances Cells were counted were calculated on day 3 andusing day the 5. Student’s The results t-test, are presented n = 3, * p as < cumulative0.05, ** p < cell0.005. number (c) Hba-a1versus mRNAdays in and culture. (d) SignificancesAlas2 mRNA were expression calculated stimulation using the after Student’s DMSOt-test, treatment n = 3, * pof< stable 0.05, ** transfectedp < 0.005.( cRNAi) Hba-a1 MEL mRNA cells. andTotal (d )RNA Alas2 was mRNA ex- expressiontracted from stimulation untreatedafter and DMSODMSO-treated treatment M ofEL stable cell transfectants transfected RNAi at different MEL cells. days Total of the RNA culture was extractedperiod. The from steady-state untreated andconcentrations DMSO-treated of Hba-a1 MEL cell (c),transfectants Alas2 (d) related at different to Gapdh days mRNAs of the culture were quantified period. The by steady-state qRT-PCR using concentrations the specified of Hba-a1ampli- (ficationc), Alas2 primers (d) related as described to Gapdh in mRNAs the Material were quantified and Methods by qRT-PCR section. ( usinge) Hemoglobin the specified synthesis amplification in stably primers transfected as described RNAi inMEL the cells Material without and ( Methods− DMSO) section. or with (e DMSO) Hemoglobin (+ DMSO) synthesis stimulation. in stably Intracellular transfected hemoglobin RNAi MEL cellslevels without at different (− DMSO) days orof withthe culture DMSO period (+ DMSO) were stimulation. quantified as Intracellular described in hemoglobin Material and levels Methods. at different (eI) Hemoglobin days of the culture content period of unstimulated were quantified MEL transfectants. (eII) Hemoglobin content of mock- transfected MEL cells (MOCK RNAi) after DMSO treatment, (eIII) He- as described in Material and Methods. (eI) Hemoglobin content of unstimulated MEL transfectants. (eII) Hemoglobin moglobin content of Gpx4 siRNA transfected MEL cells (Gpx4 RNAi) after DMSO treatment, (eIV) Hemoglobin content content of mock- transfected MEL cells (MOCK RNAi) after DMSO treatment, (eIII) Hemoglobin content of Gpx4 siRNA of DMSO treated Gpx4 deficient MEL cells (Gpx4 RNAi) and the corresponding control transfectant (MOCK RNAi). Sig- transfectednificances were MEL calculated cells (Gpx4 using RNAi) the Student’s after DMSO t-test, treatment, * p < 0.05, (eIV ** p) Hemoglobin< 0.005. content of DMSO treated Gpx4 deficient MEL cells (Gpx4 RNAi) and the corresponding control transfectant (MOCK RNAi). Significances were calculated using the Student’s t-test, * p < 0.05, ** p < 0.005.

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To put our conclusions on a broader experimentalexperimental basis, the in vitro differentiation experiments were repeated with two additional clones Gpx4 RNAi transfectants (clones 1 and 3 3 of of Figure Figure 1)1) and and the the steady steady state state concentrations concentrations of ofHba-a1 Hba-a1 mRNA mRNA on onday day 3 and 3 and day 5day of DMSO 5 of DMSO treatment treatment were were quantified quantified as re asadout readout parameter. parameter. To compare To compare the DMSO the DMSO sen- sitivitysensitivity of the of themock mock transfectants transfectants with with that that of ofthe the Gpx4 Gpx4 deficient deficient MEL MEL cells, cells, the the Hba- Hba- a1mRNA expression of the untreated cell was subtracted from the correspondingcorresponding value of the DMSO treated cell. From From Figure 55a,ba,b itit cancan bebe seenseen thatthat onon dayday 33 andand dayday 55 ofof thethe differentiation period erythroid differentiation was compromised in the additional clones of Gpx4-RNAi transfectants. These data are consistent with our previous conclusion that Gpx4-deficiencyGpx4-deficiency delays in vitro erythropoiesis of MEL cells.

