Figure 1. Cystathionine-β-synthase (CBS) expression and transsulfuration pathway activity are elevated in AKT-induced senescence.

(A) Schematic diagram illustrating that the cytoplasmic localized CBS regulates transmethylation and transsulfuration metabolic pathways, and mitochondrial localized CBS regulates oxidative phosphorylation. (B) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells were transduced with pBabe, myrAKT1, or HRAS^G12V. On day 6 post-transduction the cells were plated in either full culture medium containing 100 µM cysteine (FM) or cysteine-deficient medium (Cys-free). Western blot analysis was performed on day 10 post-transduction. Vinculin was probed as a loading control. Representative of n=3 experiments. (C) Hydrogen sulfide (H₂S) production was measured by AzMC on day 14 post-transduction. Fold changes over pBabe control are presented as mean ± SEM (n=3). One sample t-test compared to the hypothetical value 1.0 was performed (NS, not significant; *p<0.05). (D) Cells were treated with aminoxyacetate (AOAA) 30 μM on day 5 post-transduction. Cell confluency measured by IncuCyte is presented as mean ± SEM (n=3). (E) Cells were cultured in the conditions as described in (B). Cell confluency measured by IncuCyte is presented as mean ± SEM (n=3–5). Statistical significance at the last time point in (D) and (E) was determined by unpaired t-test (**p<0.01; ***p<0.001).

Figure 1—figure supplement 1. Cystathionine-β-synthase (CBS) expression and transsulfuration pathway activity are elevated in AKT-induced senescence.

(A) IMR-90 human fetal lung fibroblasts were transduced with pBabe or myrAKT1. On day 5 post-transduction, cells were transfected with either CBS siRNA (siCBS) or control siRNA (siOTP). Western blot analysis was performed on day 6 post-siRNA transfection. Actin was probed as a loading control. Representative of n=3 experiments. (B) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) human skin fibroblasts and human mammary epithelial cells (HMEC) were transduced with either pBabe or AKT1^E17K. Western blot analysis was performed on day 14 post-transduction. Vinculin was probed as a loading control. (C) BJ-TERT cells were transduced with pBabe, myrAKT1, and HRAS^G12V. Relative CBS mRNA expression measured on day 14 post-transduction by qPCR was normalized to pBabe control are presented as mean ± SEM (n=3). One sample t-test compared to the hypothetical value 1.0 was performed (NS, not significant). (D) BJ-TERT cells were transduced with pBabe, myrAKT1, and HRAS^G12V and then treated with aminoxyacetate (AOAA) 30 μM or vehicle (DMSO) on day 5 post-transduction. The percentage of cells with positive staining for SA-βGal activity was quantified on day 6 post-treatment and presented as mean ± SD (n=2). Two-way analysis of variance (ANOVA) with Holm-Šídák multiple comparisons was performed (NS, not significant).