Gata1

ChIP from ESCs was performed according to the ChIP-IT High Sensitivity kit (Active Motif). Chromatin sonication was achieved with the use of Bioruptor Plus, Diagenode, at low amplitude, pulse for 30 s on and 30 s off for 20 min in ChIP buffer. 1/20 of the chromatin preparation was extracted to generate the Input. 15–30 μg of chromatin and 4 μg of antibody per ChIP reaction were used. Binding to promoter regions was assessed via qPCR. YAP (clone 63.7; sc-101199), TEF-4 (sc-67115), β-catenin (sc-7199), GATA-1 (sc-13053), H3K4Me3 (ab8580), H3 (Cell Signalling; 14269 S), H3K27Me3 (Cell Signalling; 9733 S) and IgG (Cell Signalling; 2729 S) antibodies were used for the immunoprecipitations.

Automated immunoblot analysis was performed using Wes system (ProteinSimple, San Jose, CA, USA) according to the manufacturer’s instructions using a 12–230 kDa Separation Module (SM-W001) and the Anti-Rabbit Detection Module (DM-001). iPSCs or HSPCs were lysed in Minute Total Protein Extraction kit (Invent Biotechnologies, Plymouth, MN, USA), sonicated, and clarified by centrifugation at 16,000× g for 15 min. Supernatant was collected and protein equivalent to 50,000 cells was loaded per capillary. GATA1, STAG2, pSTAT-1, RIG-I, MDA5, and BCL2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Normalization to total protein was performed using the Total Protein Detection Module in Wes (DM-TP010).

Immunofluorescence staining was performed on paraffin sections. After quenching the endogenous peroxidases with 3% H₂O₂ in 10% methanol, targeted antigens were retrieved by boiling the slides in an antigen-unmasking solution (H-3300, Vector Laboratories). Sections were treated with blocking reagent (all supplied by Roche) before application of primary antibodies for Sertoli and Leydig cell detection: GATA-1 (1:200, Cell Signaling Technology), and P450scc (1:100, Cell Signaling Technology), respectively. The number of Sertoli cells were quantified per seminiferous tubule by averaging the number of positive GATA-1 signals for 20 tubules within each testes (n = 5). Leydig cell signals were quantified via ImageJ software by measuring fluorescence intensities of interstitial space P450scc signals and normalized to background (n = 6). Cell proliferation was assayed by immunofluorescence staining using Ki-67 antibodies (1:100, Thermo Scientific) and apoptosis was examined using immunofluorescence staining of caspase-3 antibody (1:200, Cell Signaling Technology). Dhh expression was analyzed using Dhh antibody (1:100, Santa Cruz Biotechnology Inc.). MVH staining (1:200, ab13840) was performed in order to stain spermatogonium. All primary antibodies were incubated at 4°C overnight.

The mouse myeloma cell line 5TGM1 was kindly provided by Dr. Fenghuang Zhan (University of Arkansas for Medical Sciences, Little Rock, USA)^{14 (link)}. and was cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FBS (Invitrogen), penicillin (100 IU/mL), and streptomycin (100 µg/mL) in a humidified incubator at 37 °C and 5% CO₂/95% air. CD34⁺ HSPCs were isolated from human BM biopsies with CD34 microbeads (Miltenyi Biotec, Auburn, CA) and cultured in StemSpan SFEM containing Erythroid Expansion Supplement (StemCell Technologies). The following antibodies were purchased from BD Biosciences: CD34-APC (563), CD38-PE-Cy7 (HIT2), CD45RA-FITC (L48) and CD138-PE (MI15). The antibodies CCR1-PE (5F10B29), CCR4-PE (D8SEE), CCR5-PE (NP-6G4) and CD123-PE (6H6) were purchased from eBioscience. Antibodies against p38, phospho-p38, GATA-1, GAPDH, and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (CST, USA). CCL3 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antagonists BX471, AZD2098 and Maraviroc were purchased from MCE (USA).

Whole-cell extracts or immunoprecipitates were treated with 1 × Laemmli sample buffer (Tris-HCl 125 mm, pH 7.5, 1% sodium dodecyl sulfate, 20% glycerol) and fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred to nitrocellulose membranes using an iBlot Transfer Stack and iBlot Gel Transfer Device (Thermo Fisher Scientific, Waltham, MA, USA). After incubation with StartingBlock T20 (phosphate-buffered saline) blocking buffer (Pierce Biotechnology, Waltham, MA, USA), membranes were labeled with primary antibodies overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with ImmunoStar Zeta (Wako, Osaka, Japan) and signals were detected using ImageQuant LAS-3000 (Fujifilm, Tokyo, Japan).
The following antibodies were used for western blotting analysis: CDT1 (sc-365305; Santa Cruz Biotechnology, Dallas, TX, USA), cleaved PARP (9541; Cell Signaling Technology), γH2AX (2577; Cell Signaling Technology), GAPDH (2118; Cell Signaling Technology), GATA1 (3535; Cell Signaling Technology), PU.1 (2258; Cell Signaling Technology) and c-Jun (9165; Cell Signaling Technology).