Apoptosis-Resistance of Senescent Cells Is an Intrinsic Barrier for Senolysis Induced by Cardiac Glycosides

bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423449; this version posted December 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Apoptosis-resistance of senescent cells is an intrinsic barrier for senolysis induced by

2 cardiac glycosides

3 Pavel I. Deryabin1, Alla N. Shatrova2, Irina I. Marakhova2, Nikolay N. Nikolsky2, Aleksandra V.

4 Borodkina1,*

7 1Mechanisms of cellular senescence group, Institute of Cytology of the Russian Academy of

8 Sciences, Tikhoretsky Ave. 4, 194064, Saint-Petersburg, Russia

9 2Department of intracellular signaling and transport, Institute of Cytology of the Russian

10 Academy of Sciences, Tikhoretsky Ave. 4, 194064, Saint‑Petersburg, Russia

12 Correspondence to Aleksandra Borodkina, Tikhoretsky ave. 4, 194064, St-Petersburg, Russia;

13 Tel.: +7-981-680-14-03; [email protected].

17 Running title: Apoptosis-resistance hampers senolysis

20 Keywords: stem cells, senescence, apoptosis, stress-resistance, senolysis

25 26 27 1

28 ABSTRACT

29 Targeted elimination of senescent cells – senolysis – is one of the core trends in the anti-aging

30 therapy. Cardiac glycosides were recently proved to be a broad-spectrum senolytics. Here we

31 tested senolytic properties of cardiac glycosides towards human mesenchymal stem cells

32 (hMSCs). Cardiac glycosides had no senolytic ability towards senescent hMSCs of various

33 origins. Using biological and bioinformatic approaches we compared senescence development in

34 ‘cardiac glycosides–sensitive’ A549 and ‘–insensitive’ hMSCs. The absence of senolysis was

35 found to be mediated by the effective potassium import and increased apoptosis-resistance in

36 senescent hMSCs. We revealed that apoptosis-resistance, previously recognized as a common

37 characteristic of senescence, in fact, is not a general feature of senescent cells. Moreover, only

38 apoptosis-prone senescent cells are sensitive to cardiac glycosides-induced senolysis. Thus, we

39 can speculate that the effectiveness of senolysis might depend on whether senescent cells indeed

40 become apoptosis-resistant compared to their proliferating counterparts.

42 INTRODUCTION

43 Today cellular senescence is considered as a common reaction of almost all types of

44 proliferating cells, including cancer and stem ones, as well as some post-mitotic cells to a variety

45 of stressful stimuli (Sapieha and Mallette, 2018; Lee and Schmitt, 2019; von Zglinicki et al.,

46 2020). The following types of cellular senescence can be distinguished accordingly to the origin

47 of the inducing factor: replicative (due to the DNA damage at the shortened telomere ends),

48 oncogene-induced (in response to aberrant activation of oncogenic signaling), stress-induced

49 (mediated by the DNA damage caused by the oxidative stress, heat shock, UV and γ radiation,

50 etc.), and chemotherapy-induced (activated in cancer cells in response to chemotherapeutic

51 drugs) (Campisi, 1996; Serrano et al., 1997; Toussaint, et al., 2000; Roninson et al., 2001). There

52 is a heterogeneity in markers expressed by senescent cells depending on both cell type and an

53 insult used to induce senescence. However, there are several common features typical for the

54 most types of senescent cells. The essential characteristic of senescence for any kind of dividing

55 cells is the irreversible proliferation loss (Hernandez-Segura et al., 2018). The irreversibility of

56 the cell cycle arrest is controlled by the cyclin-dependent kinase (CDK) inhibitors p16 and p21

57 and is often regulated by the tumor suppressor protein p53 (Hernandez-Segura et al., 2018;

58 Wang et al., 2020). The other important features of senescent cells are the activation of a

59 persistent DNA damage response; cell hypertrophy, which often arises as a result of impaired

60 ribosomal biogenesis and protein synthesis; disturbance of lysosomal degradation and

61 dysfunction of the rest degradation systems; increased activity of the specific lysosomal enzyme

62 senescence-associated-β-galactosidase; various mitochondrial alterations; acquisition of the

63 senescence-associated secretory phenotype, composed of pro-inflammatory factors, matrix

64 degrading enzymes, reactive oxygen species, etc; epigenetic and chromatin landscape alterations,

65 including formation of senescence-associated heterochromatic foci and senescence-associated

66 distention of satellites (Nacarelli et al., 2017; Hernandez-Segura et al., 2018; Wang et al., 2020).

67 In other words, senescent cells preserve metabolic activity and vitality, but their functioning is

68 significantly altered comparing to the origin cells.

69 It is now clear that senescent cells can modify surrounding microenvironment affecting

70 both neighboring cells and cellular niches, what may lead to tissue malfunctioning and therefore

71 may be related to the progression of aging and age-related diseases (Munoz-Espin and Serrano,

72 2014; Ovadya et al., 2018). Keeping that in mind, today more attention is focused on the

73 strategies for targeted “killing” of senescent cells (Kirkland and Tchkonia, 2020; Pignolo et al.,

74 2020; Robbins et al., 2020). To this end, a novel class of drugs termed senolytics is actively

75 developing. Senolytics target signaling pathways that contribute to the resistance of senescent

76 cells towards apoptosis, thus inducing apoptosis preferentially in senescent cells (Zhu et al.,

77 2015). The list of senolytics is constantly replenishing with the new agents. The most known

78 compounds with the stated senolytic activity are navitoclax (Bcl‐2 family inhibitor), combination

79 of dasatinib (an inhibitor of multiple tyrosine kinases) and quercetin (a natural flavonol), Hsp90

80 inhibitors, MDM2 inhibitors, FOXO4-p53 interfering peptide, a BET family protein degrader,

81 uPAR-specific CAR-T, galacto-conjugated navitoclax and various senolytic natural compounds

82 (Zhu et al., 2015; Zhu et al., 2016; Baar et al., 2017; Fuhrmann-Strossnigg et al., 2017; Jeon et

83 al., 2017; Zhu et al., 2017; Amor et al., 2020; Gonzalez-Gualda et al., 2020; Wakita et al., 2020).

84 Recently, using high-throughput drug screening two independent research groups identified

85 cardiac glycosides, particularly ouabain, digoxin and bufalin, as a broad-spectrum senolytics

86 (Guerrero et al., 2019; Triana-Martinez et al., 2019). It is worth mentioning that almost all of the

87 declared senolytics have limitations, such as undesirable side-effects or ineffectiveness towards

88 some cell types.

89 Mesenchymal stem cells (MSCs), found virtually in all post-natal organs/tissues, are

90 characterized by the capacity to self-renew (symmetric divisions) and to differentiate, thus

91 contributing to maintenance and regeneration of the residing tissue (da Silva Meirelles et al.,

92 2006). Due to these unique properties, along with the potent anti-inflammatory and

93 immunosuppressive functions, MSCs are broadly applied in the cell replacement therapy for

94 treatment of various diseases, including diabetes mellitus, multiple sclerosis, myocardial

95 infarction, and so on (Zhou and Xu, 2020). However, similar to their differentiated progenies and

96 other unipotent cells, MSCs are able to senesce either replicatively or prematurely in response to

97 oncogenes’ activation and stressful stimuli (Burova et al., 2013; Zhou et al., 2020). MSCs’

98 senescence has a plenty of undesirable aftermaths, among which are exhaustion of the pool of

99 stem cells, reduced tissue maintenance and regeneration, impaired differentiation capacity,

100 SASP-mediated senescence spreading, reduced angiogenic properties, modification of stem cell

101 niche (Griukova et al., 2019; Liu et al., 2020; Turinetto et al., 2016; Zhou et al., 2020). Taking

102 into account biological significance of the proper MSCs’ functioning, senescent MSCs might be

103 considered as the crucial target for senolysis. In line with this suggestion, a number of reviews

104 highlighting possible advantageous outcomes of senescent MSCs removal has been published

105 over the past year (Lee and Yu, 2020; Liu et al., 2020; Spehar et al., 2020; Zhou et al., 2020).

