CLCA4 suppressed colorectal cancer stem cell expansion by interacting with vimentin to suppress FAK signaling pathways. (IMAGE)
Caption
(A) Western blotting analysis of FAK and p-FAK protein levels in control and CLCA4-overexpressing colorectal cancer (CRC) cells. Right panels: Quantification of protein expression ratio. (B) Western blotting analysis of stemness-related proteins and p-FAK in CLCA4-overexpressing cells treated with or without FAK agonist. Lower panels: Quantification of protein expression ratio. (C) Tumorsphere formation assay was performed to examine the tumorsphere formation ability in CLCA4-overexpressing cells treated with or without FAK agonist. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation). (D) Immunoprecipitation and IgG samples were analyzed by mass spectrometry. Proteins with unused >1.3 were filtered out, and keratin was removed. A total of 336 proteins were identified, including 334 proteins in immunoprecipitation samples and 4 proteins in IgG samples. (E) The immunoprecipitates of CLCA4 were purified using anti-Flag antibody and separated with SDS-PAGE, and the presence of vimentin was analyzed by Western blotting. Normal IgG was used as the negative control. (F) The immunoprecipitates of vimentin were purified using anti-HA antibody and separated with SDS-PAGE, and the presence of CLCA4 was analyzed by Western blotting. Normal IgG was used as the negative control. (G) The differences in protein levels (vimentin, Bmi-1, and p-FAK) among CRC cells transfected with different plasmids were analyzed by Western blotting. Right panels: Quantification of protein expression ratio. (H) Tumorsphere formation assay was performed to examine the tumorsphere formation ability among CRC cells transfected with different plasmids. One-way ANOVA with Tukey's multiple comparisons test (mean ± standard deviation).
Credit
Fang Wei, Qi Zou, Qihui Sun, Tingting Jiang, Tian Cai, Xiaojia Li, Keping Xie
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