Journal: Genes & Diseases

Article Title: Chloride channel accessory 4 suppresses stem cell-like properties of colorectal cancer and enhances anti-PD-1 immunotherapy

doi: 10.1016/j.gendis.2025.101859

Figure Lengend Snippet: CLCA4 suppressed colorectal cancer stem cell expansion by interacting with vimentin to suppress FAK signaling pathways. (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).

Article Snippet: After 7 days, mice were intraperitoneally treated with either an in vivo blocking antibody against mouse PD-1 (Clone: 29F.1A2, BioXcell, Cat# BP0273) or a rat IgG2a isotype control antibody (Clone: 2A3, BioXcell, Cat# BP0089).

Techniques: Protein-Protein interactions, Western Blot, Control, Expressing, Tube Formation Assay, Standard Deviation, Immunoprecipitation, Mass Spectrometry, Purification, SDS Page, Negative Control, Transfection