Fig 2: Hyperosmotic conditions protect centriolar satellites from p38-mediated depletion. (A) U2OS cells were treated with the indicated stress agents (1 h) and lysates were analysed by immunoblotting with antibodies against phospho-p38, total p38, MK2 and p150 (loading control). (B) Cells treated as in (A) were immunostained with antibodies against CEP131 and ?-tubulin and counterstained for nuclear content with DAPI. (C) Quantification of (B). At least 100 cells were scored per condition in 3 independent experiments. Bars indicate the mean +/- SEM. p-values were calculated from a one-way ANOVA using Dunnett’s correction for multiple testing. (D) U2OS cells were incubated in the presence of 500 mM sorbitol for the indicated times. Cells were immunostained with antibodies against PCM1 and pericentrin and counterstained for nuclear content with DAPI. (E) Quantification of (D) performed as in (C). *, p < 0.05; ***, p < 0.0005; ****, p < 0.0001, n.s.; not significant. All scale bars, 10 µm. CS; centriolar satellites.

Fig 3: Sorbitol does not interfere with phosphorylation of MK2 targets. (A) U2OS cells were treated with anisomycin or sorbitol (1 h) in the indicated combinations. Cells were immunostained with antibodies against CEP131 and counterstained for nuclear content with DAPI. PCNT; Pericentrin. Scale bars, 10 µm. (B) Quantification of (A). At least 100 cells were scored per condition in 3 independent experiments. Bars indicate the mean +/- SEM. p-values were calculated from a one-way ANOVA using Dunnett’s correction for multiple testing. ****, p < 0.0001, n.s.; not significant. CS; centriolar satellites. (C) U2OS cells were pretreated with an MK2 inhibitor (MK2i—1 h) and incubated in the presence of sorbitol and anisomycin as indicated. Lysates were analysed by immunoblotting with antibodies against phospho-p38, total p38, MK2, NOGO-B, phospho-HSP27, phospho-SRF and p150 (loading control). (D) Lysates from cells treated as in (c) were incubated with recombinant GST or GST-14-3-3 and subjected to GST pulldown (PD). Pulldown material and whole cell extracts (WCE) were analysed by immunoblotting with antibodies against CEP131, MK2 and GST.

Fig 4: Ectopic expression of WIP1 reduces the levels of activated p38 and enhances stemness-related protein expression and CSC properties in NSCLC cells. a Western blotting was used to analyze the expression of WIP1, phospho-p38, p38, SOX2, OCT4, NANOG, and ALDH1A1 in H1299 (left panels) and H460 (right panels) cells transduced with a WIP1-overexpressing plasmid (WIP1) or vector control (pLV). Arrows indicate the positions of p38 isoforms. b Western blotting was used to analyze MK2, phospho-MK2 (Thr222), phospho-MK2 (Thr334), HSP27, and phospho-HSP27 (Ser82) in H460 cells transduced with the WIP1-overexpressing plasmid (WIP1) or vector control (pLV). c, d A sphere formation assay was performed with H1299 (top graphs) and H460 (bottom graphs) cells transduced with the WIP1-overexpressing (WIP1) or vector control (pLV) plasmid. Quantifications of sphere sizes (d) and numbers (e) are shown in bar graphs. The data are presented as the mean ± SD of three independent experiments. * indicates P < 0.05, and ** indicates P < 0.01 vs. the pLV control determined by Student’s t test. e The percentage of the side population was measured by flow cytometry following Hoechst 33342 staining of H1299 (top graph) and H460 (bottom graph) cells transduced with the WIP1-overexpressing (WIP1) or vector control (pLV) plasmid. The bar graphs show the quantifications of the percentages of the side population. The data are presented as the mean ± SD of three independent experiments. ** indicates P < 0.01, and *** indicates P < 0.001 vs. the pLV control determined by Student’s t test

Fig 5: The WIP1 inhibitor GSK2830371 enhances p38 activation and suppresses stemness-related protein expression and CSC properties in NSCLC cells. a Expression of phospho-p38, p38, and WIP1 in A549 (top panels) and H460 (bottom panels) cells evaluated by Western blotting after treatment with the indicated concentrations of GSK2830371 or DMSO for 12, 24, or 48 h. b Western blotting of stemness-related proteins and ALDH1A1 in A549 (top panels) and H460 (bottom panels) cells treated with 10 µM GSK2830371 or DMSO for 48 h. c Western blotting of MK2, phospho-MK2 (Thr222), phospho-MK2 (Thr334), HSP27, and phospho-HSP27 (Ser82) in H460 cells treated with 10 µM GSK2830371 or DMSO for 48 h. d, e Quantifications of the sizes (d) and numbers (e) of spheres formed by A549 (top graphs) and H460 (bottom graphs) cells after incubating with 10 µM GSK2830371 or DMSO for 10 days. The data are presented as the mean ± SD of three independent experiments. * indicates P < 0.05, and *** indicates P < 0.001 vs. the vehicle control (DMSO) determined by Student’s t test. f The percentage of the side population measured by flow cytometry following Hoechst 33342 staining of A549 (top graph) and H460 cells (bottom graph) after a 48-h treatment with 10 µM GSK2830371 or DMSO. The data are presented as the mean ± SD of three independent experiments. *** indicates P < 0.001 vs. the vehicle control (DMSO) determined by Student’s t test. g Western blotting of stemness-related proteins and ALDH1A1 in H1299 cells treated with 10 µM GSK2830371 or DMSO for 48 h