Skip to main content
. 2021 Aug 25;12:5058. doi: 10.1038/s41467-021-25274-3

Fig. 5. SIRT7 negatively regulates EGF-driven AKT activation.

Fig. 5

a Representative images of IHC staining showing SIRT7 and (pS473)AKT levels in tumor sections from Fig. 4e—tumors derived from murine mammary cancer 4T1 cells with Sirt7 KD (shSirt7) and transfected with empty vector (EV), human SIRT7-WT (WT), and SIRT7-2 A (2A), n = 3 mice for analysis; scale bar, 50 µm. b Gene set enrichment analysis (GSEA) showing enrichment of PI3K/AKT pathways in SIRT7 knocked down (KD7) BT-549 breast cancer cells; data represent NES = 1.574, nominal P = 0.0, FDR q = 0.16, determined by Kolmogorov–Smirnov test. c Immunoblotting analysis of cellular lysates harvested from MDA-231 breast cancer cells with the indicated manipulations of SIRT7 expression and EGF (5 ng/ml) treatment; m minutes post-EGF treatment. d Ubiquitinated AKT levels assessed in HEK293 cells transfected with the indicated plasmids. e Membrane AKT levels assessed in MDA-231 breast cancer cells with or without ectopic SIRT7 overexpression before and after EGF (5 ng/ml) treatment. Mem membrane protein, WCL whole cellular lysates. f, g Immunoblotting analysis of immunoprecipitates derived from cell lysates incubated with anti-HA or anti-SKP2 antibodies. h, i Immunoblotting analysis of SKP2 protein stability determined by CHX chase assays in MDA-231 breast cancer cells with SIRT7 knockdown and in HEK293 cells transfected as indicated plasmids. j Immunoblotting analysis of SIRT7 levels in the anti-FLAG-SKP2 immunoprecipitates. k Immunoblots showing cytoplasmic and nuclear levels of SKP2 in MDA-231 breast cancer cells with or without overexpression of SIRT7; L long exposure; S short exposure. l Immunoblots showing the protein levels in the anti-FLAG-SKP2 immunoprecipitates derived from protein lysates of HEK293 cells transfected with indicated plasmids. m, n Immunoblots of anti-FLAG-AKT immunoprecipitates of HEK293 cells transfected with empty vector (EV), human SIRT7-WT (WT), and SIRT7-2A (2A) and with or without EGF (5 ng/ml) treatment. The relevant quantification (n = 3 independent experiments) is presented in (n), error bars: means ± SEM. o Immunoblotting analysis of anti-FLAG-AMPK immunoprecipitates derived from cell lysates of HEK293 cells transfected with the indicated plasmids. p Immunoblots showing the levels of phospho-SKP2 by probing with the anti-pan p-S/T antibody in the anti-HA-SKP2 immunoprecipitates of HEK293 cells transfected with the indicated plasmids. q Ubiquitinated AKT was evaluated by immunoblotting analysis of anti-FLAG-AKT immunoprecipitates prepared from cell lysates of HEK293 cells transfected with the indicated plasmids. Note: total FLAG-AKT in immunoprecipitates are displayed as membrane stained with Fast Green solution. P values were determined by two-tailed Student’s t-test. Representative results were obtained from at least three independent experiments. Source data are provided as a Source Data file.