Figure 5. Delay of DMSO-induced erythroid differentiationdifferentiation in two additional clones of Gpx4Gpx4 deficientdeficient MELMEL cells.cells. MEL cells, stably transfected with a Gpx4 siRNA plasmid (Gpx4 RNAi) or with a corresponding control vector (MOCK RNAi) cells, stably transfected with a Gpx4 siRNA plasmid (Gpx4 RNAi) or with a corresponding control vector (MOCK RNAi) were seeded in culture dishes (104 cells/mL) and cultured at 37 °C for 5 days without or with 2% DMSO (see Material and were seeded in culture dishes (104 cells/mL) and cultured at 37 ◦C for 5 days without or with 2% DMSO (see Material and Methods). Total RNA was extracted from untreated and DMSO-treated MEL cell transfectants at different days of cultur- Methods).ing period. Total The RNAsteady-state was extracted concentrations from untreated of Hba-a1mRN and DMSO-treatedA and Gapdh MEL mRNA cell transfectants were quantified at different by qRT-PCR days of using culturing spe- period.cific amplification The steady-state primers. concentrations (a,b) Different of individual Hba-a1mRNA stably and transfected Gapdh mRNA RNAi were MEL quantified cell clones. by Significances qRT-PCR using were specific calcu- amplificationlated using the primers. Student’s (a ,tb-test,) Different * p < 0.05, individual ** p < 0.005. stably transfected RNAi MEL cell clones. Significances were calculated using the Student’s t-test, * p < 0.05, ** p < 0.005. 2.6. Delay of DMSO Induced In Vitro Erythropoiesis in MEL Cells Caused by Gpx4 Deficiency Was2.6. DelayPartially of DMSO Rescued Induced by Ebselen In Vitro Erythropoiesis in MEL Cells Caused by Gpx4 Deficiency Was Partially Rescued by Ebselen Gpx4 is an antioxidative enzyme and anemia induced by in vivo Gpx4 expression Gpx4 is an antioxidative enzyme and anemia induced by in vivo Gpx4 expression silencing in hematopoietic cells [21] can be partially rescued by vitamin E supplementa- silencing in hematopoietic cells [21] can be partially rescued by vitamin E supplementa- tion. On the other hand, in vitro erythroid differentiation studies of human CD34+ cells tion. On the other hand, in vitro erythroid differentiation studies of human CD34+ cells demonstrate a role for Gpx4 in the enucleation of human erythroblasts (late phase of demonstrate a role for Gpx4 in the enucleation of human erythroblasts (late phase of erythropoiesis). Gpx4 knockdown strongly impaired enucleation but this effect cannot be erythropoiesis). Gpx4 knockdown strongly impaired enucleation but this effect cannot reversed by the addition of alpha-tocopherol [33]. Here we explored first whether addi- be reversed by the addition of alpha-tocopherol [33]. Here we explored first whether tion of alpha-tocopherol can also rescue the compromised DMSO-induced erythropoiesis addition of alpha-tocopherol can also rescue the compromised DMSO-induced erythro- inpoiesis Gpx4 in siRNA Gpx4 siRNAtransfected transfected MEL cells. MEL For cells. this For purpose, this purpose, we induced we induced erythropoiesis erythropoiesis with DMSOwith DMSO in siRNA in siRNA transfected transfected MEL MELcells cellsin the in presence the presence or absence or absence of 100 of 100µMµ alpha-to-M alpha- copherol.tocopherol. As Asdifferentiation differentiation marker marker we wequanti quantifiedfied the thesteady steady state state levels levels of Hba-a1 of Hba-a1 and Alas2and Alas2 mRNAs mRNAs on days on days3 and 35 andafter 5 DMSO after DMSO addition addition using qRT-PCR. using qRT-PCR. Under Underthese condi- these tions,conditions, we did we not did observe not observe any rescue any rescue effect effect(data (datanot shown). not shown). Ebselen is a seleno-organic seleno-organic compound that functions as Gpx-mimetic. It reduces lipid hydroperoxides to the corresponding alcohols at the expense expense of of reduced reduced glutathione glutathione [34]. [34]. However, thethe catalyticcatalytic efficiencyefficiency of of this this non-enzymatic non-enzymatic catalyst catalyst is is much much lower lower than than that that of ofGpx-isoforms. Gpx-isoforms. We We employed employed this this seleno-organic seleno-organic compound compound to perform to perform rescue rescue experiments experi- mentsand tested and tested whether whether addition addition of ebselen of ebsele restoresn restores the impaired the impaired DMSO-induced DMSO-inducedin vitro in vitroerythropoiesis erythropoiesis in Gpx4 in deficientGpx4 deficient MEL cells. MEL For cells. this For purpose, this purpose, we first we tested first the tested cell toxicitythe cell toxicityof ebselen of forebselen Gpx4 for RNAi- Gpx4 and RNAi- MOCK-transfected and MOCK-transfected MEL cells MEL and foundcells and that found both cellthat types both celltolerated types 5toleratedµM ebselen 5 µM without ebselen major without signs ofma celljor death.signs of Next, cell wedeath. induced Next, erythropoiesis we induced with DMSO in Gpx4 RNAi- and MOCK-transfected MEL cells in the presence or absence of

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erythropoiesis with DMSO in Gpx4 RNAi- and MOCK-transfected MEL cells in the pres- 5 enceµM ebselen. or absence As differentiationof 5 µM ebselen. marker As differen we quantifiedtiation marker the steady we statequantified levels ofthe Hba-a1 steady and state Alas2levels mRNA of Hba-a1 on days and Alas2 3 after mRNA DMSO on addition days 3 usingafter DMSO qRT-PCR. addition Under using these qRT-PCR. experimental Under conditions,these experimental the DMSO conditions, sensitivity the of theDMSO Gpx4 sens RNAiitivity transfectant of the Gpx4 was RNAi significantly transfectant higher was insignificantly the presence higher of ebselen in the but presence we did of not ebselen observe but complete we did not rescue observe of the complete Gpx4-deficient rescue of phenotypethe Gpx4-deficient (Figure6 ).phenotype To improve (Figure the 6). rescue To im effectprove of the ebselen rescue we effect performed of ebselen similar we per- µ experimentsformed similar at increased experiments ebselen at increased concentrations ebselen concentrations (50 M) but observed (50 µM) but significant observed cell sig- toxicity for both cell types. nificant cell toxicity for both cell types.

Figure 6. Delay of DMSO-inducedFigure 6. erythroidDelay of DMSO-induced differentiation erythroidin Gpx4 deficient differentiation MEL cells in Gpx4 was deficient partially MEL rescued cells by was the partially se- leno-organic compound ebselenrescued that by thefunc seleno-organictions as Gpx mimetic. compound MEL ebselen cells, that which functions were stably as Gpx transfected mimetic. MEL with cells, a Gpx4 which siRNA plasmid (Gpx4 RNAi) or with a corresponding control vector (MOCK RNAi) were seeded in culture dishes (104 were stably transfected with a Gpx4 siRNA plasmid (Gpx4 RNAi) or with a corresponding control cells/mL) and cultured in the absence or presence of 5 µM ebselen. Cells were cultured4 at 37 °C for 3 days without or with 2% DMSO and total RNAvector was extracted. (MOCK RNAi) The steady-state were seeded concentrations in culture dishes of Hba-a1 (10 cells/mL) mRNA, Alas2 and culturedmRNA and in the Gapdh absence ◦ mRNA were quantified byor qRT-PCR presence using of 5 µ Mspecific ebselen. amplificat Cells wereion primers. cultured DMSO at 37 sensitivityC for 3 days in withoutthe different or with incubation 2% DMSO samples was determined andand the total DMSO RNA sensitivity was extracted. of the The Gpx4 steady-state RNAi transfectant concentrations was shown of Hba-a1 relative mRNA, to that Alas2 of the mRNA MOCK and transfectant (see Material Gapdhand Methods). mRNA wereSignificances quantified were by calculated qRT-PCR using using specific the Student’s amplification t-test, * primers.p < 0.05. DMSO sensitivity in the different incubation samples was determined and the DMSO sensitivity of the Gpx4 RNAi transfectant2.7. Heterozygous was shown Expression relative of to Catalytically that of the Inactive MOCK transfectantGpx4 in Mice (see Does Material Not Induce and Methods). a SignificancesDefective Erythropoietic were calculated Phenotype using the Student’s t-test, * p < 0.05.

