Figure 9: Critical components of the cellular response to oxPt are blocked in cells with increased miR-625-3p levels.

(a) Mean log₂ ratios of substrates groups involved in oxPt response were calculated for the 625+OX/ctrl+OX data. (b) Mean log₂ for the MAPKAPK2 substrate group. (c) The most significantly altered substrate phosphorylation motifs identified for the ctrl+OX/ctrl and 625+OX/ctrl+OX experiments identified using KSEA (displayed as fcounts, P≤0.05 indicated with "*"). Mean log₂ ratios for substrates with these motifs were calculated. Coloured boxes in the boxplots of a, b and c indicate P≤0.05, z-test; and NS, not significant. On the basis of similarity, the 16 individual motifs were grouped into the four motif groups indicated above. Note that the experimental mean log₂ ratios for clarity have been omitted in b and c. (d) oxPt treatment in HCT116.ctrl cells led to dephosphorylation of Serine 130 (S130) of Cyclin-Dependent Kinase Inhibitor 1 (CDKN1A, also known as p21^CIP1), which has been linked to increased stability of CDKN1A and inhibition of CDK/cyclin-mediated cell cycle progression⁶⁵. In contrast, increased S130 phosphorylation was seen in cells with ectopic miR-625-3p expression. As indicated, this phosphorylation may itself be mediated by elevated CDK activity⁶⁶. Increased CDK activity at the G1/S checkpoint or in early M phase was also indicated by S138/S151 phosphorylations on inactivated FZR1 (also known as CDH1) in miR-625-3p expressing cells, whereas unphosphorylated FZR1 in control cells suggested decreased CDK signalling at G0 or early G1 (ref. 67). In support of mitotic-induced nuclear lamina breakdown, increased phosphorylation was observed on multiple residues on LMNA in miR-625-3p cells; On the contrary, these became dephosphorylated after oxPt treatment in control cells indicating decreased cell cycle progression (also see Supplementary Fig. 14). (e) Western blotting against the CDK1 substrate phospho-LAMIN A/C^S22 on lysates from oxPt-treated HCT116.ctrl and HCT116.625 cells. Quantification of bands representing Lamin A and C isoforms are indicated (normalized to β-actin signal). (f) Western blotting against the phosphorylated CDK motif p-TPXK on lysates from oxPt-treated HCT116.ctrl and HCT116.625 cells. Individual substrates are indicated with a dot with red and black indicating increase or decrease/no change in intensity, respectively, in HCT116.625 as compared with HCT116.ctrl cells.