Figure EV4. SIRT1 promotes conversion of methionine to SAM through Mat2a.
- SIRT1 KO mESCs display reduced expression of Mat2b enzymes (n = 3 independent experiments).
- SIRT1 KO mESCs have reduced protein levels of MAT2A in both complete and methionine‐restricted medium. WT and SIRT1 KO mESCs were cultured in complete or methionine‐restricted medium (MR) for 24 h. The relative intensities of MAT2A were quantified using ImageJ (n = 5 independent experiments).
- SIRT1 KO mESCs have reduced Mat2a activities in both complete and methionine‐restricted medium. WT and SIRT1 KO mESCs were cultured in complete or methionine‐restricted medium (MR) for 24 h, and the activities were measured as described in Materials and Methods (n = 3 independent experiments).
- SIRT1 re‐expression rescues SIRT1 deficiency‐induced reduction in Mat2a mRNA. WT and SIRT1 KO mESCs stably infected with lentiviruses containing the empty vector (V) or a construct expressing SIRT1 (SIRT1) were cultured in complete or methionine‐restricted medium for 48 h (n = 3 independent experiments).
- Overexpression of Mat2a in mESCs. Control and SIRT1‐deficient mESCs were infected with lentiviruses containing either empty vector or mouse Mat2a gene. Stable lines were selected and analyzed for the mRNA levels of Mat2a by qPCR (n = 3 independent experiments).
- Overexpression of exogenous Mat2a rescues SIRT1 deletion‐induced mESC differentiation in ESGRO mESC maintenance medium. WT and SIRT1 KO mESCs were stably infected with lentiviruses containing either empty vector or mouse Mat2a gene and cultured in the ESGRO maintenance medium. Scale bars, 100 μm.
- Overexpression of exogenous Mat2a rescues SIRT1 deficiency‐induced mESC differentiation in complete M10 medium and ESGRO maintenance medium. Sh‐Control and sh‐SIRT1 mESCs were stably infected with lentiviruses containing either empty vector or mouse Mat2a gene and cultured in the complete M10 medium or ESGRO maintenance medium. Scale bars, 100 μm.