2.7. HeterozygousThe cellular Expression differentiation of Catalytically experiments Inactive suggested Gpx4 in that Mice a Does 50–60% Not Inducereduction a Defective of Gpx4 Erythropoieticexpression in Phenotype MEL cells delayed DMSO-induced in vitro erythropoiesis (Figures 3 and 4). To test whether such retardation can also be observed in vivo we employed heterozygous The cellular differentiation experiments suggested that a 50–60% reduction of Gpx4 Gpx4 knock-in mice, in which the Gpx4 gene locus was modified in such a way, that the expression in MEL cells delayed DMSO-induced in vitro erythropoiesis (Figures3 and4). Toanimals test whether express such heterozygously retardation can alsothe be catalytically observed in vivosilentwe employedSec46Ala heterozygousGpx4 mutant +/Sec46Ala Sec46Ala/Sec46Ala Gpx4(Gpx4knock-in). mice, Homozygous in which theGpx4Gpx4 gene locusmice was do modified not survive in such day a 6 way, of embryo that the devel- ani- malsopment express but heterozygously heterozygous allele the catalytically carriers ar silente viable, Sec46Ala reproduce Gpx4 and mutant do not (Gpx4 show+/Sec46Ala obvious). HomozygousphenotypicalGpx4 alterationsSec46Ala/Sec46Ala unless stimulatedmice do not otherwise survive [16]. day 6To of explore embryo whether development our genetic but heterozygousmanipulation allele induced carriers defective are viable, erythropoies reproduceis andwe compared do not show the obvious basic erythrocyte phenotypical pa- alterationsrameters unlessof these stimulated mice with otherwise the corresponding [16]. To explore values whether of control our animals geneticmanipulation with a similar inducedgenetic defectivebackground erythropoiesis (Figure 7a–d) we but compared did not detect the basic significant erythrocyte differences. parameters of these mice with the corresponding values of control animals with a similar genetic background (Figure7a–d) but did not detect significant differences.

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FigureFigure 7. HeterozygousHeterozygous expression expression of catalytically of catalytically inacti inactiveve Gpx4 in Gpx4 mice in does mice not does induce not a defective induce a defec- erythropoietic phenotype. 200 µL blood was withdrawn from male Sec46Ala Gpx4 mutant tive erythropoietic phenotype. 200 µL blood was withdrawn from male Sec46Ala Gpx4 mutant (Gpx4+/U46A) and corresponding wild type (wt) mice. For blood removal mice were sacrificed by cer- +/U46A (Gpx4vical dislocation) and after corresponding isoflurane anesthesia wild type and blood (wt) mice.was collected For blood by cardiac removal puncture. mice wereCoagula- sacrificed bytion cervical was inhibited dislocation by EDTA after and isoflurane the basic red anesthesia blood cell and parameters blood was were collected determined by as cardiac described puncture. Coagulationin Material and was Methods. inhibited (a) erythrocyte by EDTA and count, the (b basic) reticulocyte red blood counts, cell ( parametersc) hemoglobin, were (d) determinedhemato- as describedcrit. n = 5–8 in animals Material were and analyzed. Methods. The ( awet) erythrocyte weights of spleens count, and (b) reticulocytekidneys of male counts, Sec46Ala (c) hemoglobin, Gpx4 mutant (Gpx4+/U46A) and corresponding wild type (wt) mice were determined and the body- (d) hematocrit. n = 5–8 animals were analyzed. The wet weights of spleens and kidneys of male weight/organ weight ratios were calculated. (e) spleen/body weight ratios, (f) and kidney/body +/U46A Sec46Alaweight ratios. Gpx4 n mutant= 7–8 animals (Gpx4 were analyzed.) and corresponding (g) Renal expression wild type of erythropoietin (wt) mice were (Epo) determined mRNA. and theTotal bodyweight/organ RNA was extracted weight from kidneys ratios wereof wild calculated. type (wt) and (e) spleen/bodySec46Ala Gpx4 weight mutant ratios, (Gpx4+/U46A (f) and) kid- ney/bodymice. Epo mRNA weight expression ratios. n levels = 7–8 were animals quantified were by analyzed. qRT-PCR ( g(see) Renal “Material expression and Methods”). of erythropoietin For (Epo)these mRNA.experiments, Total n RNA= 8 animals was extracted were sacrificed from kidneys and each of sample wild typewas quantified (wt) and Sec46Ala in triplicate. Gpx4 Sig- mutant nificances+/U46A were calculated using the Mann-Whitney U test and p-values < 0.05 were considered sta- (Gpx4tistically significant.) mice. ( Epoh) Gpx4 mRNA activity expression assays. Spleen levels and were bone quantified marrow cells by qRT-PCRwere homogenized, (see “Material the and Methods”).10,000 g lysate For supernatant these experiments, was prepared n = 8 and animals the Gpx4 were activity sacrificed was assayed and each using sample RP-HPLC was quantified puri- in triplicate.fied phosphadidylcholine Significances were hydroper calculatedoxide as using substrate. the Mann-WhitneySignificances were U calculated test and p using-values the < Stu- 0.05 were considereddent’s t-test. statistically Two animals significant. of each genotype (h) Gpx4 were used activity and assays.each sample Spleen was andquantified bone marrowin triplicates. cells were homogenized, the 10,000 g lysate supernatant was prepared and the Gpx4 activity was assayed using For independent support of this conclusion we compared the relative organ weights RP-HPLC purified phosphadidylcholine hydroperoxide as substrate. Significances were calculated of spleen and kidney between the two genotypes. In mice, stress erythropoiesis proceeds using the Student’s t-test. Two animals of each genotype were used and each sample was quantified in the spleen [35] and if permanent Gpx4 deficiency challenges the erythropoietic system ina triplicates.compensatory increase in the relative spleen size would be expected. Unfortunately, we did not observe a significant difference in the relative spleen size between Gpx4+/Sec46Ala For independent support of this conclusion we compared the relative organ weights mice and corresponding controls (Figure 7e). Another possible compensation mechanism of spleen and kidney between the two genotypes. In mice, stress erythropoiesis proceeds would be an increased renal erythropoietin synthesis in Gpx4+/Sec46Ala mice. To test this op- intion the we spleen first compared [35] and if the permanent relative kidney Gpx4 weights deficiency of wildtype challenges and the Gpx4 erythropoietic+/Sec46Ala mice but system a compensatorydid not find significant increase differences in the relative (Figure spleen 7f). Next, size wouldwe quantified be expected. renal erythropoietin Unfortunately, we +/Sec46Ala didmRNA not concentration observe a significant but here again, difference we did in no thet detect relative significant spleen sizedifferences between (FigureGpx4 7g). miceFinally, and we corresponding assayed the Gpx4 controls activity (Figure in bone7e). marrow Another cells possible and spleen compensation of Gpx4+/Sec46Ala mechanism mice wouldand compared be an increased the data with renal the erythropoietin corresponding synthesisvalues obtained in Gpx4 for+/Sec46Ala wild-typemice. mice. ToHere test this optionwe neither we firstobserved compared significant the relative differences kidney between weights the two of wildtypegenotypes and (FigureGpx4 7h).+/Sec46Ala Taken mice buttogether, did not these find data significant did not differencesprovide any (Figure evidence7f). for Next, a compromised we quantified erythropoiesis renal erythropoietin in mRNAGpx4+/Sec46Ala concentration mice. but here again, we did not detect significant differences (Figure7g). Finally, we assayed the Gpx4 activity in bone marrow cells and spleen of Gpx4+/Sec46Ala mice and compared the data with the corresponding values obtained for wild-type mice. Here we neither observed significant differences between the two genotypes (Figure7h). Taken together, these data did not provide any evidence for a compromised erythropoiesis in Gpx4+/Sec46Ala mice. Int. J. Mol. Sci. 2021, 22, 7795 12 of 20

