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Supplementary Material Supplementary Material and Methods MEFs preparation and electroporation Dissected embryos were washed in cold PBS, head and entrails (heart, liver) were excised and used for genotyping. The remaining embryonic tissues were sliced, incubated in Trypsin/EDTA solution (15 min at 37°C; Sigma), further dissociated and cultured in Iscove's Modified Dulbecco's Medium [(IMDM); purchased from Sigma)] supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 UI), 2 mM L-glutamine and 1× non-essential amino acids (all chemicals were from Gibco). Cells were electroporated in buffer (125 mM KCl, 15 mM NaCl, 3 mM glucose, 25 mM Hepes pH 7.4, 1.2 mM MgCl2) supplemented with 0.66 % (w/v) ethylene glycol (PEG) 4000 in 4 mm gap cuvettes using an ECM 830 electroporator (BTX; settings: 240V, 30 ms). Luciferase assay and NF-kB pathway activation The luciferase assay was performed according to the supplier’s protocol using Dual-Glo Luciferase Assay System (Promega). To activate the NF-ĸB pathway, cells were stimulated overnight with 20 ng/ml TNFα (stock 100 µg/ml; Peprotech), 20 ng/ml lymphotoxin (LTα; stock 10 µg/ml; Invivogen) or 100 ng/ml Pam3csk4 (stock 100 µg/ml, Invivogen). PCR primers used for ddPCR The first primer is in forward and the second primer in reverse orientation: β-ACTIN, 5’- CTAGGCGGACTATGAC-3’, 5’- GACTTGGGAGAGGACT-3’; SIRT1, 5’- CTCCAGGCAGATGCCATAAC-3’, 5’- TGCCCTTGGTTAAAATTTGG-3’; TLR2, 5’- CCTCCAATCAGGCTTCTCTG-3’, 5’- TGGAGGTTCACACACCTCTG-3’. Antibodies used for IHC and Western blotting IHC: anti-β-catenin (mouse monoclonal, EM-22, Exbio), anti-chromogranin A (rabbit polyclonal, ab15160, Abcam), anti-lysozyme (rabbit polyclonal, A0099, Dako), anti-p21 (mouse monoclonal, SXM30, BD Pharmingen), anti-p53 (rabbit polyclonal, NCL-p53-CM5p, Novocastra), anti-PCNA (rabbit polyclonal, ab18197, Abcam), anti-Tlr2 (goat polyclonal, sc-16237, Santa Cruz). The activity of brush border enzyme alkaline phosphatase was visualized using Alkaline Phosphatase Magenta™ IHC Substrate Solution (Sigma). Goblet cells were stained with Periodic acid Schiff kit (Hotchkiss-McManus) purchased from DiaPath. Western blotting: anti-α-tubulin (mouse monoclonal, TU-01, Exbio), anti-CtBP (mouse monoclonal, sc-17759, Santa Cruz), anti-p21 (mouse monoclonal, SXM30, BD Pharmingen), anti-p53 (rabbit polyclonal, NCL-p53-CM5p, Novocastra), anti-p65 (rabbit monoclonal, #8242, Cell Signalling), anti-phospho-p65 (detecting phosphorylated Ser536; rabbit monoclonal, #3033, Cell Signaling), anti-Tlr2 (rabbit monoclonal, E1J2W, Cell Signalling). Supplementary Figure and Table Legends Supplementary Figure S1. Heatmaps depicting gene expression in Hic1flox/flox MEFs treated with 4- OHT when compared to MEFs treated with vehicle (ethanol) only. Genes (299 in total) displaying significantly (q < 0.05) changed expression in at least one of the indicated time points are listed. The position of Hic1 and six genes whose expression was changed more than twice (|log FC| > 1) in at least two time points is highlighted. Supplementary Figure S2. Loss of Hic1 results in increased