Fig. 5

TRIM28 induces CXCL1 via the NF-κB pathway. (A-B) Expression of p65 was determined by western blot in CMT-167 or H1299 cells overexpressing wild-type TRIM28 or TRIM28 knockdown with shRNA. The whole cell lysates and nuclear protein extracts were subjected to immunoblotting analysis. Data are representative of results obtained in three independent experiments. (C) The immunofluorescence assays of p-65 were performed in CMT-167 cells treated with the indicated plasmids. DAPI (blue) was used as a nuclear counterstain. The quantification of nuclear p-65 positive staining in at least 200 counted cells was presented as percentage ± SEM. Statistics calculated using one-way ANOVA post hoc Tukey for multiple comparisons. (D-E) RT-qPCR determination of CXCL1 mRNA expression in TRIM28-knockdown and TRIM28-overexpressed CMT-167 or H1299 cells, treated with or without NF-κB inhibitor (BAY11-7085) at 10µM for 24 h. ELISA validation of levels of CXCL1 in cell culture supernatants from CMT-167 or H1299 cell culture. Data are representative of results obtained in three independent experiments. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. (F-G) Representative IHC staining and quantification for CXCL1 in the indicated tumor models are shown. Additionally, ELISA was performed to validate the levels of CXCL1 in tumor lysates and sera from CMT-167 syngeneic tumor models, including subcutaneous tumors and blood samples collected from mice bearing CMT-167 control and CMT-167-shTRIM28 tumors. The scale bar represents 50 μm. (H-I) Representative IHC staining and quantification for CXCL1 in the indicated tumor models are displayed. Additionally, ELISA was conducted to validate the levels of CXCL1 in tumor lysates and sera from CMT-167 syngeneic tumor models, including subcutaneous tumors and blood samples collected from mice bearing CMT-167 control and CMT-167-TRIM28 tumors. The scale bar represents 50 μm. Statistical analysis was performed using a two-tailed student’s t-test. **p < 0.01