Fig 2: 3-MST silencing attenuates the restorative effects of the H2S donor NaHS on the ER stress response and autophagy. HepG2 cells previously subjected to 3-MST silencing (knockdown (KD)) or sham-silencing (si-CNTRL) were serum-starved for 8 h and treated with the vehicle or 100 nM of thapsigargin (TG), in the presence or absence of 100 µM NaHS for 16 h. Whole-cell lysate was collected and processed for protein expression of the UPR sensor–BiP, the UPR apoptotic mediator–Chop, the ER chaperone–calnexin (a,d), and of the autophagy-related proteins Atg3 and Atg7 (b,e). Representative images of the cells previously labeled with the Autophagosome Detection probe were captured under the Olympus CKX53 inverted fluorescent microscope with a DAPI channel. Autophagy is indicated by bright dot staining of autophagic vacuoles (c). Micro-cultures were assayed for XTT conversion (f) BrdU incorporation (g), and AzMC fluorochrome intensity (h) to assess cell viability, cell proliferation, and intracellular H2S levels, respectively. Each bar represents mean ± SEM from three and four independent experiments for Western blotting and cell-physiology studies, respectively. Data are expressed as a percentage of the transfection negative control, control (vehicle-treated) conditions (si-CNTRL+CC). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 compared to corresponding CC; # p ≤ 0.05, ## p ≤ 0.01 and ### p ≤ 0.001 compared to the corresponding TG-treated cells; ∂ p ≤ 0.05, ∂∂ p ≤ 0.01 and ∂∂∂ p ≤ 0.001 compared to the corresponding 3-MST KD treatment group.

Fig 3: Morphological changes during hPSC-OP9 coculture(A) Morphology of overgrown OP9 stroma at 8 days post passage, ready to be seeded with hPSCs.(B) Early hPSC (MIFF3) colony growing on OP9 stroma (day 2 of co-culture).(C) hPSC (MIFF3) colony starting to differentiate on OP9 stroma (day 7 of co-culture).(D) Differentiated hPSCs (MIFF3) at day 10 of OP9 stroma co-culture at time of harvest.Microscope Olympus, CKX53. Scale bars were added using ImageJ software.

Fig 4: C-peptide decreases decidualization in human endometrial stromal cells. (A) Human eSCs were induced to decidualize with 0.5 mM 8-Br-cAMP in the presence of an indicated concentration of C-peptide for 24 h. The cells were then lysed and subjected to qRT-PCR (n = 3). (B) qRT-PCR results for human eSCs from three different people (n = 3) treated with 0.5 mM 8-Br-cAMP with or without 50 nM C-peptide for 24 h. (C) Cells were induced to differentiate in the presence of 8-Br-cAMP with or without 50 nM C-peptide for 4 days and photographed using Nikon Eclipse Ti2 microscope (X4). Scale bar = 100 μm (n = 3). (D) Human eSCs were induced to differentiate in the presence of 0.5 mM 8-Br-cAMP for 2 days; subsequently, the 8-Br-cAMP was withdrawn, and the cells were incubated for further 2 days. The cells were lysed and analyzed using qRT-PCR (n = 3). (E) Cells were induced to decidualize using 0.5 mM 8-Br-cAMP in the presence or absence of 50 nM C-peptide for 24 h and stained for detecting EdU incorporation. The cells were photographed using Olympus CKX53 microscope (X20). Scale bar = 50 μm (n = 3). (F) Cells were treated with 50 nM C-peptide for 24 h and then subjected to western blot analysis (n = 3). (G) Cells were treated as in (E), stained with PI, and analyzed using flow cytometry (n = 3). *P < 0.05, **P < 0.01 versus an undifferentiated control; #P < 0.05 vs. a differentiated control. Human eSCs from two to three different people were used.

Fig 5: Morphology of hPSCs(A) Photo of an undifferentiated MIFF3 IPS clone on Matrigel and in StemFit media.(B) Photo of an undifferentiated MIFF3 IPS clone on Matrigel and in StemFit media just before passage.(C) Differentiated MIFF3 hIPS colony unsuitable for use in differentiation.Microscope Olympus, CKX53. Scale bars were added using ImageJ software.