Fig 2: p38 inhibitor restores cell growth and invasion in PPM1D-knockdown K-1 cells. (A) Cell growth was determined using a CCK-8 assay in cells with diminished PPM1D expression treated with or without SB203580. Each treatment was assayed in triplicate every 24 h for a total of 3 days. (B and C) The invasive ability of the cells was assessed using a Transwell cell invasion assay. (B) Representative images and (C) quantitative results. (D) Western blot analysis of PPM1D-silenced cells treated with or without p38 inhibitor for 24 h. The drug was used at a concentration of 100 nM. The relative quantification of protein bands as the ratio to their loading control are presented at the bottom of the western blotting. Values are expressed as the mean ± SEM of three independent experiments. P-values were determined using ANOVA test followed by Tukey's post hoc test. **P<0.01, ***P<0.001. N.S., no significance; PPM1D, protein phosphatase, Mg2+/Mn2+ dependent, 1D; CCK-8, Cell Counting Kit-8.

Fig 3: PPM1D silencing-induced K-1 cell apoptosis is suppressed by p38 inhibition. (A) K-1 cells treated with or without SB203580 were collected after transfection with siRNA specific to PPM1D. Apoptosis of K-1 cells was detected by flow cytometry. (a-c) Representative plots presenting Annexin V/PI staining. (a) NC, (b) PPM1D-1 siRNA-transfected, (c) PPM1D-1 siRNA-transfected + SB203580. (B) Analysis of apoptotic (early and late) cells from A. Values are expressed as the mean ± SEM of three independent experiments. P-values were determined using ANOVA test followed by Tukey's post hoc test. ***P<0.001. (C) Western blot analysis was performed to confirm the protein knockdown and pharmacological inhibition of p38. Actin was served as a loading control. The relative quantification of protein bands as the ratio to their loading control were presented at the bottom of the western blotting. PPM1D, protein phosphatase, Mg2+/Mn2+ dependent, 1D; NC, negative control (scrambled siRNA); siRNA, small interfering RNA; PI, propidium iodide.

Fig 4: PPM1D suppresses p38 MAPK and p53 signaling pathways. p-p38 MAPK, p53 and Bax protein levels were upregulated in siPPM1D-treated thyroid cancer cells. (A and B) Western blot analysis of protein levels in TPC-1 and K-1 cells transfected with siPPM1Ds or scrambled RNA for 24 h (upper panel) and relative quantitative analysis of the protein levels in each group (lower panel). Values are expressed as the mean ± SEM of three independent experiments. P-values were determined using ANOVA test followed by Tukey's post hoc test. *P<0.05, **P<0.01, ***P<0.001. N.S., no significance; PPM1D, protein phosphatase, Mg2+/Mn2+ dependent, 1D; p-p38, phosphorylated p38; MAPK mitogen-activated protein kinase; WT, wild-type; NC, negative control; siPPM1D-1/siPPM1D-2: siRNAs against PPM1D; siRNA, small interfering RNA.

Fig 5: PPM1D protein expression is increased in thyroid cells. (A) The protein expression of PPM1D in PTC (tumor) and adjacent non-tumor tissues (para-tumor) tissues was examined by immunohistochemistry. Yellow-brown represents positive staining for PPM1D, localized in the cytosol (scale bars, 50 µm in the upper images and 20 µm in lower images). (B) PPM1D expression was quantified by determining the IOD scores. Values are expressed as the mean ± SEM from three independent experiments. P-values were determined using the independent-samples t-test, **P<0.01. (C) Cells transfected with the indicated siRNAs were harvested and subjected to western blot analysis. GAPDH served as a loading control. The relative quantification of protein bands as the ratio to their loading control were presented at the bottom of the western blotting. PPM1D, protein phosphatase, Mg2+/Mn2+ dependent, 1D; PTC, papillary thyroid carcinoma; IOD, integrated optical density; siRNA, small interfering RNA.