Extended Data Fig. 7: CtBP-TRIM28 complex represses autophagy to promote metastasis.
From: Reciprocal stabilization of CtBP and TRIM28 represses autophagy to promote metastasis
(a) Pearson correlation between CtBP1, CtBP2 or TRIM28 protein levels and Macro-autophagy score (REACTOME, R-HSA-1632852). Data from CPTAC2 (PDC000120) (n = 149). Two-tailed, Pearson correlation coefficient test. (b) WB of TRIM28 in MDA-MB-231 with CtBP2, CtBP2 (G189A), TRIM28 or TRIM28 (V638S) overexpression. (c) Transwell assay of MDA-MB-231 cells overexpression indicated lenti-vectors were cultured in suspension for indicated time scale. Migration cells were stained with crystal violet after 24 h. (d) Flow cytometry analysis of apoptosis in MDA-MB-231 cells transduced with indicated lenti-vectors after culturing in suspension (4 days) (n = 3). (e) WB of TRIM28 in MDA-MB-231 with CtBP2, CtBP2 (G189A), TRIM28 or TRIM28 (V638S) overexpression. (f, g) WB of ITGA5, ITGB1 and P62 in MDA-MB-231 treated with HBSS (f) or CtBP KD (g) for indicated time scale. (h) WB of TRIM28 and CtBP2 in MDA-MB-231 with CtBP2 and TRIM28 overexpression. (i) WB of TRIM28, t-ULK1 and CtBP2 in MDA-MB-231 with stable CtBPKD, TRIM28 KD or ULK1 KD. (j) WB of ITGA5, ITGB1 and P62 in MDA-MB-231 with CtBP or TRIM28 overexpression after suspension culture (24 h or 48 h). (k) WB of RB1CC1, ULK1, TRIM28 and CtBP2 in MDA-MB-231 with stable RB1CC1 KD, ULK1 KD, TRIM28 KD or CtBP2 KD. (l) Lung metastasis of MDA-MB-231 cells with indicated shRNAs (n = 3) (left). Representative images of lungs from nude mice (black arrows indicate metastatic tumors), HE staining of lung tumor sections (dash line indicates metastatic tumor areas, scale bar: 500 μm) are shown at right. Middle, top is luminescence quantitation signal of lung metastatic tumors per mice (n = 3 mice per condition). Bottom is the quantitation of tumor area in lung sections (n = 3 sections per condition). Tumor area was quantitated by image J. (m) WB of TRIM28 and CtBP2 in MDA-MB-231 with stable TRIM28 and CtBP2 overexpression. Data are shown as the mean with s.d. (d,l); n = 3 biologically independent replicates (f,g,j). Statistical analysis was performed using a two-tailed, unpaired Student’s t-test (d,l); n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001.