106 Nevertheless, there are only few experimental data regarding this issue (Grezella et al., 2018;

107 Peng et al., 2020; Sharma et al., 2020; Zhang et al., 2020).

108 Within the present study we demonstrate for the first time that cardiac glycosides, namely

109 ouabain and bufalin, fail to display senolytic activity towards human MSCs (hMSCs) derived

110 from endometrium (END-MSCs), adipose tissue (AD-MSCs), dental pulp (DP-MSCs) and

111 Warton jelly (WJ-MSCs). Additionally, we confirm that both cardiac glycosides are able to

112 induce apoptosis preferentially in senescent A549, as it was previously described in the pilot

113 studies (Guerrero et al., 2019; Triana-Martinez et al., 2019). By assessing alterations in ionic

114 homeostasis caused by the Na+/K+-ATPase blocking and expression levels of the related genes

115 we reveal that the absence of ouabain-induced senolysis might be mediated by the enhanced

116 effectiveness of the compensatory K+ import in senescent END-MSCs compared to senescent

117 A549. Furthermore, using advanced bioinformatics we demonstrate that senescence of END-

118 MSCs, resistant to ouabain-induced senolysis, is accompanied by the acquisition of apoptosis-

119 resistant phenotype, while senescence of ouabain-sensitive A549 is not. Therefore, we provide

120 clear evidence that apoptosis-resistance is not a general feature of senescent cells. Based on the

121 data obtained we conclude that the effectiveness of senolytic approaches might depend on

122 whether cells indeed became apoptosis-resistant during senescence development.

123

124 RESULTS

125

126 1. H2O2 –treated END-MSCs enter the premature senescence

127 Within the present study we used human mesenchymal stem cells isolated from

128 desquamated endometrium (END-MSCs), which satisfy the minimum criteria suggested by the

129 ISCT for defining hMSCs (Deryabin et al., 2019). Previously, we have developed the reliable

130 experimental model to study various aspects of the premature senescence of END-MSCs

131 (Burova et al., 2013). Namely, we have shown that END-MSCs subjected to sublethal oxidative

132 stress gradually acquired all the typical features of senescent cells, including persistent DNA

133 damage foci and active DNA damage response, irreversible cell cycle arrest mediated by the

134 classical p53/p21/Rb pathway, proliferation loss, cell hypertrophy, appearance of SA-β-Gal

135 staining and development of senescence-associated secretory phenotype (Burova et al., 2013;

136 Borodkina et al., 2014; Griukova et al., 2018). Here we applied the designed model to study

137 senolytic effect of ouabain as well as to reveal the underlying intracellular alterations in ion

138 homeostasis. Initially, by estimating the most common parameters we confirmed that single-dose

139 (1 h, 200 μM) H2O2 treatment was sufficient to induce senescence in END-MSCs. Importantly,

140 stressed END-MSCs were considered senescent in two weeks after the oxidative stress;

141 therefore, all the senescence markers were assessed not earlier than 14 days after H2O2 treatment.

142 Indeed, H2O2-treatment of END-MSCs led to proliferation block, cell hypertrophy as indicated

143 by the increased cell size, accumulation of the lipofuscine detected by the elevation of

144 autofluorescence, appearance of the senescence-associated β-galactosidase staining (SA-β-Gal),

145 activation of the p21/Rb pathway, and loss of HMGB1 together supporting senescence

146 establishment under the chosen experimental conditions (Figure 1A–C,E,F). The most harmful

147 effects of senescent cells at tissue and organismal levels are believed to be the consequence of

148 their prolonged vitality. In line with this point, H2O2-treated END-MSCs retained high viability

149 even at the late stages of senescence development (viability of senescent END-MSCs was

150 assessed in 17 days (14 days + 3 days) after the initial oxidative stress) (Figure 1D). To sum up,

151 sublethal H2O2-treatment of END-MSCs is the appropriate model of the premature senescence

152 and is relevant to investigate the effects of senolytic compounds on END-MSCs.

153

154 155 Figure 1. Validation of oxidative stress induced END-MSCs premature senescence model. Senescent 156 END-MSCs (A) lose proliferation, (B) undergo hypertrophy, (C) acquire elevated autofluorescence, 157 retain high cell viability (D) and (E) display SA-β-Gal activity compared to the young ones. (F) 158 Phosphorylation levels of p53 and Rb and expression levels of p21 and HMGB1 proteins in young and 159 senescent END-MSCs. Values presented are mean ± s.d. For multiple groups comparisons at (A) and (D) 160 one-way ANOVA was applied, n = 3, ns – not significant, *** p < 0.001. For pair comparisons at (B), 161 (C), and (E) Welch’s t-test was used, n = 3 for b and c, n = 50 for e, *** p < 0.001. Scale bars for images 162 are 500 μm. GAPDH was used as loading control. 163

164 2. Cardiac glycoside ouabain has no senolytic activity towards senescent END-MSCs in a

165 wide concentration range

166 Recent evidence suggest that cardiac glycosides, including ouabain, digoxin, bufalin,

167 represent a family of compounds with senolytic activity (Guerrero et al., 2019; Triana-Martinez

168 et al., 2019). Even though today cardiac glycosides are considered as the broad-spectrum

169 senolytics, the data regarding their effects towards senescent hMSCs are lacking. Therefore, here

170 we tested whether ouabain has the potential to induce death selectively in senescent END-MSCs.

171 To do so, we assessed viability of young and senescent END-MSCs after treatment with ouabain

172 at the wide concentration range (from 10-7 to 10-5 M). Interestingly, neither concentration applied

173 led to the noticeable decrease in the viability of both young and senescent cells on the first day

174 after ouabain application (Figure2–figure supplement 1). However, on the third day after ouabain

175 treatment we revealed significant dose-dependent decline in the viability of young END-MSCs

176 (Figure 2A, Figure2–figure supplement 1). Unexpectedly, senescent cells turned out to be more

177 resistant towards ouabain at each concentration tested (Figure 2A, Figure2–figure supplement 1).

178 In line with this result, 10-6 M ouabain led to more prone apoptotic death in young cells (20.36 %

179 An+/PI- and 34.99 % An+/PI+) compared to senescent ones (15.69 % An+/PI- and 12.42 %

180 An+/PI+) (Figure 2B). Obtained results clearly demonstrate that in context of senescent END-

181 MSCs ouabain has no senolytic activity.

182

183 184 Figure 2. Ouabain has no senolytic activity towards senescent END-MSCs in a wide concentration range. 185 (A) Relative cell viability (%) of young and senescent END-MSCs in 3 days after treatment with 10-7, 10- 186 6, 10-5 M ouabain. (B) Apoptosis induction in END-MSCs upon 10-6 M ouabain assessed by Annexin 187 V/DAPI double staining. n = 3 independent experiments. All data correspond to the mean ± s.d. Statistical 188 significance was assessed by the Welch’s t-test: ns – not significant, ***p < 0.05.

189 3. Ouabain is able to induce apoptosis selectively in senescent A549 cells

190 Taking into account the fact that our results do not correspond with the recently published

191 evidences regarding broad-spectrum senolytic action of cardiac glycosides, we decided to

192 reproduce this effect using cellular model described in the relevant studies (Guerrero et al., 2019;

193 Triana-Martinez et al., 2019). Thus, we performed series of experiments using young and

194 senescent A549 lung carcinoma cells. Senescence in A549 cells was induced by etoposide

195 treatment. Etoposide-induced senescence of A549 cells is a frequently used and thus well

196 characterized model of therapy-induced senescence (Wang et al., 2017). Also, ouabain was

197 shown to selectively kill etoposide-treated senescent A549 cells (Guerrero et al., 2019). To prove

198 senescence in A549, we assessed proliferation rate, cell size, accumulation of lipofucine, SA-β-

199 Gal staining, activation status of the p53/p21/Rb pathway and expression level of HMGB1

200 (Figure 3A–E).