counts of goblet and enteroendocrine cells. A,B, goblet (A) and enteroendocrine (B) cell distribution in the indicated segments of the small intestine. Specimens obtained from four Hic1flox/flox and four Hic1flox/flox Villin-Cre+ mice were stained using Periodic acid Schiff (PAS) and an anti-chromogranin A antibody to visualize goblet and enteroendocrine cells, respectively. Stained cells (indicated by black arrowheads in the histology images on the right) were in several different fields indicated by numbers on the X axis. C, no changes in enterocytes were noted in the Hic1-deficient small intestine. Left, qRT-PCR analysis of enterocyte- specific markers hairy and enhancer of split-1 (Hes1) and sucrase-isomaltase (SI). The expression level of the respective gene in wt mice (upon normalization to Ubb) was arbitrarily set to 1. Right, staining of brush border enzyme alkaline phosphatase (AP) produced by differentiated enterocytes in the small intestine. Scale bar: 0.15 mm; error bars: SDs. Supplementary Figure S3. DSS-induced transcriptional response in the colon of Hic1flox/flox and Hic1flox/flox Villin-Cre+ mice six and nine days after DSS withdrawal. Epithelial lining of the colon obtained from four animals of each genotype was analyzed using qRT-PCR. The results were normalized to the Ubiquitin B (Ubb) housekeeping gene; the relative expression of another housekeeping gene, β-actin, is also shown. The expression level of the corresponding gene in mice without DSS treatment was arbitrarily set to 1. Error bars: SDs. Supplementary Table S1 Primers used for qRT-PCR analysis Supplementary Table S2. List of genes differentially expressed in Hic1flox/flox MEFs treated with 4- OHT for 48, 72, and 120 hours when compared to control MEFs treated (for the given time periods) with vehicle. Selection criterion: q < 0.05. Of note, none of the genes passed the selection criterion for the 24-hour time point. Supplementary Table S3. The most different ‘Gene Ontology Biological Processes’ (GO BPs) and WikiPathways categories in expression profiles of Hic1flox/flox MEFs 72 hours after addition of 4-OHT when compared to MEFs treated with vehicle only. In total, 268 gene probes passed the significance criterion (q-value < 0.05). Corresponding annotated genes (listed in Supplementary Table S2) were analyzed using the GO BP and WikiPathways 2015 Enricher datasets. The results were sorted according to the p-value; GO BPs and WikiPathways with p < 0.05 are listed. Supplementary Figure S1 24 h 48 h 72 h 120 h ILMN_2822825 Wfdc2 Hspb6 Wfdc2 Eno3 Anxa8 Ly6c1 Hspb6 AI646023 Sfrp2 Tlr2 Crabp1 Dpt Tlr2 Gas6 Tceal6 Ccl9 Prl2c3 Fhdc1 Igfbp2 Apol9b Dlk2 ILMN_1227579 Bace2 Sema4f Adssl1 Pon3 BC100451 Cox4i2 Sod3 Adamts2 ILMN_2791059 ILMN_2653725 Capn5 Klc3 Rhof Frmd8 Pdlim2 Bmp4 Hebp2 Nme5 Trappc3 Btd Elmo2 Pik3r3 Dad1 Mfap2 Acpl2 Cdc42ep2 Slc25a1 Dnajc12 Pqlc3 Taf11 Prss22 Nme7 H2−Ab1 Hoxc6 Atg9b Arpc5 Hoxc9 Zfyve21 Nanos1 5430435G22Rik Mrgprf Sema3f ILMN_2863332 Ltc4s Chst12 Nacc2 Itga3 Dyrk1b ILMN_2727536 Cbr3 Abca7 ILMN_1245221 Copz2 Tspyl3 4930583H14Rik Haghl Rnf167 Slpi Edem2 Lcmt1 C85492 Magee1 Fam110a ILMN_2590923 