Figure 3. Depletion of cystathionine-β-synthase (CBS) in AKT-induced senescence (AIS) cells does not affect cysteine and glutathione (GSH) abundance in cysteine-replete conditions.

(A) Schematic of [3–13C] serine isotope tracing. Gray circles indicate 13C carbon atoms. Clear circles indicate unlabeled carbon atoms. (B–E) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells were transduced with pBabe or myrAKT1. After 6 days cells were transfected with either CBS siRNA (siCBS) or control siRNA (siOTP). On day 3 post-siRNA transfection, cells were cultured in either full medium (FM) or cysteine-deficient medium (Cys-Free) for 48 hr. Six hours before harvest, the culture medium was replaced with the basal isotope labeling medium containing 400 μM [3–13C] serine. The thiol redox metabolome was assessed by targeted liquid chromatography mass spectrometry (LC/MS). The abundances of labeled and unlabeled metabolites including (B) serine, (C) cystathionine, (D) NEM-Cys, and (E) NEM-GSH normalized with cell number are presented as mean ± SEM (n=4). Statistical significance was determined by one-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons (for the total metabolite levels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; and for the M+1 metabolite levels, ##p<0.01; ###p<0.001; ####p<0.0001 compared to pBabe-siNTP_FM cells). (F) BJ-TERT cells expressing doxycycline-inducible shCBS were transduced with myrAKT1, and treated with doxycycline (1 μg/ml) on day 5 post-transduction. Hydrogen sulfide (H₂S) production was measured by AzMC on day 14 post-transduction. Fold changes over doxycycline-untreated group are presented as mean ± SEM (n=3). One sample t-test compared to the hypothetical value 1.0 was performed (NS, not significant). (G and H) Gene set enrichment analysis of RNA-seq data showing downregulation of hallmark of oxidative phosphorylation and reactive oxygen species pathways in myrAKT1-shCBS cells compared with myrAKT1-shCtrl cells.

Figure 3—figure supplement 1. Depletion of cystathionine-β-synthase (CBS) in AKT-induced senescence (AIS) cells does not affect cysteine and glutathione (GSH) abundance in cysteine-replete conditions.

(A) The effects of siRNA knockdown of genes involved in the transsulfuration and transmethylation pathways on the cell number of AIS cells with Z score in blue, as measured by the functional genomic siRNA screen for AIS escape. (B) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells were transduced with pBabe or myrAKT1. After 4 days cells were transfected with either CBS siRNA (siCBS) or control siRNA (siOTP). Western blot analysis on day 6 post-siRNA transfection. Actin was probed as a loading control. (C) BJ-TERT cells were transduced with pBabe or myrAKT1 followed by transduction with pGIPZ-shCBS (myrAKT1-shCBS) or control vector (pBABE-shCtrl, myrAKT1-shCtrl) on day 6 post-transduction. Metabolic profiling by gas chromatography mass spectrometry (GC/MS) was performed on day 14 post-transduction. n=6 in both pBabe-shCtrl and myrAKT1-shCBS groups and n=5 in myrAKT1-shCtrl group, with one sample excluded due to a technical issue during the sample processing. Principal component analysis (PCA) plot, x-, y-, and z-axis represent individual principal components. (D) Heatmap of the top 50 metabolites analyzed by MetaboAnalyst 4.0. (E) The abundance of serine, glycine, glutamine, and homocysteine measured by GC-MS. Metabolite abundance normalized with cell number is presented as mean ± SEM (N=6 in both pBabe-shCtrl and myrAKT1-shCBS groups and n=5 in myrAKT1-shCtrl group). One-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons was performed (NS, not significant; *p<0.05; **p<0.01). (F) BJ-TERT cells expressing doxycycline-inducible shCBS were treated with erastin 5 µM for 3 days. Cell viability was calculated using the cell number measured by IncuCyte relative to the untreated control group and is expressed as mean ± SEM (N=3). Two-way ANOVA with Holm-Šídák multiple comparisons was performed (*p<0.05; **p<0.01). (I) RNA-seq was performed on day 14 post-transduction. Volcano plot showing differentially expressed genes (FC ≤ –2 or FC ≥ 2, FDR ≤ 0.05) between cells expressing myrAKT1-shCBS vs. myrAKT1-shCtrl cells, which are downregulated (blue), upregulated (red), or not significant (black) (n=3 biological replicates). The top 10 significantly upregulated and downregulated genes are indicated.