201 202 Figure 3. Validation of etoposide induced A549 senescence model. Senescent A549 cells display (A) loss 203 of proliferation, (B) acquire elevated cell size, (C) autofluorescence level and (D) SA-b-Gal activity 204 compared to the young ones. (E) Phosphorylation levels of p53 and Rb and expression levels of p21 and 205 HMGB1 proteins in young and senescent A549. Values presented are mean ± s.d. For multiple groups 206 comparisons at (A) one-way ANOVA was applied, n = 3, ns – not significant, *** p < 0.001. For pair 207 comparisons at (B), (C), and (D) Welch’s t-test was used, n = 3 for (B) and (C), n = 50 for (D), *** p < 208 0.001. Scale bars for images are 500 μm. GAPDH was used as loading control. 209 210 To verify senolytic activity of ouabain towards senescent cancer cells, we first estimated

211 dose-dependent cell viability. In line with our results described above, we were not able to detect

212 any significant decline in the number of viable young or senescent A549 cells within 24 h after

213 ouabain application (Figure 4––figure supplement 1). However, in 3 days after treatment ouabain

214 significantly reduced viability of senescent A549 cells in a dose-dependent manner, while the

215 number of young A549 cells decreased to a much lesser extent (Figure 4A, Figure 4––figure

216 supplement 1). Namely, approximately 90 % of young cells preserved viability at 10-6 M ouabain

217 compared to 50 % of senescent A549 cells treated with the same dose (Figure 4A).

218 According to the published data, ouabain triggered caspase-3-dependent apoptosis in

219 senescent A549 cells (Triana-Martinez et al., 2019). Indeed, we revealed significant increase in

220 the double positive AnV/PI-fraction in senescent cancer cells treated with 10-6 M ouabain (Figure

221 4B). Furthermore, using fluorescent assay we observed activation of caspase-3 in ouabain-treated

222 senescent A549 cells (Figure 4C). Taken together, these results are completely consistent with

223 the data described by other authors, and confirm senolytic activity of ouabain towards A549.

224

225 226 Figure 4. Ouabain induces caspase-dependent apoptosis selectively in senescent A549 cells. (A) Relative 227 cell viability (%) of young and senescent A549 in 3 days after treatment with 10-7, 10-6, 10-5 M ouabain. 228 (B) Apoptosis induction in A549 upon 10-6 M ouabain assessed by Annexin V/DAPI double staining. (C) 229 Activity of Caspase-3/7 estimated in young and senescent A549 treated with 10-6 M ouabain. n = 3 230 independent experiments. All data correspond to the mean ± s.d. Statistical significance was assessed by 231 the Welch’s t-test: * ns – not significant, p < 0.05, **p < 0.01.

232 233 4. Cardiac glycoside bufalin is able to induce senolysis in A549 cells, while fails to kill

234 senescent END-MSCs

235 To verify the opposite senolytic action of cardiac glycosides towards A549 and END-

236 MSCs, we applied bufalin, another compound with the stated senolytic activity belonging to the

237 cardiac glycosides family (Guerrero et al., 2019). Bufalin had almost no effect on the viability of

238 young and senescent END-MSCs in a wide concentration range (from 10-7 to 10-5 M) (Figure

239 5A, Figure 5–figure supplement 1A). Contrarily, both types of A549 cells were sensitive to

240 bufalin (Figure 5B, Figure 5–figure supplement 1B). Moreover, senescent A549 cells were more

241 prone towards bufalin-induced cell death than their young counterparts (Figure 5B). The results

242 described above confirm that cardiac glycosides indeed have senolytic activity towards A549,

243 but these compounds turned to be ineffective for targeted death induction in senescent END-

244 MSCs.

245 246 Figure 5. Cardiac glycoside bufalin is able to induce senolysis in A549 cells and fails to kill senescent 247 END-MSCs. (A) Relative cell viability (%) of young and senescent END-MSCs in 3 days after treatment 248 with 10-7, 10-6, 10-5 M bufalin. (B) Relative cell viability (%) of young and senescent A549 in 3 days after 249 treatment with 10-7, 10-6, 10-5 M bufalin. All data correspond to the mean ± s.d. Statistical significance 250 was assessed by the Welch’s t-test: ns – not significant, *p < 0.05.

251 5. Ouabain has no senolytic action towards human mesenchymal stem cells of various

252 origins

253 To broaden our observations regarding the absence of ouabain-induced senolysis in END-

254 MSCs, we analyzed ouabain effects on hMSCs isolated from other sources including adipose

255 tissue, dental pulp and Wharton’s jelly. To additionally strengthen our data, we applied different

256 models of senescence – replicative senescence for AD-MSCs, doxorubicine-induced senescence

257 for DP-MSCs and oxidative stress-induced senescence for WJ-MSCs (Figure 6A–F).

258 259 Figure 6. Validation of the three additional hMSCs senescence models, replicative for AD-MSCs and 260 premature for DP-MSCs (doxorubicin induced) and WJ-MSCs (oxidative stress induced). (A) Loss of 261 proliferation ability and acquisition of elevated (B) cell size, (C) autofluorescence levels and (E) and (F) 262 SA-β-Gal activity by senescent hMSCs compared to the young cells. (D) Phosphorylation levels of p53 263 and Rb and expression levels of p21 and HMGB1 proteins in young and senescent hMSCs. Values are 264 mean ± s.d. For multiple groups comparisons at a one-way ANOVA was applied, n = 3, ns – not 265 significant, ** p < 0.01, *** p < 0.001. For pair comparisons at (A), (B), (C), and (E) Welch’s t-test was 266 used, n = 3 for (A), (B), (C) n = 50 for (F), ** p < 0.01, *** p < 0.001. Scale bars for images are 500 μm. 267 GAPDH was used as loading control. 268 269 As shown in Figure 7A–C, various types of hMSCs differed in the viability upon ouabain

270 treatment, for example both young and senescent DP-MSCs were much more resistant to

271 ouabain action than WJ-MSCs (Figure 7B,C, Figure 7––figure supplement 1B,C). Nevertheless,

272 ouabain was not able to induce senolysis in either type of hMSCs (Figure 7A–C, Figure 7––

273 figure supplement 1A–C). Together, the obtained data demonstrate that the absence of cardiac

274 glycoside-induced senolysis is a common feature for various types of hMSCs.

275

276 277 Figure 7. Ouabain has no senolytic action towards AD-MSCs, DP-MSCs and WJ-MSCs. (A), (B), (C) 278 Relative cell viability (%) of young and senescent AD-MSCs, DP-MSCs and WJ-MSCs after treatment 279 with 10-7, 10-6, 10-5 M ouabain, respectively. All data correspond to the mean ± s.d. Statistical significance 280 was assessed by the Welch’s t-test: ns – not significant, *p < 0.05, **p < 0.01.

281 282 6. Disturbance of K+/Na+ homeostasis caused by ouabain leads to different physiological

283 reactions in young and senescent A549 and END-MSCs

284 Having established the fact that senolytic effect of ouabain is cell type-dependent, further

285 we tried to elucidate what may underlie the revealed differences in responses of hMSCs and

286 A549. The main molecular mechanism of action of various cardiac glycosides is inhibition of

287 Na+/K+-ATPase (Skou, 1988). Na+/K+-ATPase is a plasma membrane enzyme that pumps

288 sodium out of the cell while pumping potassium into the cell against concentration gradients, and

289 thus participating in maintenance K+ and Na+ homeostasis. Therefore, we asked whether there

290 might be any differences in basal and ouabain-induced ion homeostasis between young and

291 senescent cells of two types. We first estimated intracellular contents of K+ and Na+ in young

292 and senescent END-MSCs and A549 cells in normal culture conditions and upon ouabain

293 application. Of note, within the present study the measured ions were normalized per total

294 protein in the same cells to quantify intracellular ion content. Young and senescent cells of both

295 types displayed typical ion distribution, namely high intracellular K+ and low intracellular Na+

296 (Figure 8A,B). As expected, ouabain shifted intracellular ion contents, leading to the vice versa

297 intracellular ionic ratios in both cell lines (Figure 8A,B). Importantly, the overall tendency of

298 ionic changes induced by ouabain was similar for END-MSCs and A549, indicating the absence

299 of any significant variations in the primary response of both cell types to ouabain.