Gale ILMN_3144575 Ptp4a3 Rab3d Ctsw Ddit4l Gale Rassf7 Fbxo16 Sc5d Plin4 Atp6v1d Commd4 Tlcd2 Akr1b10 Tob1 Dhh ILMN_1227627 ILMN_1228783 Scx Gm347 Akr1b10 Izumo4 Enpp5 ILMN_2778799 ILMN_2607529 Insig1 Pmvk ILMN_2613923 Nudt7 Nsdhl ILMN_2706906 Dhcr24 Gjb4 Slc9a3r1 Pdlim2 Aldoc Dapk2 Tlcd2 Dapk2 Copz2 Angptl7 Ephx1 Angptl7 Cbr2 Dclk1 Uqcrh Cbr2 Ssh3 Tspan17 St3gal5 BC028528 Ly6a Angptl2 Emp3 ILMN_3162895 Gpnmb Mustn1 Pcyt2 Dbi Mvd Snn 2810432D09Rik ILMN_2731550 Fam50a Snca Hsd17b7 Arl16 Elovl6 ILMN_2654952 Nsdhl Sc4mol Mxd4 Agfg2 Hoxa5 Trappc1 Traf4 Zcchc18 Pcyt2 ILMN_2614161 Nsdhl Fdps Nsdhl Sqle Cyp51 Sqle 1500031L02Rik Myrip Cxcl12 Tbc1d17 Pcsk9 ILMN_2634083 Kbtbd4 Nde1 Zfp30 Mgst1 Shisa4 ILMN_2853342 Arf2 Asns Trim44 Zfp598 Mgp Arv1 Tgm2 Gzmd Ergic2 Pdgfb Atf4 Nppb Fam129a Chka Tmem74 Eif4enif1 Gjb2 Atf3 Rin1 Otub2 Inpp5f Myo1b Prkab1 Ift172 Gar1 Clcn3 Socs2 Krt19 Strap Lrp12 Fem1b Tgfbr1 Mcm10 Eln Rgs16 Hic1 Ndrg1 Hic1 Stmn4 Mmp9 Osmr Serpinb2 Picalm Otub2 Cdc42ep3 ILMN_2898917 Dgat2 Klf2 Fam82a2 Egr2 Ciapin1 Gfpt2 ILMN_2711948 Lrp12 ILMN_2685088 Arntl Ets2 Trit1 ILMN_1242170 ILMN_3008406 Uprt Color Key Ddx20 Stc2 Lrrc8c Crls1 Thumpd3 and Density Plot Asns 2410042D21Rik Lsm14a Lass6 Shmt2 Dhx33 Dhrs7 Ndrg1 Txnip 1500012F01Rik Zfp639 Wdr75 Slc6a9 Mpp6 0.4 ILMN_1214036 Cdh3 Agfg1 Wdr3 Fhl2 ILMN_2595732 Ch25h Slmo2 Trib3 Slc7a3 Cideb Taf5l Prpf38b 0.2 Slc1a4 Plk3 ILMN_1249579 Pparg Density Areg Ube2d2 Nat10 Zfp238 Kctd9 Trmt61a ILMN_2959330 0 Nup98 Hbegf Itfg2 H3f3b Leprotl1 Fgf21 Pole Gphn −1 0 0.5 1 Riok1 Rai14 Elavl2 Rars ILMN_2862111 Ankrd54 Cth Row Z−Score Slc7a11 Zcchc5 Supplementary Figure S2 A PAS 450 - Hic1loxP/loxP Villin-Cre+ 400 - Hic1loxP/loxP 350 loxP/loxP (per 1 slide) 300 Hic1 250 + 200 PAS 150 100 illin-Cre V Goblet cell count 50 0 loxP/loxP 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 --- Hic1 duodenum jejunum ileum B 50 Chromogr. A - Hic1loxP/loxP Villin-Cre+ - Hic1loxP/loxP 40 (per 1 slide) loxP/loxP 30 Hic1 + 20 Chromogr. A 10 illin-Cre V 0 loxP/loxP Enteroendocrinne cell count 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 --- Hic1 duodenum jejunum ileum C AP 2.0 in intestine loxP/loxP + 1.5 Hic1 illin-Cre + V 1.0 abundance AP loxP/loxP 0.5 Hic1 illin-Cre V vs. 0.0 Relative mRNA loxP/loxP loxP/loxP SI Hic1 β-actin Hes1 Hic1 Hic1 Supplementary Figure S3 Day 6 Day 9 40 40 in 35 in 35 30 30 ed intestine 25 ed intestine 25 Hic1flox/flox abundance abundance treat treat flox/flox + - 20 - 20 Hic1 Villin-Cre 15 15 10 10 Relative mRNA 5 Relative mRNA 5 control vs. DSS control vs. DSS 0 0 α α IL6 Tlr2 IL6 Tlr2 β-actin Cox2 TNF β-actin Cox2 TNF Supplementary Table S1 Primers for qRT-PCR Gene symbol Organism Sequence Actb Mouse Forward: 5'-GATCTGGCACCACACCTTCT-3' Reverse: 5'-GGGGTGTTGAAGGTCTCAAA-3' Atoh1 Mouse Forward: 5'-GCTGTGCAAGCTGAAGGG-3' Reverse: 5'-TCTTGTCGTTGTTGAAGG-3' Cox2 Mouse Forward: 5'-CGGAGAGAGTTCATCCCTGA-3' Reverse: 5'-ACCTCTCCACCAATGACCTG-3' Hic1 Mouse Forward: 5'-GAGGCTGCTGAGGTGGCTGC-3' Reverse: 5'-CTCTTGTCGCAGGACGCGCA-3' Hes1 Mouse Forward: 5'-GCTCACTTCGGACTCCATGTG-3' Reverse: 5'-GCTAGGGACTTTACGGGTAGCA-3' IL6 Mouse Forward: 5'-GATGGATGCTACCAAACTGGA-3' Reverse: 5'-GGAAATTGGGGTAGGAAGGA-3' SI Mouse Forward: 5'-TTCAAGAAATCACAACATTCAATTTACCTAG-3' Reverse: 5'-CTAAAACTTTCTTTGACATTTGAGCAA-3' Sirt1 Mouse Forward: 5'-CGGAGGGCCAGAGAGGCAGT-3' Reverse: 5'-CTCTTGCGGAGCGGCTCGTC-3'