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2.8. Heterozygous Expression of Catalytically Inactive Gpx4 in Mice Does Not Impact Ex Vivo Erythropoiesis of Embryonic Liver Derived Erythroblasts 2.8. HeterozygousIn early stages Expression of mammalian of Catalytically embryogenesis, Inactive Gpx4 in the Mice liver Does plays Not Impact a major Ex Vivo role in erythro- Erythropoiesis of Embryonic Liver Derived Erythroblasts poiesis [36,37]. Primary mouse erythroblasts can be prepared from fetal mouse liver and erythropoiesisIn early stages can of be mammalian induced in embryogenesis, these cells by the the liver addition plays ofa major erythropoietin role in erythro- (EPO) [38,39]. poiesis [36,37]. Primary mouse erythroblasts can be prepared from fetal mouse liver and Since our in vivo studies with Gpx4+/Sec46Ala mice did not support our conclusion (Gpx4 erythropoiesis can be induced in these cells by the addition of erythropoietin (EPO) deficiency[38,39]. Since delays our in erythropoiesis) vivo studies with drawn Gpx4 from+/Sec46Ala our micein did vitro notdata support we carried our conclusion out additional ex vivo(Gpx4 experiments deficiency delays to explore erythropoiesis) the possibility drawn from that our ex vivoin vitro erythropoiesis data we carried of out primary ad- mouse +/Sec46Ala erythroblastsditional ex vivo prepared experiments from to Gpx4explore the possibilitymice might that ex be vivo compromised. erythropoiesis For of pri- this purpose wemary prepared mouse erythroblasts hepatocytes prepared from embryos from Gpx4 of+/Sec46Ala wildtype miceand mightGpx4 be compromised.+/Sec46Ala mice, For expanded thethis primary purpose we mouse prepared erythroblasts, hepatocytes and from induced embryosin of vitro wildtypeerythropoiesis and Gpx4+/Sec46Ala by themice, addition of EPOexpanded [38,39 the]. Asprimary readout mouse parameters erythroblasts, for an erythroidd induced differentiation in vitro erythropoiesis we compared by the the pro- liferationaddition of kinetics EPO [38,39]. of the As tworeadout cell parameters types and for the erythroid expression differentiation of Hba-a1 we as compared erythroid marker (Figurethe proliferation8). Unfortunately, kinetics of the here two we cellneither types and observe the expression significant of Hba-a1 differences as erythroid between the marker (Figure 8). Unfortunately, here we neither observe significant differences between two genotypes. the two genotypes.