300 The disturbance in K+ and Na+ homeostasis caused by ouabain should ultimately lead to

301 the dissipation of the plasma membrane potential. Thus, further we used specific fluorescent

302 probe DiBAC4(3) to measure transmembrane potential of non-excitable cells. Increased

303 fluorescence of this dye reflects membrane depolarization. In each cell type tested ouabain

304 induced predictable membrane depolarization, confirming ionic disbalance (Figure 8C). Notably,

305 we observed comparable levels of membrane depolarization in young and senescent END-

306 MSCs, while senescent A549 characterized by significantly more depolarized membrane

307 compared to young A549 cells (Figure 8C).

308 Other important consequences of ionic deregulation are changes in mitochondrial

309 membrane potential (MMP). Thus, using fluorescent probe JC-1 we assessed MMP in young and

310 senescent cells upon ouabain treatment. It should be specifically highlighted that senescent cells

311 are commonly characterized by decreased MMP reflecting malfunctioning of mitochondria,

312 alterations in energy metabolism and increased intracellular reactive oxygen species levels

313 (Passos et al., 2010). As expected, MMP in senescent END-MSCs and A549 cells was lower

314 than in the young ones, though this decrease did not affect viability of senescent cells (Figure

315 1D, Figure 3A). Similar to the above results, ouabain induced pronounced MMP depolarization

316 in senescent A549 compared to their proliferating counterparts, while effect on MMP of young

317 and senescent END-MSCs was minimal (Figure 8D).

318 Summarizing the results described within this part, we can conclude that blocking

319 Na+/K+-ATPase by ouabain causes comparable alterations in intracellular ions in young and

320 senescent cells of both types, however, in case of senescent A549, contrarily to senescent END-

321 MSCs, these alterations lead to the dramatic depolarization of plasma membrane and drop of

322 MMP.

323

324 325 Figure 8. Ouabain-resistant senescent END-MSCs are able to restore disturbance of K+/Na+ homeostasis 326 caused by ouabain via effective K+ import, while ouabain-sensitive senescent cells lack this ability. (A), 327 (B) Basal and ouabain-modified intracellular levels of K+ and Na+ in young and senescent END-MSCs 328 and A549, respectively. (C) Membrane potential determination of young and senescent END-MSCs and 329 A549 cells before and 24 h after ouabain treatment, using fluorescent probe DiBAC4(3). (D) 330 Mitochondrial membrane potential evaluation of young and senescent END-MSCs and A549 cells before 331 and 24 h after ouabain treatment, using fluorescent probe JC-1. (E) Design of the comparative 332 bioinformatic analysis of three independent RNA-Seq datasets. (F) Principal Component Analysis for the 333 subset of genes related to the GO term “cellular senescence” (GO 0090398) based on the combined data 334 from three independent RNA-Seq datasets. (G) GSEA results for the differentially expressed genes 335 between the cells resistant to ouabain-mediated senolysis (END-MSCs) and ones sensitive to ouabain- 336 mediated senolysis (A549 and IMR-90) for ‘Positive regulation of cation transmembrane transport’ and 337 ‘Potassium ion import’ biological processes. (H) Heatmap reflecting the core enrichment genes for 338 ‘Positive regulation of cation transmembrane transport’ and ‘Potassium ion import’ biological processes. 339 Values are mean ± s.d. Statistical significance was assessed by the two-tailed Student's t-test: ns – not 340 significant, *p < 0.05, **p < 0.01, ***p < 0.001.

341

342 7. Alterations in the mechanisms of K+ import mediate ouabain-resistance of senescent

343 END-MSCs

344 To clarify mechanisms mediating ouabain-induced changes in membrane polarization and

345 MMP between senescent A549 and END-MSCs, further we employed advanced bioinformatic

346 approach. The aim of the analysis was to reveal possible transcriptomic features mediating cell

347 type dependent ouabain-resistance or -sensitivity of senescent cells. In other words, we asked the

348 question: how senescence of END-MSCs (ouabain-resistant cells) differs from senescence of

349 A549 (ouabaine-sensitive cells)? To answer this question, we utilized three independent RNA

350 sequencing (RNA-Seq) datasets. The first one was RNA-Seq analysis of young and senescent

351 END-MSCs performed by us. The second one was the dataset for young and senescent A549

352 downloaded from GEO (Wang et al., 2017). Of note, senescence inducing conditions coincided

353 with those applied in the present study and in the pilot studies of cardiac glycosides-induced

354 senolysis. And the third dataset was for young and senescent IMR-90 obtained directly from the

355 study on cardiac glycosides as senolytic compounds (Guerrero et al., 2019). Importantly, IMR-

356 90 were proved to be prone for ouabaine-induced senolysis. It should be noted that comparing

357 only two datasets relevant for END-MSCs and A549 would ultimately lead to improper results,

358 as in such analysis the sought-for difference between senescence process in END-MSCs and

359 A549 would be indistinguishable from the variations mediated by diverse technical batch effects

360 (i.e. type of sequencing machine, the conditions of the sample ran). To minimize any batch

361 effects, we compared one dataset for ouabain-resistant cells (END-MSCs) and two for ouabain-

362 sensitive cells (A549, IMR-90). To validate the relevance of the further comparative analysis, we

363 performed a Principal Component Analysis for the subset of genes related to the GO term

364 “cellular senescence” (GO 0090398) based on the summary dataset containing samples from all

365 three described experiments. Indeed, for each cell type samples clustered into two separate

366 groups – the young and the senescent ones (Figure 8F).

367 To perform analysis of differentially expressed genes (DEGs) during senescence of

368 ouabain-resistant and -sensitive cells, we generated the statistical model that summed up two

369 predictors and their interaction. The schematic presentation of the analysis is displayed in Fig.

370 8e. In brief, the first predictor in the model divided all samples by young and senescence

371 subgroups, the second – by ouabain-resistantce or ouabain-sensitivity, and the last component in

372 the model reflected the interaction of both predictors. The result of differential expression testing

373 for the last variable is presented in Supplementary table 1. Importantly, there was no significant

374 difference in the expression levels of Na+/K+-ATPase subunits during senescence of ouabain-

375 resistant or -sensitive cells (Supplementary Table 2). This result correlated well with the

376 similarity in intracellular K+ and Na+ shifts upon Na+/K+-ATPase inhibition described in the

377 previous part (Figure 8A,B).

378 We then conducted gene set enrichment analysis (GSEA) in Gene Onthology (GO) terms

379 for Biological Processes (BP) (Supplementary Table 3). Notably, among the significantly

380 enriched processes we found ‘Potassium ion import’ and ‘Positive regulation of cation

381 transmembrane transport’ to be up-regulated during senescence of ouabain-resistant END-MSCs

382 compared to senescence of ouabain-sensitive cells (Figure 8G). The core enrichment gene lists

383 related to these processes included KCNJ2, KCNJ14, KCNJ8, SLC12A7, SLC12A8, WNK4 which

384 were significantly upregulated in senescent END-MSCs (Figure 8H). The proteins encoded by

385 KCNJ genes are inward-rectifier type potassium channels, which have a greater tendency to

386 allow potassium to flow into a cell rather than out of a cell. SLC12 is a family of cation-coupled

387 chloride transporters. WNK4 gene encodes for serine/threonine kinase that acts as an activator of

388 sodium-coupled chloride cotransporters and inhibitor of potassium-coupled chloride

389 cotransporters.

