Extended Data Fig. 5: Smurf1 is upregulated via KRAS/AP-1 axis.
From: Targeting Smurf1 to block PDK1–Akt signaling in KRAS-mutated colorectal cancer
a, Representative images of HE and IHC staining against indicated proteins in colon tissues from KrasG12D/+ Villin-Cre and Kras+/+ Villin-Cre mice. Scale bars, 10 μm. b, Quantification of IHC of Smurf1, p-Akt (T308) and Ki-67 of colon tissues from KrasG12D/+ Villin-Cre and the control mice by Image-Pro Plus 6 (n = 3 mice). c, Protein extracts of intestinal tissues from KrasG12D/+ Villin-Cre and the control mice were immunoprecipitated with PDK1 antibody and analyzed by IB. d, Relative Smurf1 mRNA level in colon tissues from KrasG12D/+ Villin-Cre and the control mice (n = 5 mice). e,f, KRAS G12D inhibitor MRTX1133 reduced Smurf1 protein (e) and mRNA (f) levels in KRAS G12D mutant HCT116 cells (n = 4 technical repeats from one of three independent experiments). Cells treated with MRTX1133 for indicated times (10 mM). g–i, c-Fos or c-Jun was stably knocked down in HCT116 cells, followed by transfection with WT or G12D mutant KRAS plasmids. qPCR analysis was performed to measure the mRNA level of c-Fos (g), c-Jun (h) and Smurf1 (i) in indicated cells (n = 4 technical repeats from one of three independent experiments). Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t test (b,d,f,g–i).
