Autophagic Flux Unleashes GATA4-NF-κB Axis to Promote Antioxidant Defense-Dependent Survival of Colorectal Cancer Cells under Chronic Acidosis

Figure 7

ICAM-1 secreted by colon cancer cells promotes survival under acidic microenvironment. (a) First row, HCT15 cells were cultured in pH 7.4 or pH 6.5 medium; second row, HCT15 cells were cultured in pH 6.5 medium with 1/3 pH 7.4 or pH 6.5 condition medium and measured with colony-forming assay after 12 d. (b) HCT15 cells were treated as in (a), and apoptosis was measured by flow cytometry. (c) Soluble ICAM-1 concentration was measured by ELISA. (d) Expression of ICAM-1 was determined by Western blotting. (e) Flow cytometric analysis of ICAM-1 levels in CRC-AA and their parental cells. (f) Knockdown of ICAM-1. HCT15 and HCT15-AA cells were transfected with siRNA for 48 h; RNAi efficiency was determined by Western blotting. GAPDH was used as a loading control. (g) HCT15 cells were cultured in pH 7.4 or pH 6.5 medium, with addition of CRC-AA conditioned medium, CRC-AA siICAM-1 conditioned medium, or ICAM-1 recombinant protein. The level of apoptosis was measured by flow cytometry. (h) DCF intensity in CRC and CRC-AA cells transfected with ICAM-1 siRNA for 24 h. (i) HCT15 cells were cultured in pH 7.4 or pH 6.5 medium, with addition of CRC-AA conditioned medium or ICAM-1 recombinant protein; ROS levels were measured by flow cytometry analysis of DCF. (j) NF-κB-dependent expression of ICAM-1. p65 was depleted by siRNA for 48 h in HCT15 and HCT15-AA cells. ICAM-1 and p65 protein levels were determined by Western blotting. (k) Schematic model showing the role of the GATA4-NF-κB pathway driven by autophagy in CRC-AA cells. ; ; ns, .

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