390 Together, these findings allowed suggesting that senescent END-MSCs can restore

391 depleted intracellular K+ levels more effectively than senescent A549 cells and thus can manage

392 with ouabain-induced ionic disbalance.

393

394 8. Senescent END-MSCs display elevated apoptosis-resistance compared to the young ones,

395 while senescent A549 do not

396 Our data demonstrating the difference in the maintenance of K+ homeostasis in senescent

397 END-MSCs and A549 cells together with the established role of intracellular K+ in the regulation

398 of cell death prompted us to compare the overall stress resistance acquired during senescence of

399 both cell types. It is well known that exogenous stresses are accompanied by the disturbance in

400 ionic homeostasis, leading to a rapid exchange of various ions, including K+ between the cell and

401 its environment (Kondratskyi et al., 2015). The outcomes of stressful influences are largely

402 dependent on the ability of cells to restore the appropriate intracellular ionic balance. As shown

403 in Figure 8G,H, senescence of END-MSCs was accompanied by the upregulation of K+ import

404 suggesting that during senescence development END-MSCs enhance their ability to restore K+

405 levels. Interestingly, we were not able to reveal similar tendency for A549 senescence, thus,

406 A549 seemed not to acquire any specific features related to the K+ import during senescence

407 progression. According to the literary data, stress-induced decrease in intracellular K+ and

408 inability to restore its level favor activation of caspases and nucleases and thus are proposed to

409 be a pro-apoptotic factors (Kondratskyi et al., 2015).

410 In line with this suggestion, using bioinformatic analysis of the above RNA-seq datasets

411 we revealed significant down-regulation of the processes related to apoptosis during senescence

412 of END-MSCs compared to A549 and IMR-90 (Figure 9A). Moreover, senescence of A549 and

413 IMR-90 characterized by significant up-regulation of pro-apoptotic genes, including BAD, BAX,

414 BOK, BAK-1, NOXA (Figure 9B). These data indicate that END-MSCs acquire apoptosis-

415 resistant phenotype during senescence, while senescent IMR-90 and A549 became apoptosis-

416 prone (Figure 9B).

417 We next tested whether the observed distinctions in apoptosis background during

418 senescence of END-MSCs and A549 may have an impact on the stress resistance of senescent

419 cells. To do so, we assessed cell viability upon oxidative stress and staurosporine, typically

420 applied to induce apoptosis. Indeed, senescent END-MSCs turned out to be significantly more

421 resistant towards both stresses compared to their young counterparts (Figure 9C). Contrarily,

422 A549 cells demonstrated vice versa situation, as senescent cells displayed tendency to be more

423 sensitive towards stress-induced cell death (Figure 9D). This observation shifts the existing

424 paradigm that enhanced death resistance is a common feature of any kind of senescent cells,

425 demonstrating its obvious cell specificity.

426

427

428 Figure 9. Senescence of ouabain-resistant END-MSCs is accompanied by acquisition of apoptosis- 429 resistant phenotype, while ouabain-sensitive cells become apoptosis-prone during senescence. (A) GSEA 430 results for the DEGs between the cells resistant to ouabain-mediated senolysis (END-MSCs) and the ones 431 sensitive to ouabain-mediated senolysis (A549 and IMR-90) in the apoptosis-related terms. (B) Heatmap 432 reflecting expression of the core enrichment genes for apoptosis-related terms from a. The common pro- 433 apoptotic genes are marked with asterisks. (C), (D) Relative cell viability (%) of young and senescent

434 END-MSCs or A549 treated either with H2O2 or staurosporine at indicated concentrations. Values are 435 mean ± s.d. Statistical significance was assessed by the two-tailed Student's t-test: *p < 0.05, **p < 0.01, 436 ***p < 0.001, ns – not significant.

437 438 Several crucial conclusions and the outflowing assumptions can be done based on the

439 data presented within this study. Сardiac glycosides have no senolytic effects on hMSCs of

440 different origin. The absence of this effect is probably mediated by the upregulation of the

441 systems supporting ionic balance in this type of senescent cells. Other less evident reason for

442 ouabain-resistance of hMSCs might be connected with the increase in overall resistance to stress-

443 induced death during their senescence. Senescent A549 cells are ouabain-sensitive, however, this

444 type of senescent cells is less resistant to cell death induced by either stress compared to the

445 young ones. We can speculate that preferential killing of senescent cells by senolytics might

446 work for those types of senescent cells that in fact did not become more resistant to cell death

447 (Figure 10).

448 449 Figure 10. Proposed model suggesting apoptosis-resistance of senescent cells to be an intrinsic barrier for 450 senolysis induced by cardiac glycosides. Becoming senescent heterogeneous cell types acquire apoptosis- 451 prone, apoptosis-resistant or mixed phenotypes. Senolytics, e.g. cardiac glycosides, can effectively 452 eliminate only apoptosis-prone senescent cells.

453 454 DISCUSSION

455 Within the present study we tested senolytic effects of cardiac glycosides, namely

456 ouabain and bufalin, towards hMSCs. Both compounds were recently identified to have selective

457 cytotoxic effect on senescent cells (oncogene- /therapy-induced senescence models) of different

458 origins, including primary cells IMR-90, HUVEC, ARPE-19, T/C-28, and cancer cells SKHep1,

459 A549, SK-Mel-5, MCF7, HCT116, HaCat, H1299, U373-MG, H1755 (Guerrero et al., 2019;

460 Triana-Martinez et al., 2019). Unexpectedly, we were not able to reveal any preferential

461 cytotoxic effects neither of ouabain nor of bufalin on senescent END-MSCs compared to the

462 young ones. Importantly, the absence of senolysis was verified using hMSCs obtained from

463 adipose tissue, dental pulp and Warton jelly, and besides various senescence models. The latter

464 led us to the suggestion that resistance to cardiac glycoside-induced senolysis might be the

465 common feature for hMSCs. At the same time we were able to induce apoptosis preferentially in

466 senescent A549, thus reproducing senolytic effect of cardiac glycosides described in the relevant

467 articles (Guerrero et al., 2019; Triana-Martinez et al., 2019). With cells resistant and sensitive to

468 ouabain-induced senolysis (ouabain-resistant/ouabain-sensitive) available, we tried to elucidate

469 fundamental causes underlying this difference.

470 The common molecular mechanism of cardiac glycosides’ action is binding to the

471 Na+/K+-ATPase and blocking its activity. By hydrolyzing ATP this enzyme ensures pumping

472 Na+ out of the cell and importing K+ into the cells, and thus maintains physiological

473 electrochemical gradient, ionic homeostasis, cellular pH and cell volume that are essential for

474 cell survival and functioning (Chen et al., 2014). Therefore, we speculated that the senolytic

475 ability of ouabain might depend on the severity of K+/Na+ disbalance in the treated cells.

476 However, we did not reveal any significant differences in cationic shifts between ouabain-

477 resistant and -sensitive models, as the dynamics of ouabain-induced alterations in intracellular

478 K+ and Na+ were similar between both types of young and senescent cells. This similarity can be

479 partially explained by the absence of any significant alterations in the expression levels of

480 Na+/K+-ATPase subunits during senescence of ouabain-resistant or -sensitive cells as indicated

481 by the bioinformatic analysis of the RNA-seq data.