FigureFigure 8. HeterozygousHeterozygous expression expression of a ofcatalytically a catalytically inactive inactive Gpx4 in Gpx4 mice does in mice not doesimpact not ex impactvivo ex vivo erythropoiesis of embryonic liver derived erythroblasts. Fetal livers of day 12.5 mouse embryos erythropoiesis(wild type and Gpx4 of embryonic+/U46A) were liver prepared, derived homogenized erythroblasts. and primary Fetal livers erythroblasts of day 12.5 were mouse expanded embryos (wild +/U46A typefor 5 anddaysGpx4 in STEMPRO)TM were-34 SFM prepared, medium homogenized containing 50 ng/mL and primary recombinant erythroblasts murine stem were cell fac- expanded for 5tor, days 1 U/mL in STEMPRO human erythropoietin-alpha,TM-34 SFM medium and containing 1 µM dexamethasone. 50 ng/mL Terminal recombinant erythroid murine differenti- stem cell factor, 1ation U/mL was human induced erythropoietin-alpha, in continuously proliferating and 1 µerMythroblasts dexamethasone. by exposure Terminal to 5 U/mL erythroid erythropoi- differentiation etin-alpha and 0.5 mg/mL iron loaded human transferrin as described in Material and Methods. (a) wasCumulative induced cell in continuouslynumber of proliferating proliferating erythroblasts erythroblasts derived by from exposure Gpx4+/U46A to 5and U/mL the correspond- erythropoietin-alpha anding wild-type 0.5 mg/mL controls iron loaded(n = 3). ( humanb) Cumulative transferrin cell number as described after induction in Material of terminal and Methods. erythroid ( adif-) Cumulative cellferentiation number (n of = proliferating 3). (c) Morphological erythroblasts and histochemical derived from analysisGpx4 of+/U46A proliferatingand the and corresponding differentiated wild-type wild type (upper panels) and Gpx4+/U46A (lower panels) mouse erythroblast. Proliferating erythro- controls (n = 3). (b) Cumulative cell number after induction of terminal erythroid differentiation blasts (5 days) and erythroblasts that have been differentiated for 60 h in the presence of erythro- (npoietin = 3). (werec) Morphological HE stained and and subsequently histochemical counterstained analysis stained of proliferating with neutralized and differentiated benzidine to wild type (upperdetect hemoglobin panels) and expressionGpx4+/U46A (arrows).(lower (d panels)) Hba-a1 mouse mRNA erythroblast. expression du Proliferatingring terminal differentia- erythroblasts (5 days) andtion erythroblastsin wildtype (Gpx4 that+/+) haveand Gpx4 been deficient differentiated (Gpx4+/U46A for) cells. 60 h Total in the RNA presence was extracted of erythropoietin at different were HE time points of the culturing period. The steady-state concentrations of Hba-a1 mRNA was related stained and subsequently counterstained stained with neutralized benzidine to detect hemoglobin expression (arrows). (d) Hba-a1 mRNA expression during terminal differentiation in wildtype (Gpx4+/+) and Gpx4 deficient (Gpx4+/U46A) cells. Total RNA was extracted at different time points of the culturing period. The steady-state concentrations of Hba-a1 mRNA was related to Gapdh mRNA

as described in the Material and Methods section. n = 5. Significances were calculated using the Mann-Whitney U test and p-values < 0.05 were considered statistically significant. Int. J. Mol. Sci. 2021, 22, 7795 13 of 20

3. Discussion 3.1. Gpx4 Isoforms Are Expressed in MEL Cells To contribute to the discussion on the potential role of Gpx4 isoforms in erythropoiesis we selected the frequently employed in vitro erythropoiesis model of cultured MEL cells and explored whether expression silencing of this enzyme impacts the differentiation ki- netic of these cells. To find out whether these cells are a suitable model for our experimental strategy we first quantified the expression of Gpx4 on mRNA, protein, and activity levels. As indicated in Figure1a cGpx4 mRNA is the most abundant isoform. The expression levels of the different Gpx4-isoforms (cGpx4, mGpx4, and nGpx4) in MEL cells are comparable to those in mouse somatic tissues [16].

3.2. Gpx4 and Erythropoiesis When the Gpx4 gene was specifically deleted in hematopoietic cells mice develop ane- mia and mechanistic studies employing these tissue specific knockout mice suggested that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necropto- sis in erythroid precursor cells [21]. In fact, disruption of the Gpx4 gene induces functional inactivation of caspase 8 by glutathionylation. This post-translational modification of caspase 8 induces necroptosis, which proceeds independently of tumor necrosis factor α activation. Genetic ablation of Rip3 normalizes erythropoiesis and prevents anemia. These results demonstrate that erythroid-specific Gpx4 deficiency activates RIP3-dependent necroptosis in erythroid precursor cells [21]. In a more recent study the potential role of Gpx4 in the later stage of erythropoiesis, in particular in reticulocyte-erythrocyte transition was explored [29]. Quantification of the basic erythrocyte parameters in the peripheral blood and the bone marrow of chimeric mice with Gpx4 deficiency in erythropoietic cells revealed anemia with a compensatory increase of early erythroid precursors and reticulocytes. Additional dietary depletion of vitamin E aggravated the anemic phenotype. Interestingly, peripheral reticulocytes failed to mature normally but the cells accumulated large autophagosomes with engulfed mito- chondria. Moreover, Gpx4-deficiency induced systemic hepatic iron overload, increased iron demand in the erythropoietic system, and strongly elevated erythropoietin levels in blood plasma. From these data the authors concluded that Gpx4 deficiency disturbs reticulocyte-erythrocyte transition, which constitutes the last step of erythropoiesis [29]. In addition to the role of Gpx4 in murine erythropoiesis, a role during humane erythroid maturation has also been demonstrated. Inhibition of GPX4 was shown to impair enucle- ation of human erythroblasts in a ferroptosis-, mitophagy-, and necroptosis-independent manner [33]. Taken together these studies suggested a role of Gpx4 in early [21] and late erythro- poiesis [29,33]. To contribute to the discussion on the potential role of Gpx4 in erythro- poiesis we employed the frequently used MEL cell system. This cellular in vitro model mirrors the second phase of erythroid differentiation, which involves the maturation of nu- cleated proerythroblasts to erythroblasts [1,28]. This phase is characterized by hemoglobin biosynthesis, a decrease in cell size and nuclear condensation. Resting MEL cells do not express hemoglobin but stimulation with DMSO induces the expression of erythroid spe- cific genes such as hemoglobin and δ-ALA synthase [28,30]. To induce Gpx4 deficiency we stably transfected MEL cells with a RNAi construct and this technique induced a more than 50% reduction of Gpx4 expression (Figure1). Interestingly, the transfection procedure did not induce major alterations in basic cell characteristics. These Gpx4-deficient cells were viable and showed similar growth kinetics as corresponding mock-transfected con- trols (Figure2d ). When erythropoiesis was induced by incubating the cells with DMSO we observed a temporal delay of erythropoiesis and these results suggested that Gpx4 may also be important for the 2nd phase of erythropoiesis. Together with the above men- tioned in vivo studies [21,29] our in vitro data suggest a role of Gpx4 in all three stages of erythropoiesis. Int. J. Mol. Sci. 2021, 22, 7795 14 of 20

