Fig. 4: Hyper-acetylation impairs P5CS enzymatic activity induced by SLC25A51 knockdown.

A HEK293T cells were transfected with plasmids as indicated, and P5CS acetylation was analyzed by immunoblot. B P5CS-Flag plasmid was co-transfected with different amounts of HA-SIRT3 plasmids into HEK293T cells, and the acetylation level of P5CS was measured by western blot. C The cell lysates of SIRT3 knockout U2OS cells were immunoprecipitated with anti-acetyllysine agaroses, and SLC25A51 acetylation level was detected. D HEK293T cell lysates were immunoprecipitated with control IgG or anti-SIRT3 antibody, and then detected by anti-SIRT3 and anti-P5CS antibodies. E Representative immunofluorescence staining of endogenous SIRT3 (red), P5CS (green) and TOM20 (grey) in LoVo cells. Scale bars (bottom right), 10μm. F Microscale thermophoresis assay of SIRT3-His and P5CS-Flag proteins. G Cell lysates of SIRT3 knockout HCT116 cells expressing P5CS-Flag were treated with 0.025% glutaraldehyde and analyzed by Western blot analysis. H Cell lysates of SLC25A51 knockdown LoVo cells expressing P5CS-Flag were treated with 0.025% glutaraldehyde and analyzed by Western blot analysis. I The enzymatic activity of P5CS in wild type and SLC25A51 knockdown LoVo (left) and H1299 (right) cells. J Cellular amino acids contends of wild type and SLC25A51 knockdown LoVo cells were measured and exhibited as the heat map. K The cellular proline content of wild type and SLC25A51 knockdown LoVo (left) and H1299 (right) cells. *P < 0.05, **P < 0.01. Data are representative of three independent experiments with similar results. Bars, mean ± SD.