Fig 2: PKC and ROCK regulate MLC phosphorylation.(A) Immunoblot depicts MLC phosphorylation at Ser19 in wild-type mouse platelets pre-incubated with vehicle, the PKC inhibitor BIM (15 µM), and/or the ROCK inhibitor Y27632 (50 µM) for 30 min prior to stimulation with thrombin (0.1 U/ml) for 10 min. Total MLC is shown as a loading control. (B) Bar graph represents the densitometric quantification of p-MLC2 (Ser19) relative to total MLC in thrombin-activated platelets pre-treated with vehicle (TR, orange), BIM (BIM, green bar), Y27632 (Y27632, blue bar), or BIM plus Y27632 (BIM + Y27632, purple bar). Resting platelets serve as the negative control (NT, red bar). Data are mean ± SEM and represent a minimum of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, based on one-way ANOVA and Tukey post hoc multiple comparison tests. (C) Immunoblot depicts MYPT1 phosphorylation at Thr853 in wild-type mouse platelets pre-incubated with vehicle, the PKC inhibitor BIM (15 µM), and/or the ROCK inhibitor Y27632 (50 µM) for 30 min prior to stimulation with thrombin (0.1 U/ml) for 10 min. Total MYPT1 is shown as a loading control. (D) Bar graph represents the densitometric quantification of p-MYPT1 (Thr853) relative to total MYPT1 in thrombin-activated platelets pre-treated with vehicle (TR, orange), BIM (BIM, green bar), Y27632 (Y27632, blue bar), or BIM plus Y27632 (BIM + Y27632, purple bar). Resting platelets serve as the negative control (NT, red bar). Data are mean ± SEM and represent a minimum of four independent experiments. *P < 0.05, ****P < 0.001, based on one-way ANOVA and Tukey post hoc multiple comparison tests.

Fig 3: Changes in cellular morphology and stress fiber formation in TR-MUL5 cells treated with acrolein and ripasudil. Representative fluorescent staining patterns of F-actin (green, Acti-stainTM 488) and p-MLC2 (red, Alexa Fluor® 546) in TR-MUL5 cells treated with acrolein (25 μM) and/or ripasudil (2 μM). Blue, nuclei counterstained with DAPI. Scale bar, 50 μm.

Fig 4: myCAF activation induced by S100A8 is triggered via CD147-RhoA-ROCK-MLC2-MRTF-A pathway(A) KEGG pathway enrichment analysis of distinct CAF subtypes.(B) Spearman correlation between the S100A8 RNA level and the pathway signature score. The gray area represents 95% CI (n = 60).(C) Western blot analysis of RhoA-associated protein markers in CAFs treated with indicated concentration of rS100A8 proteins.(D) Western blot analysis of RhoA-associated and myCAF-related protein markers in CAFs transfected with siRNAs targeting RhoA, ROCK1, MLC2, or negative control and treated with rS100A8 proteins or PBS.(E) Representative immunofluorescence images of MRTF-A nuclear translocation in CAFs treated with rS100A8 proteins or PBS. White arrows indicate MRTF-A nuclear translocation in cells. Scale bar, 100 μm.(F) Quantification of the percentage of MRTF-A nuclear translocation in (E) (n = 6 biological replicates). Data are presented as mean ± SD. ∗∗∗∗p < 0.0001 of two-tailed Student’s t test.(G) Western blot analysis of the indicated proteins in CAFs transfected with MRTF-A or control siRNA and treated with indicated concentration of rS100A8 proteins.(H) Representative immunofluorescence images of CD147 and αSMA in control and CD147-knockout CAFs. Scale bar, 100 μm.(I) Western blot analysis of the indicated proteins in control and CD147-knockout CAFs treated with indicated concentration of rS100A8 proteins.(J) Western blot analysis of the indicated proteins in control and CD147-knockout CAFs cocultured with control or S100A8-overexpression KYSE450 cells.Each assay for western blot had three biological repeats. See also Figures S4 and S5.

Fig 5: Changes in phosphorylation of MYPT1 and MLC2 in TR-MUL5 cells treated with acrolein and ripasudil. Phosphorylation levels of MYPT1 and MLC2 in TR-MUL5 cells after acrolein stimulation. Ripasudil suppressed the increase of phosphorylated forms of MYPT1 and MLC2 induced by acrolein stimulation in TR-MUL5 cells.