Extended Data Fig. 3: CtBP represses autophagy via TRIM28 retention in nucleus.
From: Reciprocal stabilization of CtBP and TRIM28 represses autophagy to promote metastasis
(a) Quantitation of TRIM28 H-score in MG sections from CtBP2flox/flox or MG-specific CtBP2-/- mice by pathologists based on individual MG (n = 15) from 3 mice. (b) WB of TRIM28 in MDA-MB-231 and MCF-7 with CtBP KD (3 or 5days). (c) The mRNA level of TRIM28 within 7 days after CtBP1/2 KD. (d–g) WB of TRIM28, P62, LC3B, CtBP in MDA-MB-231 and MCF-7 with CtBP OE (3 days) (d), in MDA-MB-231 with CtBP KD extend to 9 days (e), in A549, SKOV3, HEPG2 cells with CtBP KD (48 h) (f), in MDA-MB-231 and MCF-7 with CtBP KD, followed by 10 μM MG132 treatment (8 h) (g). (h, i) Stable GFP-LC3B OE cells were treated with Rapamycin (24 h) or CtBP KD in combination with TRIM28 KD (48 h). GFP-LC3B puncta (green) and DAPI-stained nuclei (blue) imaged (h). Scale bars=10 µm. Quantitated puncta per cell using Image J (n = 9) (i). (j) WB of P62, GFP-LC3B, CtBP and TRIM28 in MDA-MB-231 (GFP-LC3B OE) cells with TRIM28 KD or CtBP KD for 48 h or treated by Rapamycin (24 h). (k, l) MDA-MB-231 cells with stable mCherry-GFP-LC3B OE were treated with HBSS (8 h) or with CtBP KD in combination with TRIM28 KD (48 h). mCherry (red), mCherry-GFP-LC3B(yellow) puncta and DAPI-stained nuclei (blue) imaged (k). Scale bars=10 µm. Quantitated puncta per cell using Image J (n = 13) (l). (m) Flag-TRIM28-FL or Flag-TRIM28-ΔNLS plasmid was transfected in HEK-293T cells(48 h). Flag tag (green) and DAPI-stained nuclei (blue) imaged. Scale bars=10 µm. (n, o) QRT-PCR for LAMP2A (n) or EI24 (o) with siRNA treatment (48 h) (n = 3). (p, q) MDA-MB-231 cells were treated with HBSS or Rapamycin in combination with CQ (8 h). TRIM28 (green) and EI24 (red) and DAPI-stained nuclei (blue) imaged (blue) (p). Scale bars=10 µm. Quantitated colocalized puncta per cell using Image J (n = 15) (q). (r) QRT-PCR for EI24 with EI24 siRNA treatment (48 h) in MDA-MB-231 cells (n = 3). Data are shown as the mean with s.d. (a,c,i,l,n,o,q,r); n = 3 biologically independent replicates (b,d,e-g,j). Statistical analysis was performed using a two-tailed, unpaired Student’s t-test (a,c,i,l,n,o,q,r); n.s. P > 0.05, *P < 0.05, **P < 0.01 and ***P < 0.001.