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  • Targeting Glioblastoma Stem Cells Through Disruption of the Circadian Clock

    Targeting Glioblastoma Stem Cells Through Disruption of the Circadian Clock

    Published OnlineFirst August 27, 2019; DOI: 10.1158/2159-8290.CD-19-0215 RESEARCH ARTICLE Targeting Glioblastoma Stem Cells through Disruption of the Circadian Clock Zhen Dong1, Guoxin Zhang1, Meng Qu2, Ryan C. Gimple1,3, Qiulian Wu1, Zhixin Qiu1, Briana C. Prager1,3, Xiuxing Wang1, Leo J.Y. Kim1,3, Andrew R. Morton3, Deobrat Dixit1, Wenchao Zhou4, Haidong Huang4, Bin Li5, Zhe Zhu1, Shideng Bao4, Stephen C. Mack6, Lukas Chavez7, Steve A. Kay2, and Jeremy N. Rich1 Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 27, 2019; DOI: 10.1158/2159-8290.CD-19-0215 ABSTRACT Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immu- noprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenu- ated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1–CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryp- tochrome and REV-ERB agonists induced synergistic antitumor effi cacy. Collectively, these fi ndings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms.
  • BMC Genomics Biomed Central

    BMC Genomics Biomed Central

    BMC Genomics BioMed Central Research article Open Access Histone deacetylase inhibitors: A new mode for inhibition of cholesterol metabolism Sridar V Chittur*1, Niquiche Sangster-Guity2 and Paulette J McCormick1 Address: 1Center for Functional Genomics, University at Albany, State University of New York, Cancer Research Center, One Discovery Drive, Rm 310, Rensselaer, NY 12144, USA and 2Johns Hopkins University, School of Medicine, 1550 Orleans St, CRBII Rm 456, Baltimore, MD 21231, USA Email: Sridar V Chittur* - [email protected]; Niquiche Sangster-Guity - [email protected]; Paulette J McCormick - [email protected] * Corresponding author Published: 29 October 2008 Received: 6 March 2008 Accepted: 29 October 2008 BMC Genomics 2008, 9:507 doi:10.1186/1471-2164-9-507 This article is available from: http://www.biomedcentral.com/1471-2164/9/507 © 2008 Chittur et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Eukaryotic gene expression is a complex process involving multiple cis and trans activating molecules to either facilitate or inhibit transcription. In recent years, many studies have focused on the role of acetylation of histone proteins in modulating transcription, whereas deacetylation of these same proteins is associated with inactivation or repression of gene expression. This study explores gene expression in HepG2 and F9 cell lines treated with Trichostatin A (TSA), a potent histone deacetylase inhibitor. Results: These experiments show that TSA treatment results in clear repression of genes involved in the cholesterol biosynthetic pathway as well as other associated pathways including fatty acid biosynthesis and glycolysis.