482 Generally speaking, the impact of Na+/K+-ATPase inhibiting on cell survival was shown

483 to be cell-type specific. For example, ouabain was shown to potentiate apoptosis in lymphocytes,

484 Jurkat cells, canine epithelial cells, while it failed to induce death in epithelial cells from rat

485 aorta, rat cerebral granule cells and porcine renal proximal tubular LLC-PK1 lymphocytes

486 despite similar modulation of the cationic ratio (Ledbetter et al., 1986; Olej et al., 1998; Isaev et

487 al., 2000; Bortner et al., 2001; Orlov et al., 2001; Zhou et al., 2001; Pchejetski et al., 2003). One

488 of the proposed mechanisms of proapoptotic ouabain action is depletion of intracellular K+ that

489 favors apoptotic shrinkage, activation of caspases and initiation of apoptotic programming

490 (Kondratskyi et al., 2015; Chen et al., 2014). Considering comparable K+ loss but opposite effect

491 on death induction obtained for ouabain-resistant and ouabain-sensitive models, we suggested

492 the differences in cation’s compensatory systems. Drop of intracellular K+ level should

493 essentially lead to the activation of the K+ import from the extracellular space to compensate the

494 lack of this cation. Therefore, the effectiveness of K+ restoration might underlie apoptosis

495 predisposition upon ouabain treatment. To test this suggestion, we applied complex

496 bioinformatic analysis comparing alterations in transcriptomic signatures during senescence of

497 ouabain-resistant cells (END-MSCs) and ouabain-sensitive cells (A549 and IMR-90). We

498 observed strong upregulation of the processes related to potassium ion import during END-

499 MSCs senescence, while during senescence of ouabain-sensitive A549 and IMR-90 these

500 processes remained unchanged or even decreased. Based on that we can speculate that senescent

501 END-MSCs can effectively cope with ouabain-induced K+ depletion via active cation importing

502 systems, what prevents apoptosis induction. Contrarily, ouabain-sensitive senescent A549 and

503 IMR-90 seem to be less able to overcome K+ loss, thus are apoptosis-prone. In favor of this

504 assumption, ouabain induced more significant plasma and mitochondrial membranes

505 depolarization in senescent A549 cells, demonstrating pronounced ionic disbalance typical for

506 dying cells.

507 Rather than being the unique feature of ouabain-induced apoptosis, drop of intracellular

508 K+ content is probably the common characteristic of apoptotic death triggered by various

509 stresses, e.g. staurosporine treatment and oxidative stress (Kondratskyi et al., 2015).

510 Accordingly, decreased ability of senescent A549 to restore cytoplasmic K+ should ultimately

511 lead to decline in the overall stress resistance. However, this notion contradicts with the modern

512 definition of cell senescence, which states that senescent cells are highly resistant to apoptosis

513 (Zhu et al., 2015). It should be specifically highlighted that enhanced apoptosis resistance

514 formed the basis for senolytics development (Zhu et al., 2015; Zhu et al., 2016).

515 The first mention of the increased stress resistance of senescent cells dates back to 1995,

516 when replicatively senescent fibroblasts were found to be more resistant to serum withdrawal

517 compared to the young ones (Wang, 1995). This initial study was followed by the limited

518 number of investigations indicating that senescent cells are more resistant to UV-light,

519 staurosporine, thapsigargin and other stresses than their proliferating counterparts (Marcotte et

520 al., 2004; Ryu et al., 2007). However, we are far from the first to raise the question of the

521 enhanced apoptosis-resistance as the general feature of senescent cells. Thus, in the review

522 published in 2003 it was reported “… apoptosis resistance is not a general feature of senescent

523 cells, which may also be apoptotic prone depending on cell type and apoptotic stimuli…” (Soti et

524 al., 2003). Indeed, several data demonstrated higher sensitivity of senescent cells to stress-

525 induced apoptosis than of young cells (Hoffmann et al., 2001; Hampel et al., 2004; Jeon and

526 Boo, 2013). For example, senescent HUVECs were more prone to apoptosis induced by oxidized

527 LDL or TNFα compared to young cells (Hoffmann et al., 2001). Despite of the obvious

528 controversy, the last doubt appeared in 2015 and sounded as follows: “It seems doubtful that

529 global apoptosis resistance is a general feature of senescence cells” (Burton and Faragher, 2015).

530 In the same year the first study uncovering senolysis was published (Zhu et al., 2015). Within

531 this study the authors highlighted increased expression of pro-survival gene networks in

532 senescent cells that contributed to their enhanced resistance to apoptosis. Thus, initially

533 senolytics represented the drugs to target proteins that protected senescent cells from apoptosis.

534 The discovery of senolytics together with the impressive results of senescent cells’ removal

535 using INK-ATTAC transgenic mice triggered a flurry of studies searching for the new

536 compounds with senolytic activity (Zhu et al., 2015; Baker et al., 2016; Zhu et al., 2016; Baar et

537 al., 2017; Fuhrmann-Strossnigg et al., 2017; Jeon et al., 2017; Zhu et al., 2017; Guerrero et al.,

538 2019; Triana-Martinez et al., 2019; Amor et al., 2020; Gonzalez-Gualda et al., 2020; Wakita et

539 al., 2020). Over the last two years a plenty of reviews on senolytics was published (Paez-Ribes et

540 al., 2019; Soto-Gamez et al., 2019; Kirkland and Tchkonia, 2020; Pignolo et al., 2020; Robbins

541 et al., 2020; Wang et al., 2020). However, since 2015 enhanced apoptosis resistance of senescent

542 cells was not the matter of debate, though, in fact, it was not tested within these studies.

543 Interestingly, when comparing transcriptomic signatures of ouabain-resistant cells (END-

544 MSCs) and ouabain-sensitive cells (A549 and IMR-90), we found that only senescence of END-

545 MSCs was accompanied by acquisition of noticeable anti-apoptotic profile. On the contrary,

546 senescence of A549 and IMR-90 characterized by significant up-regulation of pro-apoptotic

547 genes, including BAD, BAX, BOK, BAK-1, NOXA and so on. On the one hand, these results

548 provide additional molecular explanation for the absence of ouabain-induced senolysis in END-

549 MSCs, and, on the other hand, clearly demonstrate that A459 and IMR-90 become predisposed

550 to apoptosis during senescence. Of note, our data regarding transcriptomic alterations in genes

551 related to apoptosis in senescent IMR-90 coincided with the results presented, yet not discussed,

552 in the article published by Guerrero et al., as the datasets for young and senescent IMR-90 cells

553 originated from this study (Guerrero et al., 2019). The precise analysis of the heatmap provided

554 within this study revealed upregulation of BAX, BID, BAD, BAK1 and NOXA in senescent IMR-

555 90 compared to the young cells. Furthermore, Baar et al. also revealed “unexpected”

556 upregulation of proapoptotic and downregulation of antiapoptotic genes in senescent IMR-90,

557 mentioning that senescent IMR-90 should be primed to undergo apoptosis (Baar et al., 2017).

558 In order to strengthen our observation, we compared resistance of young and senescent

559 END-MSCs and A459 to the most common apoptosis-inducing stimuli – oxidative stress and

560 staurosporine. According to the results of our bioinformatic analysis, senescent END-MSCs

561 turned out to be far more stress-resistant than young cells. At the same time, senescent A549

562 demonstrated the opposite reaction being slightly more susceptible than young cells to both types

563 of stressful stimuli. Therefore, senolytic action of cardiac glycosides, at least for A549, might be

564 mediated by the predisposition of senescent cells of this origin to apoptosis compared to their

565 young counterparts, whereas the absence of ouabain-induced senolysis in hMSCs might be

566 explained by the increase in overall apoptotic resistance during senescence of these cells. If the

567 latter is true, then senescent hMSCs are expected to be resistant towards other senolytic

568 compounds. Indeed, by analyzing the existing evidence we found that various compounds with

569 the stated senolytic activity, including navitoclax, nicotinamide riboside, danazol geldanamycin,

570 ganetespib, fisetin, BCL-XL inhibitors, quercetin turned to be ineffective in the targeted removal

571 of senescent human preadipocytes (fibroblast like precursor cells derived from human adipose

572 tissue) and hMSC from bone marrow (Zhu et al., 2016; Fuhrmann-Stroissnigg et al., 2017; Zhu

573 et al., 2017; Grezella et al., 2018).