The molecular basis for the Gpx4-deficiency induced delay of in vitro erythropoiesis has not been explored in this study. Gpx4 expression silencing is likely to impact the redox state of the MEL cells and thus may modify the expression of redox regulated genes. Such genes have been implicated in erythropoiesis [28,40,41] and their catalytic activity might be modulated by Gpx4 expression silencing. As many permanent cell lines MEL cells are cancer cells. Nevertheless, they have frequently been used as model systems for in vitro erythropoiesis since they adequately mirror the second phase of mouse erythropoiesis. We cannot exclude that the retardation effect we observed may be related cancer character of these cells and this is a limitation of this study. However, similar limitations are also applicable to any other studies using cancer cell lines including previous erythropoiesis studies with MEL cells [28,30].

3.3. Heterozygous Expression of a Catalytically Inactive Gpx4 Mutant Does Not Compromise In Vivo Erythropoiesis in Mice To translate our in vitro findings into the in vivo situation we explored Gpx4+/U46A mice. These knock-in mice express heterozygously the catalytically silent Sec46Ala Gpx4 mutant. The animals express significantly lower amounts of catalytically active Gpx4 and comparative activity assays with testis and brain indicated a 50% reduction of spe- cific Gpx4 activity [16]. A similar reduction in the catalytic activity was observed when Gpx4-RNAi-transfected MEL cells were compared with corresponding mock-transfectants (Figure2c). From this activity comparison we hypothesized that the Gpx4+/U46A knock-in mice might also exhibit a defective erythropoietic phenotype. To test this hypothesis, we compared the basic erythrocyte parameters of Gpx4+/U46A mice and corresponding wild type controls (Figure7a–d) but did not observe significant differences. To exclude major compensation reactions, we also compared spleen weights (upregulated extramedullary erythropoiesis) and kidney weights (elevated erythropoietin production) but neither ob- served significant differences between Gpx4+/U46A mice and wildtype controls (Figure7e,f). Moreover, we found that renal erythropoietin synthesis was not upregulated in Gpx4+/U46A mice (Figure7e ). Taken together, these data suggest that heterozygous expression of a catalytically silent Gpx4 mutant did not compromise in vivo erythropoiesis. It looks like that this genetic manipulation may not be severe enough to induce defective red cell development in either of its three stages.

4. Materials and Methods 4.1. Chemicals and Reagents The chemicals were from the following sources: DMSO from AppliChem (Darm- stadt, Germany), Bradford Reagenz from Roth (Karlsruhe, Germany), glutathione, glu- tathione reductase, tetramethylbenzidine, hemoglobin, NADPH and α-tocopherol from Sigma-Aldrich (Steinheim, Germany), ebselen from Rhone-Poulenc Rorer GmbH (Cologne, Germany) and isoflurane from AbbVie (Ludwigshafen, Germany). Oligonucleotide syn- thesis was performed at BioTez (Berlin, Germany). Other chemicals were purchased from Invitrogen (Darmstadt, Germany).

4.2. Animals, Breeding, and Isolation of Spleen and Bone Marrow All the mice were bred and maintained in a specific pathogen-free (SPF) animal facility, on 12 h/12 h LD cycle, with food and water ad libitum. Procedures were performed accord- ing to the EU Directive 2010/63/EU, Federation of Laboratory Animal Science Associations and the local guidelines. All animal experiments were performed in compliance with the German animal welfare law and have been approved by the institutional committee on animal experimentation. Gpx4+/U46A mice were created as described before [16] and corre- sponding wildtype control animals were outbred by mating heterozygous allele carriers. Separate colonies of Gpx4+/U46A mice and outbred wild-type controls were maintained. All individuals were genotyped. Int. J. Mol. Sci. 2021, 22, 7795 15 of 20

4.3. PCR Genotyping Genomic DNA was prepared from ear punch using Invisorb Spin Tissue Mini Kit (Invisorb, Berlin, Germany) and genomic PCR was carried out with MyTaqTMRed Mix (BIOLINE, Heidelberg, Germany) system. Primers used for analyzing the Gpx4 gene locus were as follows: up- 50- GACAGATGGCTCTGGACCTGGGTG-30, do-: 50-TAATCTGGCG TGGTAGGGGCA GAC-30. The wildtype allele showed up as a 412 bp fragment but the Sec46Ala mutant allele gave an amplification product of higher molecular weight (587 bp). The higher molecular weight was due to a residual LoxP/FRT sequence in the genome after deletion of the neo-cassette. Heterozygous allele carriers showed two bands in PCR.

4.4. MEL Cell Culture Murine erythroleukemia cells (MEL-745A cl.DS19) were obtained from the German Collection of Microorganisms and Cell Cultures (Leibniz Institut, Braunschweig, Germany) and clone 19 is described to be DMSO sensitive, resulting in erythroid differentiation. Cells were grown in RPMI-medium (PAN BIOTECH, Aidenbach, Germany) containing 10% fetal ◦ calf serum (PAN BIOTECH) at 37 C with 5% CO2.

