All cell culture materials were obtained from Gibco Inc. Recombinant Human Activin-A (GFH6) was purchased from Cell Guidance Systems. RepSox (#72392), a cell permeable, selective inhibitor of the TGF-β type 1 receptor (TGFβRI) ALK5 was obtained from Stemcell Technologies. Recombinant forms of FSH (Gonal-F) and hCG (Ovitrelle) was purchased from Merck Global (Darmstadt, Germany). SAPK/JNK Kinase Assay Kit (#8794, nonradioactive), Hoechst 33342 (#4082), anti c-Jun (#9165), anti-phospho-c-JunSer73 (#3270S), Anti-Phospho-SAPK/JNKThr183/Tyr185 (#9251), Smad2 (#3122) and Phospho-Smad2Ser465/Ser467 (#18338) antibodies were obtained from Cell Signaling. Oil Red O was purchased from Sigma Inc. (USA). All western blotting buffers and reagents were purchased from Bio-Rad. Anti-Vinculin Antibody (V9264) was purchased from Sigma-Aldrich. Mouse antihuman monoclonal antibodies were purchased from Santa Cruz Biotechnology for the detection of human 3β-HSD Type II (sc-100466), 17β-HSD type-I (sc-376719), StAR (sc-166821), and P450 SCC (CYP11A1, sc-292456). Aromatase (CYP19A1, ab34193) monoclonal mouse antibody was from Abcam Inc. YO-PRO®-1 Iodide (491/509) was obtained from Life Technologies. Texas Red™-X Phalloidin was obtained from Thermo Fisher Scientific.
Repsox
Manufactured by STEMCELL
RepSox is a small molecule inhibitor that selectively targets the transforming growth factor beta (TGF-β) pathway. It functions by inhibiting the TGF-β receptor I (ALK5) kinase activity.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Cell Signaling Technology (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti vinculin antibody, by Merck Group (1 mentions)
Mouse antihuman monoclonal antibodies, by Santa Cruz Biotechnology (1 mentions)
Sc 166821, by Santa Cruz Biotechnology (1 mentions)
Anti phospho c jun ser73, by Cell Signaling Technology (1 mentions)
Yo pro 1 iodide, by Thermo Fisher Scientific (1 mentions)
Phospho smad2 ser465 ser467, by Cell Signaling Technology (1 mentions)
Sapk jnk kinase assay kit, by Cell Signaling Technology (1 mentions)
Anti phospho sapk jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
Oil red o, by Merck Group (1 mentions)
Anti c jun, by Cell Signaling Technology (1 mentions)
Fibroblast growth factor 2 (fgf2), by STEMCELL (1 mentions)
Neurocult nsc basal medium, by STEMCELL (1 mentions)
Y 27632, by STEMCELL (1 mentions)
Epidermal growth factor (egf), by STEMCELL (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Heparin, by STEMCELL (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
4 protocols using repsox
1
Granulosa Cell Culture Protocols
2
In vitro Expansion of Purified GBCs
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To expand purified GBCs in vitro, we modified our previously detailed culture protocol (Goldstein et al., 2016 (link)) slightly. For “engraftable cultures,” cells were selected based on c-KIT expression as described above and were plated onto gelatin-coated culture dishes at approximately 105 cells per well in NeuroCult NS-C Basal Medium, EGF 20 ng/mL, FGF2 10 ng/mL, heparin 2 μg/mL (all from STEMCELL Technologies), and penicillin-streptomycin (Invitrogen, Carlsbad, CA). RepSox (25 μM, STEMCELL Technologies), a TGF-β type 1 receptor (ALK5) inhibitor, was added along with Y27632 (10 μM, STEMCELL Technologies) overnight. Cultures were then maintained in base medium without inhibitors and were split 1:3 when 80% confluent, collecting both floating spheres and adherent cells. For “non-engraftable cultures,” additional cultures were prepared following our published protocol exactly, maintained in NS-C medium with SB431542 (10 μM) and BMP4 (10 ng/mL). For both culture preparations, >3 biological replicates were prepared using cells pooled from three mouse noses for each sample.
3
Stepwise Differentiation of Pluripotent Cells
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Differentiation sphere cultures were established as described in Supplemental Experimental Procedures . Basal differentiation medium consists of DMEM high glucose, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Supplements were added as follows: days 1–4 (1× B27 supplement, 50 ng/mL EGF, 1 μM RA [days 1–2 only], 50 ng/mL FGF7 [days 3–4 only]); days 5–10 (1× B27 supplement, 500 nM LDN-193189 [STEMCELL Technologies, 72142], 30 nM TPB [EMD Millipore, 565740], 1 μM RepSox [STEMCELL Technologies, 72392], 25 ng/mL FGF7); and days 11-17 (DMEM low glucose [Thermo Fisher Scientific, 12320-032], 2 mM L-glutamine, 1× MEM non-essential amino acids [Thermo Fisher Scientific, 11140-050]).
4
Neuroprotective Effects of RepSox
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The small molecule inhibitor of ALK5, RepSox (STEMCELL), was dissolved in dimethyl sulfoxide and sterile filtered. Stock solutions were then diluted in 0.9% NaCl and mice were injected with 10 mg/kg by i.p. injection 1 h before intrahippocampal injection of kainic acid and then every other day for 7 days. Mice were sacrificed 24 h or 7 days later and processed for immunohistochemistry.
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