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. 2017 Jul 17;292(37):15408–15425. doi: 10.1074/jbc.M117.784090

Figure 5.

Figure 5.

CXCL10 production in metastatic IECs is elicited independent of poly(I:C) internalization but requires endosomal acidification. A, confocal microscopy image of HT29 cells treated with poly(I:C)Rhodamine (red, 5 μg/ml) for 2 h before cells were washed and fixed, and the plasma membrane (PM) was stained with an antibody against Na,K-ATPase (PMA488, green). Images (top) show co-localization (Coloc, left) and single tracks of poly(I:C)Rhodamine (center) and plasma membrane PMA488 staining (right) of the area denoted by the square in the main image. Scale bar = 5 μm. B and C, SW620 (B) or HT29 (C) cells were treated with poly(I:C) (Added Poly(I:C)) or double-stranded DNA dA:dT (Added dA:dT) added in solution or by plating cells in wells precoated with poly(I:C) (Coated Poly(I:C)) or dA:dT (Coated dA:dT) with the given concentrations for 24 h before CXCL10 release was determined by ELISA. D, CXCL10 production in HT29 cells exposed to HMW poly(I:C) or LMW poly(I:C) (2 μg/ml) either added in solution (Added) or by plating cells in wells precoated with poly(I:C) (Coated) for 21 h. E, CXCL10 release in HT29 cells pretreated with anti-TLR3 (15 or 5 μg/ml) or control goat IgG (15 μg/ml) for 1 h prior to stimulation with poly(I:C) (5 or 2 μg/ml) for 10 h. CXCL10 content in the supernatant was assessed by ELISA. **, p < 0.01; *, p < 0.05 versus cells pretreated with control IgG (two-way ANOVA, Bonferroni post-test). The results in A–D show mean ± S.D. of triplicates and are representative of three independent experiments. #, below detection. F, CXCL10 expression in HT29 cells treated with the dynamin inhibitor Dynasore (80 or 40 μm) or the DMSO control for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). The results show the mean of triplicates from three independent experiments ± S.D. ns, not significant (two-way ANOVA, Bonferroni post-test). #, below detection. G, CXCL10 production in HT29 cells transfected with NS RNA (20 nm) or two siRNAs against clathrin heavy chain 1 (siC2 and siC3, 10 + 10 nm) in two rounds for 26 h and 20 h prior to stimulation with poly(I:C) (2 μg/ml) for 8 h. The results show mean ± S.D. of triplicates and are representative of three independent experiments. **, p < 0.01 versus cells transfected with non-silencing siRNA (two-way ANOVA, Bonferroni post-test). Inset, Western blots of lysates of HT29 cells treated in parallel as described for siRNAs against clathrin heavy chain 1 (siC2 or siC3) or non-silencing RNA (N), stained with antibody against clathrin heavy chain 1 (CHC, top blot) or against α-tubulin (Tubulin, bottom blot). H, CXCL10 production in HT29 cells pretreated with bafilomycin A (2–1 μm) or the DMSO control for 30 min prior to stimulation with poly(I:C) (2 or 1 μg/ml) for 10 h. The supernatant was assayed for CXCL10 content by ELISA. The results show mean ± S.D. of triplicates and are representative of two independent experiments. **, p < 0.01; *, p < 0.05 versus untreated cells (two-way ANOVA, Bonferroni post-test).