4.5. Antisense Transfection and Selection Procedure The siRNA plasmids were kindly provided by Dr. Nicolai Savaskan [32]. Briefly, for Gpx4 mRNA silencing the pSuper plasmid vector system was used (NKI, Amsterdam, the Netherlands). The target sequence for murine GPx4 50-TGGTCTGCCTGG ATA AGT A-30 was cloned into the pSuper-GFP (Gpx4 siRNA) vector according to the manufacturer’s recommendations (NKI) as described in [42]. As control empty pSuper-GFP vector was used (MOCK siRNA). For stable transfection, MEL cells were transfected with either the Gpx4 si RNA plasmid or the corresponding control plasmid using TransIT-LT1 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer’s recommendations. Individual clones were selected with 400 µg/mL geneticin (G418, Roth) and the degree of Gpx4 mRNA silencing was quantified by quantitative real-time PCR (see Section 4.7) Three representative subclones of Gpx4 siRNA and mock-transfected MEL cells were selected for further analysis. The cell viability was routinely examined by the trypan blue staining.

4.6. DMSO Induced Erythroid Differentiation in Stable MEL Cell Transfectants Stable MEL cell transfectants were seeded at 104 cell/mL. Erythroid differentiation of the cell culture was induced by adding DMSO to the final concentration of 2%. Stimulated and untreated control cells were cultured for up to 6 days. Cells were kept in a concentration of 0.5 × 106 cells/mL. The cell viability was routinely examined by the trypan blue staining. Aliquots were harvested for RNA isolation (see Section 4.7) and hemoglobin determination (see Section 4.8). For growth analysis cells were counted every day and doubling time was calculated in the ex- ponential phase of the growth curves using the following formula: Td = Tx × ln (2)/ln (Nx/N0), were Td—cell population doubling time, Tx—cultivation time, Nx—number of cells at Tx time point, and N0—the initial number of cells [43]. For rescue experiments we added 100 µM α-tocopherol or 5 µM ebselen to the culture medium before erythroid differentiation was in- duced by adding DMSO to the final concentration of 2%. The steady-state concentrations of Hba-a1 mRNA, Alas2 mRNA and Gapdh mRNA were quantified by qRT-PCR using specific amplification primers (Table1). For the determination of the DMSO sensitivity of the Gpx4 RNAi transfectant, the normalized expression of Hba-a1 and Alas2 in the DMSO-untreated sample was subtracted from the DMSO-treated sample and then related to the corresponding mock transfectant.

4.7. RNA Isolation and Quantitative Real-Time PCR Total RNA was extracted using the NucleoSpin RNA Plus (Macherey-Nagel, Düren, Germany). Synthesis of cDNAs was performed with 0.1–2 µg of the total RNA prepara- tions using oligo (dT) primers and Tetro Reverse Transcriptase (BIOLINE, Luckenwalde, Int. J. Mol. Sci. 2021, 22, 7795 16 of 20

Germany). Quantitative real-time PCR (RT-PCR) was carried out with a Rotor Gene 3000 (Corbett Research, Sydney, Australia) using the SensiFast SYBR PCR Kit (BIOLINE). The following primer sets were employed to quantify the Gapdh, mGpx4, m + cGpx4, nGpx4, Epo, Hba-a1, Alas2 (Table1). The Rotor-Gene Q software was used to analyze the PCR raw data. Specific amplicons were prepared as external amplification standards for all target and reference (Gapdh) genes and this procedure allowed exact quantification of the mRNA copy numbers. Gapdh mRNA was used as an internal amplification standard and expression of the target genes was normalized to the Gapdh content. Analyses were performed at least in triplicate and mean ± standard deviations are given.

Table 1. Primer sets used for quantitative real-time PCR.

Amplicon Forward/Reverse Gene ID 50-CCATCACCATCTTCCAGGAGCGA-30 Gapdh 14433 50-GGATGACCTTGCCCACAGCCTTG-30 50-GAGATGAGCTGGGGCCGTCTGA-30 mGpx4 625249 50-ACGCAGCCGTTCTTATCAATGAGAA-30 50-CGCCTGGTCTGGCAGGCACCA-30 m + cGpx4 625249 50-ACGCAGCCGTTCTTATCAATGAGAA-30 50-AGTTCCTGGGCTTGTGTGCATCC-30 nGpx4 625249 50-ACGCAGCCGTTCTTATCAATGAGAA-30 50-CACCCTGCTGCTTTTACTCTCCTT-30 Epo 13856 50-CTTCTGCACAACCCATCGTGACAT-30 50-ATGGTGCTCTCTGGGGAAGACAAA-30 Hba-a1 15122 50-TCATCGAGGTGGCCTGCAGCATT-30 50-ATGGTGGCAGCAGCTATGTTGCTA-30 Alas2 11656 50-CTTGAACTTCTGGAGCTGCCCTC-30

4.8. Photometric Hemoglobin Determination Hemoglobin concentrations in the MEL cell lysates were measured by the tetram- ethylbenzidine assay [44]. Small amounts of cell culture were removed to different time points of incubation. Pellets of MEL cells were lysed by the addition of 5% acetic acid and centrifuged at 10,000× g for 10 min. The supernatant was transferred to a new tube and total protein concentration was determined by the Bradford method [45]. Tetramethylben- zidine assay was adapted to 96 well plates and the absorbance was measured at 600 nm. Values obtained from triplicates were normalized to the total protein content of the cell lysate. Hemoglobin concentration was determined by comparing the absorbance values to a standard curve prepared by similar incubation of pure human hemoglobin solutions.

4.9. Immunoblotting Protein extracts were prepared from stable MEL cell RNAi transfectants (mock- transfected and Gpx4 RNAi). Aliquots with an equal amount of total protein (100 µg) were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The proteins were transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with Blotting Grade Blocker (Bio-Rad, Feldkirchen, Germany) and incubated overnight with the monoclonal anti-human Gpx4 antibody (#MABF1969, Merck, Darmstadt, Germany) [31], or anti-GAPDH (Sigma, Taufkirchen, Germany). The membrane was washed with PBS/0.3% Tween 20 and incubated with secondary antibodies (anti-mouse: Sigma or anti-rabbit: Santa Cruz, Heidelberg, Germany) for 30 min. Blots were developed with an ECL kit (PerkinElmer, Rodgau, Germany).

