Figure 7. Loss of cystathionine-β-synthase (CBS) synergizes with PI3K/AKT pathway to promote gastric cancer pathogenesis.

(A–B) GES-1 gastric epithelial cells were stably transfected with doxycycline-inducible myrAKT1 and pGIPZ-shCBS or non-silencing small hairpin RNA (shRNA) control (shCtrl). (A) Western blot analysis showing HA-tagged myrAKT1 and CBS after doxycycline (0.75 μg/ml) induction for 3 days. Vinculin was probed as a loading control. (B) Relative colony formation assessed by soft agar colony formation assay on day 21 post-doxycycline induction was normalized to doxycycline-untreated shCtrl group and presented as mean ± SEM (n=3). The representative images of colonies formed in the soft agar. Scale bar = 500 μm. (C–D) GES-1 cells with PTEN knockout by CRISPR or Cas9 control were transduced with doxycycline-inducible CBS shRNA#1. (C) Western blotting after treatment with doxycycline (0.75 μg/ml) for 3 days. Vinculin was probed as a loading control. (D) Relative colony formation assessed by soft agar colony formation assay on day 21 post-doxycycline induction was normalized to doxycycline-untreated shCtrl group and presented as mean ± SEM (n=3). The representative images of colonies formed in the soft agar. Scale bar = 500 μm. (E–F) AGS gastric cancer cells were stably transfected with doxycycline-inducible CBS^wt or CBS^I278T. Cells were cultured in the presence or absence of doxycycline induction (0.08 μg/ml for CBS^wt or 1 μg/ml for CBS^I278T). (E) Western blot analysis on day 3 post-treatment. Vinculin was probed as a loading control. (F) Fold changes of hydrogen sulfide (H₂S) production over doxycycline-untreated CBS^wt group were presented as mean ± SEM (n=4). Statistical significance in (A, C, and F) was determined by one-sample t-test compared to the hypothetical value 1.0 (NS, not significant; *p<0.05; **p<0.01; ***p<0.001) and two-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons (#p<0.05; ##p<0.01; ###p<0.001). (G–I) AGS gastric cancer cells expressing doxycycline-inducible CBS^wt were subcutaneously implanted in BALB/c nude mice. On day 38 post-implantation, mice were supplied with water containing 0.2% (W/V) doxycycline and 600 mg doxycycline/kg food. (G) Tumor volume was presented as mean ± SEM (n=6 mice per group). Statistical significance at each time point was determined by unpaired Student’s test (*p<0.05; **p<0.01). (H) Western blot analysis of CBS and senescence-associated protein expression in tumor tissues. Actin was probed as a loading control. (I) IHC of Ki67 on the tumor tissue. Scale bar: 50 μm.

Figure 7—figure supplement 1. Loss of cystathionine-β-synthase (CBS) synergizes with the PI3K/AKT pathway to promote gastric cancer pathogenesis.

(A–C) GES-1 gastric epithelial cells were stably transfected with doxycycline-inducible myrAKT1 and pGIPZ-shCBS or non-silencing small hairpin RNA (shRNA) control (shCtrl). Cells were treated with doxycycline (0.75 μg/ml). Fold changes of (A) hydrogen sulfide (H₂S) production and (B) total glutathione (GSH) level over doxycycline-untreated shCtrl group are presented as mean ± SEM. One-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons was performed (NS, not significant). (C) Cell confluency measured by IncuCyte is presented as mean ± SEM (n=4). (D) GES-1 cells with PTEN knockout by CRISPR or Cas9 control were transduced with doxycycline-inducible CBS shRNA#1. Cells were treated with doxycycline (0.75 μg/ml). Cell confluency measured by IncuCyte is presented as mean ± SEM (n=4). (E–F) AGS gastric cancer cells were stably transfected with doxycycline-inducible CBS^wt or CBS^I278T and treated with doxycycline (0.08 μg/ml for CBS^wt or 1 μg/ml for CBS^I278T). (E) Fold changes of total GSH level measured on day 3 post-doxycycline induction are presented as mean ± SEM (n=3). NS, not significant by one sample t-test. (F) Cellular confluency measured by IncuCyte is presented as mean ± SEM (n=3).