Extended Data Fig. 4: CtBP-TRIM28 activates autophagy by promoting AKT-mTOR signaling pathway.
From: Reciprocal stabilization of CtBP and TRIM28 represses autophagy to promote metastasis
(a) WB of P62, LC3B-II and LC3B-I in MDA-MB-231 cells transfected with TRIM28-FL or TRIM28-ΔNLS mutant for 72 h. (b–d) WB of P62, LC3B AMPKα1 or p-AMPKα1 in MDA-MB-231 and MCF-7 cells with TRIM28 KD (3 or 5 days) (b), in MDA-MB-231 with CtBP KD or TRIM28 KD (3 d) (c), in MDA-MB-231 with CtBP KD in combination with compound C treatment (24 h) (d). (e) Cytoplasmic Acetyl-coA (Ac-CoA) quantitation in MDA-MB-231 and MCF-7 cells with CtBP KD or TRIM28 KD (48 h) (n = 3). (f, g) WB of whole cell Acetylated-lysine (Ac-lys), Acetylated histone H3 (Ac-H3) or total-H3 in MDA-MB-231 with CtBP KD (48 h) (f), with CtBP KD or TRIM28 KD (48 h) (g). (h) Protein level Pearson correlation between CtBP1, CtBP2 or TRIM28 and P62, LC3B, BECN1, ATG14 and ATG16L in clinical breast samples (n = 222) retrieved from TCGA (ID: TCGA_Breast_BI_Proteome). Two-tailed, Pearson correlation coefficient test. (i) Kaplan-Meier DSS survival analysis of breast cancer patients from TCGA by P62, LC3B, BECN1, ATG14, ATG16L expression from TCGA datasets (TCGA_Breast_BI_Proteome). Patients were classified to high expression and low expression groups. Log-rank based on Mantel-Cox test. (j) Gene Ontology (GO) analysis of the up-regulated genes in MCF-7 cells with CtBP KD or TRIM28 KD by Metascape. (k) GSEA analysis of the differentially expressed genes (DEGs) in CtBP KD versus Ctrl in MCF-7 cells. Top hits include KEGG-autophagy (hsa04140) and Reactome-autophagy (R-HSA-9612973) datasets. (l, m) QRT-PCR validation of autophagy related gene (ATGs) expression in MCF-7 cells with CtBP KD, TRIM28 KD (l), CtBP OE or TRIM28 OE (m). (n) Quantification of t-AKT, p-AKT (Ser473), t-mTOR, p-mTOR, t-ULK1, p-ULK1(S758), t-S6K and p-S6K (Thr389) in MDA-MB-231 with CtBP KD in combination with PTEN KD by Image J (n = 3). Data are shown as the mean with s.d. (e,l-n); n = 3 biologically independent replicates (a-d,f,g). Statistical analysis was performed using a two-tailed, unpaired Student’s t-test (e,l-n); n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001.
