Fig. 3

Oxaliplatin-mediated membrane CLDN1 expression is dependent on MAPKp38,/GSK3β/ Wnt-βcat signaling cascade (A) Analysis of MAPK p38 phosphorylation (Pp38) by western botting in SW620 cells after incubation or not (UT) with oxaliplatin for 14 and 24 h (1.2 µM) Top: representative western blotting image; bottom: quantification of MAPK p38 phosphorylation level in the different conditions. (B) CLDN1 expression at the membrane (g-mean quantification) of SW620 cells incubated (+) or not (-) with oxaliplatin and/or LY2228820 (p38 inhibitor) (1 µM) for 72 h p ≤ 0.001 (Student’s t-test). (C) Western blotting showing the expression of GSK3β, GSK3βSer9 and β-catenin in SW620 cells incubated or not with oxaliplatin (5µM for 24 h). (D-E) GSK3βSer9 expression evaluated (D) by flow cytometry and (E) by immunofluorescence using the Celigo™ imaging cytometer. Incubation with H2O2 was used as positive control because it increases GSK3β phosphorylation at Ser9 (20). (F) GSK3β silencing. Left, western blot analysis of GSK3β expression in SW620 cells in which GSK3β was silenced (shGSK3β) or not (WT). Right, effect of GSK3β silencing on CLDN1 membrane expression after incubation with oxaliplatin (1.2 µM for 72 h) evaluated by FACS relative to non-silenced cells ** p = 0.001 (Student’s t-test). (G) Proximity ligation assay (PLA) with oligonucleotide-conjugated antibodies against p38 and GSK3β in SW620 cells incubated or not with oxaliplatin (5µM for 24 h). Left: Fluorescence images, nuclei were counterstained with DAPI (blue). Right: PLA dot counts per cell in the corresponding fluorescence images p ≤ 0.001 (Student’s t-test) (H) Expression and localisation of total and inactive (phosphorylated) βcatenin by immunofluorescence staining in SW620 cells after incubation or not with oxaliplatin (5µM for 72 h). In treated cells, total β-catenin expression increases and inactive β-catenin translocates to the nucleus (arrow) (I) The TOP/FOP Flash luciferase assay shows the transcriptional activation of the Wnt/β-catenin signaling pathway in SW620 cells after oxaliplatin incubation (5µM for 72 h) compared with untreated cells. * p = 0.01 (paired t-test) (J) Oxaliplatin (5µM for 72 h) effect on the mRNA expression of Wnt /β-catenin target genes in SW620 cells compared with untreated cells (Student’s t-test). (K) Oxaliplatin-induced (1.2µM for 72 h) CLDN1 membrane expression increase is reduced when SW620 cells are co-incubated with 7.5 µM XAV939 (small molecule inhibitor of tankyrase) ****p < 0.0001 (one-way ANOVA test)