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. 2018 Sep 11;7(2):457–473. doi: 10.1016/j.jcmgh.2018.09.002

Figure 5.

Figure 5

MDX induces IRE1β expression via P38 MAPK. (A) MDX induces Ern-2 expression in the HT29-MTX cell line. Cells were either left untreated (UT) or cultured with increasing concentrations of MDX for 1 hour. Ern-2 RNA transcripts were assessed by real-time PCR. Data are means ± SD of 10 samples per group derived from 4 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (**P ≤ .01, *P ≤ .05). (B) HT29-MTX cells were either left UT or stimulated with 5% MDX for the indicated time points. p-p38, p38, phosphorylated extracellular signal–regulated kinase 1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and β-actin (β-act) expression were analyzed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with densitometry analysis (lower panel). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*P ≤ .05, **P ≤ .01, ***P ≤ .001). (C) Effect of the p38 inhibitor (p38i) on MDX-mediated p38 activation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or dimethyl sulfoxide (DMSO) (vehicle). p-p38 and p38 expression was assessed by Western blot. One of 4 representative experiments in which similar results were obtained is shown together with the densitometry analysis (lower panel). Data are means ± SD. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (***P ≤ .001). Right: Effect of p38i on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were stimulated or not with MDX for 1 hour in the presence or absence of p38i (10 μmol/L) or DMSO (vehicle) and Ern-2 RNA transcripts evaluated by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*P ≤ .05). (D) Effect of p38 knock-down on MDX-mediated Ern-2 up-regulation. HT29-MTX cells were transfected with either control or p38 siRNA (CTR or p38 siRNA, respectively) for 18 hours and then stimulated or not with 5% MDX for 1 hour. Representative Western blot for p-p38, p38, and β-actin expression are shown in the left panels together with the densitometry analysis. Data are means ± SD of 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (**P ≤ .001). Right: Expression of Ern-2 RNA transcripts assessed by real-time PCR. Data are means ± SD of 5 independent experiments. Differences among groups were compared using 1-way analysis of variance followed by the Bonferroni post hoc test (*P ≤ .05). (E) Immunofluorescence analysis of p-p38 (green) in colon samples isolated from MDX-fed mice and controls killed on day 45. The figure is representative of 3 separate experiments in which similar results were obtained. Right panels show the number and intensity of p-p38–expressing cells per field of colon section. Data are expressed as means ± SD and were generated using 3–5 mice per group from 3 independent experiments. Differences between groups were compared using the 2-tailed Student t test (**P ≤ .01). mRNA, messenger RNA; p38 tot, total p38.