574 The data regarding the absence of senolysis in hMSCs somewhat contradict with the

575 inspiring results obtained using in vivo mice models, demonstrating positive outcomes upon

576 systemic application of senolytics (Peng et al., 2020; Zhang et al., 2020). For example, it was

577 shown that clearance of senescent salivary gland stem cells using ABT263 may prevent

578 radiotherapy-induced xerostomia (Peng et al., 2020). Furthermore, removal of senescent bone

579 marrow MSCs by quercetin improved bone marrow formation (Zhang et al., 2020). Importantly,

580 it was shown that senescent mouse and rat MSCs unlike hMSCs are responsive to senolytics. For

581 example, quercetin, quercetin + dasatinib, ABT263 significantly removed senescent mouse

582 MSCs (Zhu et al., 205). Also, 17-DMAG has been shown to greatly reduce senescent bmMSCs

583 in a progeroid mouse model (Fuhrmann-Stroissnigg et al., 2017). Therefore, the improvements in

584 various organs and systems functioning described within these studies are speculated to be the

585 consequence of the targeted removal of senescent mouse stem cells. However, these senolytics

586 have not yet shown any functional rejuvenation of hMSCs. Such an obvious distinction between

587 the responsiveness of mice and human MSCs towards senolysis suggests that improvements

588 from senolytic therapies may not be conserved across the species. The results obtained for

589 senolytic compound UBX0101 can be considered as a good illustration confirming the last

590 statement. This agent has been shown to clear senescent cells in a mouse model of osteoarthritis,

591 which significantly reduced pain and promoted repair of the damaged cartilage (Jeon et al.,

592 2017). Unfortunately, UBX0101 failed to attenuate disease progression and pain in patients with

593 moderate-to-serve painful osteoarthritis during phase II clinical trial (NCT04129944; Dolgin,

594 2020; Garth, 2020). The similar concerns are raised about senolytic combination

595 dasatinib+quercetin (Garth, 2020).

596 To sum up, two important conclusions flow out from the obtained data. The former

597 demonstrated that cardiac glycosides are unable to clear senescent hMSC. The absence of

598 senolysis might be mediated by effective K+ cellular import and increased apoptosis resistance in

599 senescent hMSCs. The latter is more fundamental and reveals that apoptosis resistance is not a

600 general feature of senescent cells, as during senescence some cells acquire ‘apoptosis-resistant’

601 phenotype, while others do not. Importantly, only apoptosis-prone senescent A549 cells could be

602 effectively cleared by cardiac glycosides. Based on that we can speculate that the effectiveness

603 of other senolytic approaches might depend on whether senescent cells are indeed apoptosis-

604 resistant compared to their proliferating counterparts. Finally, though ‘senolytics’ is a hot topic

605 and a lot of inspiring data are published, conclusions regarding the effectiveness of senolysis

606 should be taken with caution as heterogeneity of cell senescence still remains a ‘puzzle’.

607

608 METHODS

609 Cells

610 END-MSCs were previously isolated from desquamated endometrium (Burova et al.,

611 2013). The study was reviewed and approved by the Local Bioethics Committee of the Institute

612 of Cytology of the Russian Academy of Sciences. Written informed consent was obtained from

613 all patients who provided tissue. WJ-MSCs, DP-MSCs, AD-MSCs and A549 were obtained from

614 the Russian Collection of Cell Cultures (Institute of Cytology, Saint-Petersburg, Russia). A549

615 line was authenticated by karyology, tumorigenicity, isoenzyme (LDH and G6PD) tests, and

616 STR analysis. Cells were cultured in complete medium DMEM/F12 (Gibco BRL) supplemented

617 with 10 % FBS (HyClone), 1 % penicillin-streptomycin (Gibco BRL) and 1 % glutamax (Gibco

618 BRL). All cells were routinely tested for mycoplasma contamination.

619

620 Senescence- and stress-inducing conditions

621 For oxidative stress-induced senescence END-MSCs/WJ-MSCs were treated with 200

622 µM/100 µM H2O2 (Sigma) for 1 h. For doxorubicine-induced senescence DP-MSCs were treated

623 with 100 nM of doxorubicine (Veropharm) for 3 days. In each case cells were considered

624 senescent not earlier then 14 days after treatment. For replicative senescence AD-MSCs earlier

625 than 4th passage were identified as young cells and later than 10th as senescent ones. For

626 chemotherapy-induced senescence A549 were treated with 2 µM etoposide (Veropharm) for 3

627 days and analyzed not earlier than 7 days after senescence induction. In this case, treatment

628 design completely coincided with those described in the study from which RNA-seq data

629 originated (Wang et al., 2017). Two cardiac glycosides we applied as senolytic compounds –

630 Ouabain (Sigma), Bufalin (Calbiochem).

631 In order to compare stress-resistance between young and senescent END-MSCs or A549,

632 cells were treated either with 400/800 µM H2O2 (Sigma) for 1 h or with 0.3/1 µM staurosporine

633 (Sigma). Cell viability was analyzed in 48 h after stress induction.

634

635 Flow cytometry analysis

636 Measurements of cell viability, proliferation, cell size, autofluorescence, apoptosis rates,

637 caspase 3/7 cleavage, mitochondrial membrane potential, membrane depolarization were carried

638 out by flow cytometry. Flow cytometry was performed using the CytoFLEX (Beckman Coulter)

639 and the obtained data were analyzed using CytExpert software version 2.0. Adherent cells were

640 rinsed twice with PBS and harvested by trypsinization. Detached cells were pooled and

641 resuspended in fresh medium and then counted and analyzed for autofluorescence. In order to

642 access cell viability, 50 μg/ml propidium iodide (Life Technologies) was added to each sample

643 just before analysis. The cell size was evaluated by cytometric forward light scattering of PI-

644 negative cells. Apoptosis induction was verified using Annexin-V-APC (Invitrogen) and DAPI

645 (Sigma) co-staining following manufactures instructions. Caspase activity was assessed using

646 CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen) following manufactures

647 protocol. Loss of mitochondrial membrane potential was assessed using the ratiometric dye JC-1

648 (Invitrogen). The staining procedure was carried out in accordance with the manufacture’s

649 protocol. For membrane depolarization we used DiBAC4(3) fluorescent probe (Invitrogen).

650 Gating strategy for flow cytometry analysis is provided in Supplementary Figure 1.

651

652 Senescence-associated β-Galactosidase staining

653 Senescence-associated β-Galactosidase staining was performed using senescence β-

654 galactosidase staining kit (Cell Signaling Technology) according to manufacturer’s instructions.

655 The kit detects β-galactosidase activity at pH 6.0 in cultured cells that is present only in

656 senescent cells and is not found in pre-senescent, quiescent or immortal cells. Quantitative

657 analysis of images was produced with the application of MatLab package, according to the

658 algorithm described in the relevant paper (Shlush et al., 2011). For each experimental point not

659 less 50 randomly selected cells were analyzed.

660

661 Western blotting

662 Western blotting was performed as described previously41. SDS-PAGE electrophoresis,

663 transfer to nitrocellulose membrane and immunoblotting with ECL (Thermo Scientific) detection

664 were performed according to standard manufacturer’s protocols (Bio-Rad Laboratories).

665 Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase

666 (GAPDH) (clone 14C10), phospho-p53 (Ser15), p21 (clone 12D1), phospho-Rb (Ser807/811),

667 HMGB1 (clone D3E5), as well as horseradish peroxidase-conjugated goat anti-rabbit IgG. All

668 antibodies were purchased from Cell Signaling.

669

670 Determination of cell ions

671 Cellular K+ and Na+ contents were measured by emission photometry in an air–propane

672 flame using a Perkin-Elmer AA 306 spectrophotometer as described previously (Marakhova et

673 al., 1998). In summary, the cells were pelleted in RPMI medium, washed five times with MgCl2

674 solution (96 mM) and treated with 5% trichloroacetic acid (TCA). TCA extracts were analyzed

675 for ion content. Intracellular K+, Na+, and Rb+ contents were determined by flame emission on

676 a Perkin-Elmer AA 306 spectrophotometer. TCA precipitates were dissolved in 0.1 N NaOH and

677 analyzed for protein by Lowry procedure. The cell ion content was expressed as amount of ions

678 per amount of protein in each sample analyzed.