4.10. Gpx4 Activity Assay Bone marrow, spleen or MEL cells were lysed in 0.5 mL of lysis buffer (50 mM Tris-HCl, 0.3 mM KCl, 1 mM EDTA, 10% glycerol, 5 mM TCEP, 1% Triton X100, protease inhibitory Int. J. Mol. Sci. 2021, 22, 7795 17 of 20

mix (SERVA, Heidelberg, Germany), pH 7.5). After centrifugation at 10,000× g for 10 min the supernatants were used for Gpx4 activity assays. Gpx4 activity was assayed spec- trophotometrically employing the coupled optical test [16]. The assay mixture consisted of 1 mL 0.1M Tris/Cl buffer, pH 7.4, containing 5 mM ethylenediaminetetraacetic acid, 0.1% Triton X-100, 0.2 mM NADPH, 3 mM glutathione, and 1 U glutathione reductase. Aliquots of 10,000 g lysate supernatants of MEL cell homogenates (mock-transfected or Gpx4-deficient cells), bone marrow cells or spleen (wild-type or Gpx4+/U46A mice) were added, and the assay mixture (absence of substrate) was preincubated at 37 ◦C for 5 min. Then, the reaction was started by the addition of 25 µM phosphatidylcholine hydroperox- ide, and the decrease in absorbance at 340 nm was measured (molar extinction coefficient for NADPH of 6.22 × 106 (M × cm)−1).

4.11. Ex Vivo Erythropoiesis 4.11.1. Cultivation of Murine Erythroid Progenitors Mouse erythroblasts were grown from fetal livers as described by von Lindern [38]. Fe- tal livers of day 12.5 mouse embryos (wild type Gpx4+/+ or Gpx4+/U46A) were resuspended in 1 mL serum-free stem cell expansion medium (STEMPROTM-34 SFM Complete Medium; Life Technologies GmbH, Darmstadt, Germany). Cells were passed through a 70 µm Nylon cell strainer (BD Biosciences, Erembodegem, Belgium). The washed cells were seeded into STEMPROTM-34 SFM medium containing 50 ng/mL recombinant murine stem cell factor (SCF, PromoCell GmbH, Heidelberg, Germany), 1 U/mL human erythropoietin-alpha (PromoCell GmbH), and 1 µM dexamethasone (PromoCell GmbH). The proerythroblast culture was expanded for five days keeping 0.7–1 × 106 cells/mL by daily partial medium changes with the addition of fresh factors.

4.11.2. Induction and Analysis of Terminal Differentiation Terminal differentiation was performed as described by van Lindern [38]. Erythrob- lasts were washed twice in PBS and seeded at 1.5 × 106 cells/mL in STEMPROTM-34 SFM medium containing 5 U/mL erythropoietin-alpha and 0.5 mg/mL iron-loaded hu- man Transferrin, (Sigma-Aldrich). Differentiating erythroblasts were maintained between 2–4 × 106 cells/mL. Erythroblasts at various stages of differentiation were counted, cy- tocentrifuged onto glass slides, and stained with 3,30-dimethoxybenzidine (Sigma) for hemoglobin [46]. The cell morphology was analyzed by staining with histological dyes (Giemsa stain, Roth).

4.12. Blood Analysis and Organ Collection For diagnostic blood analysis and organ preparation mice were sacrificed by cervical dislocation after isoflurane anesthesia. Blood was collected by cardiac puncture into EDTA- containing tubes. The quantification of the basic blood parameters was carried out at the Institute for Veterinarian Diagnostics (Berlin, Germany) using a hematology analyzer (XT-2000iV, Sysmex, Landskrona, Sweden). To isolate bone marrow cells, the femur bones were prepared. Both the ends of the bones were cut off using a scalpel, and the bone marrow cells were eluted from the bone marrow cavity with 10 mL phosphate-buffered saline (PBS). Cells were washed once with 1 mL of PBS and pelleted by centrifugation (800× g).

4.13. Statistical Analysis Statistical analyses were carried out with SPSS 23 (IBM, Armonk, NY, USA) software package and the results are presented as medians. For experiments, in which the data are not normally distributed two-tailed Mann/Whitney U test was employed. Significance was accepted at p < 0.05. Within the boxplots the black vertical lines indicated the median. The 25th and the 75th percentile are visualized as upper and lower box limits. For normally distributed data the Students t-test was used. Int. J. Mol. Sci. 2021, 22, 7795 18 of 20

5. Conclusions RNAi-induced expression silencing of Gpx4 in mouse erythroleukemia cells delays in vitro erythropoiesis and these data indicate that Gpx4 plays a role in this process. On the other hand, mice expressing heterozygously a catalytically inactive mutant of Gpx4 (Gpx4+/Sec46Ala knock-in mice) do not show a defective erythropoietic phenotype and similar results were obtained in ex vivo erythropoiesis studies using embryonic erythroid precursors. Thus, heterozygous expression of a catalytically inactive Gpx4 mutant does not compromise in vivo erythropoiesis.

Author Contributions: Conceptualization, H.K. and A.B.; methodology and investigation, M.R. and A.B.; visualization, A.B.; writing—original draft preparation, H.K. and A.B.; writing—review and editing, M.R., H.K. and A.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by in part by research grants from Deutsche Forschungsgemein- schaft to H.K., grant number Ku 961/13-1, Ku 961/14-1. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki with official approval by the local animal care committee (T 0437/08). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Marieke von Lindern (Sanquin Research, Ams- terdam, The Netherlands) for valuable advice for performing the ex vivo erythropoiesis experiments. Conflicts of Interest: The authors declare no conflict of interest.

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