Figure 5: miR-625-3p regulates resistance to oxPt through MAP2K6 and MAPK14. | Nature Communications

Figure 5: miR-625-3p regulates resistance to oxPt through MAP2K6 and MAPK14.

From: miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cells

Figure 5

(a) Western blotting using antibodies against the phosphorylated forms of MAPK14T180/Y182, HSPB1Ser82, 4EBP1Ser64 and CDC25cS216 in HCT116.625 and SW620.625 cells 48 h after DOX induction. β-Actin and tubulin was used as loading control (left). Quantification of HSPB1Ser82, 4EBP1Ser64 and CDC25cS216 western blot bands from three to five western blots normalized to α-tubulin and β-actin and displayed as log2(625/ctrl)±s.e.m. In one case the HSPB1Ser82 signal in SW620.625 was below detection level, and for this sample the median value for the two other replicates was used (right). (b) Changes in phosphorylation of activated pT180/Y182-MAPK14 and downstream substrates in HCT116.ctrl.mock, HCT116.625.mock and HCT116.625.map2k6 cells after 48 h of DOX induction followed by 30 min of 64 μM oxPt treatment (left). Quantification of HSPB1Ser82, 4EBP1Ser64 and CDC25cS216 substrate phosphorylation from three to five western blots normalized to α-tubulin and β-actin and displayed as oxPt-induced phosphorylation change compared with untreated cells (log2(64 μM/0 μM)±s.e.m. (right). (c) Same as b for the HCT116.ctrl.map2k6 cells. (d) Cells were DOX-induced for 48 h and treated with 0–64 μM oxPt for 48 h before cell death was determined (LDH assay). Bars represent the mean percentage of cell death±s.e.m. (n=3). Significant difference between HCT116.625.mock and HCT116.625.map2k cells is indicated (*P≤0.05, t-test). (e) Control HCT116 cells (ctrl) and cells expression a dominant-negative version of MAP2K6 (DN) were induced for 48 h and treated with 64 μM oxPt or left untreated for 48 h before the increase in cell death (64 μM/0 μM) was determined by LDH. Results are displayed relative to control cells (set to 1; mean±s.e.m. from n=4 experiments; *P≤0.05; t-test). Western blot against MAP2K6 (f) Correlation between MAP2K6 mRNA levels and mir-625-3p in clinical samples (P=0.212, Pearson’s correlation). (g) MAP2K6 mRNA was downregulated in tumours from mCRC patients not responding (NR, n=9) compared with responders (R, n=17) to first-line oxPt-based therapy (t-test).

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