Fig. 5: HMGCS1 mediates the moonlighting function of MAT2A in senescence regulation.

A Effect of endogenous Mat2a knockdown followed by restoration of Mat2a WT, MUT1 or SAM (500 μM) on pericyte senescence, as determined by SAHF formation (H3K9me3 staining) and SA-β-gal staining (n = 3). B Western blot assessment of the expression levels of P21 and Lamin B1 in the indicated pericytes. C–E OCR measurement, maximal respiration analysis and cellular ATP assessment of pericytes following endogenous Mat2a knockdown with Mat2a WT, MUT1 or SAM (500 μM) restoration (n = 3). F Scheme displaying the procedure used for identifying the specific targets of MAT2A through proteomic and IP-MS analysis. The workflow was created with BioRender.com. G Heatmap showing the change direction of differential proteins in pericytes with or without Mat2a knockdown. H Display showed the differentially regulated proteins, categorized per known or predicted function(s), literature and sequence similarity. Circle size was proportional to the number of differentially expressed proteins. I Intersection of the results from the proteomics and IP-MS analyses. J Scheme displaying the HMGCS1-mediated MVA pathway. K IP and WB analyses showing the interaction of MAT2A and HMGCS1 in 293T cells with indicated transfections. L In vitro binding analysis of MAT2A and HMGCS1 with GST pull-down assays. M Design of MAT2A and HMGCS1 truncations. N IP and WB analysis representing the interactions between Flag-tagged truncated MAT2A and His-tagged PRMT1 proteins in 293T cells. O IP and WB analysis representing the interactions between His-tagged truncated HMGCS1 and Flag-tagged MAT2A proteins in 293T cells. P Molecular docking showing the interaction between MAT2A truncation (slate) and HMGCS1 truncation (cyan). Q Docked positions of MAT2A and HMGCS1 and design of the mutations of binding sites between MAT2A and HMGCS1. R IP and WB analysis of the interactions between FLAG-tagged MAT2A mutation (MUT2) and His-tagged HMGCS1 mutation in 293T cells. Data were shown as mean ± SD. n = 3 biologically independent samples (A, C, D, E). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (A, D, E). n.s. no significance. Source data are provided as a Source Data file.