679

680 RNA-Seq analysis

681 Samples from the following three Gene Expression Omnibus datasets were used in the

682 analysis: GSE102639 (GSM2742113 - GSM2742114 and GSM2742121 - GSM2742122 for

683 young and senescent A549 cells, accordingly); GSE122081 (GSM3454482 - GSM3454484 and

684 GSM3454500 - GSM3454502 for young and senescent IMR-90 cells, accordingly); and our

685 dataset GSE160702 (GSM4877895 - GSM4877898 and GSM4877907 - GSM4877910 for young

686 and senescent END-MSCs, respectively). Data for all the datasets were processed in the same

687 way.

688 Raw reads data underwent quality filtering and adapter trimming via FilterByTile and

689 BBDuk scripts from the BBtools package (version 38.75) using the default options. The

690 remaining reads were additionally filtered and trimmed with the use of trimFilter script from the

691 FastqPuri package (version 1.0.7). Trimming operation was applied for both ends of reads if they

692 contained N’s or their quality was below the quality threshold set to 27, all reads shorter than 25

693 bases were discarded. The quality control of trimming was held with the FastQC software

694 (version 0.11.7) and FastqPuri scripts. The reads, having passed all operations, comprised no less

695 than 90% of the initial data.

696 Transcript abundances were estimated using the Salmon lightweight-mapping (version

697 1.1.0) running in the selective alignment mode. The list of decoys was generated based on the

698 Gencode human reference genome GRCh38.p13 (release 33) and used further for building the

699 index on concatenated transcriptome and genome Gencode reference files (release 33) using k-

700 mer size of 21. Mapping operations were run with additional flags --numBootstraps 30 --seqBias

701 --gcBias --validateMappings. Resulting mapping rates were around 70%.

702 Further data processing was performed using R version 3.6.3 with the Tidyverse

703 collection of packages (version 1.3.0). Estimated gene counts, metadata and transcript ranges

704 were loaded into R and summarized to a gene level using tximeta (version 1.4.5). Resulting

705 count matrix was filtered to contain rows having at least 5 estimated counts across all samples,

706 the resulting matrix contained 20400 genes.

707 For the PCA and heatmap representation the read counts were normalized using rlog

708 transformation from the DESeq2 package (version 1.26.0). Heatmaps were constructed with the

709 use of genefilter (version 1.38.0) and pheatmap (version 1.0.12) R packages. Validation of

710 division samples by senescence variable was conducted via subsetting normalized read counts by

711 genes related to the Gene Ontology term “Cellular senescence” (GO 0090398). Differential

712 expression analysis and log fold changes estimation were computed using DESeq2 for the last

713 variable in the design formula controlling for cells senescence status, ouabain treatment reaction

714 and an interaction of the indicated factors. To strengthen differential expression testing, log fold

715 changes correction using combination of adaptive shrinkage estimator from the apeglm package

716 (version 1.8.0) and specifying additional log fold change threshold equal to 0.667 was applied.

717 Resulting shrunken estimates were used further for gene ranking and running Gene Set

718 Enrichment Analysis using clusterProfiler (version 3.14.3) and fgsea (version 1.12.0) R packages

719 with p-values adjustment for multiple comparisons according to the Benjamin-Hochberg method.

720

721 Statistical analysis

722 To get significance in the difference between two groups Student’s t-test or Welch’s t-test

723 was applied. For multiple comparisons between groups, ANOVA with Tukey HSD was used.

724 Unless otherwise indicated, all quantitative data are shown as mean ± s.d., and the asterisks

725 indicate significant differences as follows: ns, not significant, * p < 0.05, ** p < 0.01, *** p <

726 0.001. Statistical analysis was performed using R software.

727

728 Funding

729 This study was funded by the Russian Science Foundation (# 19-74-10038).

730

731 Acknowledgments

732 The authors are thankful to Maria Sirotkina for the assistance in the figures design.

733

734 Author Contributions

735 A. V. B. supervised the work, wrote and edited the manuscript. A. V. B. and P. I. D. performed

736 and designed most of the experiments. P. I. D. designed and conducted bioinformatic analysis,

737 performed statistical analysis of the obtained data and edited the manuscript. A. N. S. performed

738 FACS. I. I. M. determined intracellular ion contents. N. N. N. assisted in manuscript editing.

739

740 Competing interests

741 The authors declare no competing interests.

742

743 Materials and correspondence

744 Correspondence to Aleksandra Borodkina.

745

746 Data Availability Statement

747 All data generated or analysed during this study are included in the manuscript and supporting

748 files. Source data files have been provided for all Figures. Sequencing data have been deposited

749 in GEO under accession codes GSE160702.

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960 Supplementary Information

961 962 Figure 2–figure supplement 1. Standard growth curves of young and senescent END-MSCs treated with 963 ouabain. Values are mean ± s.d. Statistical testing was performed using one-way ANOVA with Tukey 964 HSD (results displayed are for Day 3 treatment outcomes against control at Day 0), n = 3, ns – not 965 significant, ** p < 0.01, *** p < 0.001. 966

967 968 Figure 4–figure supplement 1. Growth curves of young and senescent A549 cells treated with ouabain. 969 Values are mean ± s.d. Statistical testing was performed using one-way ANOVA with Tukey HSD 39

970 (results displayed are for Day 3 treatment outcomes against control at Day 0), n = 3, ns – not significant, 971 *** p < 0.001.

972 973

974 975 Figure 5–figure supplement 1. (A), (B) Standard growth curves of young and senescent END-MSCs and 976 A549 treated with bufalin, respectively. Values are mean ± s.d. Statistical testing was performed using 977 one-way ANOVA with Tukey HSD (results displayed are for Day 3 treatment outcomes against control at 978 Day 0), n = 3, ns – not significant, * p < 0.05, *** p < 0.001. 979 980

981 982 Figure 7–figure supplement 1. (A), (B), and (C) Standard growth curves of young and senescent AD- 983 MSCs, DP-MSCs, and WJ-MSCs treated with ouabain, respectively. Values are mean ± s.d. Statistical 984 testing was performed using one-way ANOVA with Tukey HSD (results displayed are for Day 3 985 treatment outcomes against control at Day 0), n = 3, ns – not significant, * p < 0.05, *** p < 0.001.

986 987 Supplementary Figure 1. Flow cytometry gating strategy, related to the Figures of the main text. a 988 Elimination of debris measurements identified as FSC-A low and SSC-A low. b Elimination of doublet 989 measurements by gating along the region FSC-A/FSC-H. c One-parametric histogram reflecting viable 990 and dead cells based on the PI (PC5.5-A) fluorescence intensity. d Dot plot reflecting viable and dead 991 cells PI (PC5.5-A) fluorescence against FSC-A. f Time-resolution of sample flow. 992 993 Supplementary Table 1. The results of differential expression analysis for the cells resistant to ouabain- 994 mediated senolysis (END-MSCs) against ones sensitive to ouabain-mediated senolysis (A549 and IMR- 995 90). 996 997 Supplementary Table 2. Subset from the results of differential expression analysis for the cells resistant 998 to ouabain-mediated senolysis (END-MSCs) against ones sensitive to ouabain-mediated senolysis (A549 999 and IMR-90) for genes encoding Na+/K+-ATPase subunits. 1000 1001 Supplementary Table 3. GSEA results for the differentially expressed genes between the cells resistant 1002 to ouabain-mediated senolysis (END-MSCs) and ones sensitive to ouabain-mediated senolysis (A549 and 1003 IMR-90) in the Gene Ontology Biological Processes terms.

42 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423449; this version posted December 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423449; this version posted December 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.18.423449; this version posted December 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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