Fig. 3

IGFBP2 protects esophageal adenocarcinoma cells from acidic bile salts-induced DNA double strand breaks and apoptosis. a, OE33 cells with IGFBP2 overexpression (IGFBP2) and control cells were treated with ABS (pH 4, 200 μM) for 20 min and then recovered for the designated time points. Western blot analysis was used to detect levels of IGFBP2, caspase3, PARP and H2AX. b, FLO1 cells with IGFBP2 knockdown (si-IGFBP2) and control cells (si-control) were treated with ABS for 20 min and then recovered for designated time points. Western blot analysis was used to detect levels of IGFBP2, caspase3, PARP and H2AX. c and d, FLO1 cells with IGFBP2 knockdown and control cells were treated with ABS for 20 min and then recovered for 3 h. Immunofluorescence staining for p-H2AX (Ser139, red) was performed. DAPI (blue) was used to stain cell nucleus. Quantification of positive p-H2AX cells was shown in D. e and f, OE33 cells with IGFBP2 overexpression and control cells were treated with ABS for 20 min and then recovered for 3 h. Immunofluorescence staining for p-H2AX (Ser139, red) was performed. DAPI (blue) was used to stain cell nucleus. Quantification of positive p-H2AX cells was shown in f. g and h, flow cytometry analysis of Annexin V positive cells in FLO1 cells with IGFBP2 knock down or control siRNA, treated with ABS for 30 min and recovered for 3 h. g shows representative flow cytometry profiles and h displays bar graph of live and apoptotic cells. ABS, acidic bile salts; UT, untreated with ABS. * p < 0.05, ** p < 0.01, **** p < 0.0001