Fig 2: TME5C increases the levels of p-ERK, p-AKT, p-p38, and Mcl-1 in endothelial cells. (A and C). HUVECs or HHSECs were exposed to control diluents (PBS as control) or TM mutants (500 nM). After 48 h, proteins were extracted and subjected to western blot analyses. The membrane was sequentially probed with the indicated antibodies. (B and D). Relative quantifications of p-ERK, p-AKT, p-p38, and Mcl-1. ImageJ software was used to measure the band intensities after western blotting. All experiments were performed three times. Results represent the mean ± SD. *P<0.05. TM: thrombomodulin; HUVECs: human umbilical vein endothelial cells; HHSECs: human hepatic sinusoidal endothelial cells.

Fig 3: MLKL oligomerization induces p38 activation to mediate the phosphorylation of Ser473 TRIM28.A HT-29 cells were treated with the indicated inhibitors followed by TSZ treatment for 4 h. The cell lysates were analyzed by western blotting using indicated antibodies. B p38 KO cells were engineered by CRISPR–Cas9 technology and p38 expressing vector was reconstituted in HT-29 cells. WT and p38 KO HT-29 cells were treated with TSZ in the presence or absence of Nec-1s at indicated time points. The cell lysates were analyzed by western blotting using indicated antibodies. C WT and p38 reconstituted p38 KO HT-29 cells were treated with TSZ in the presence or absence of Nec-1s as indicated. The cell lysates were analyzed by western blotting using indicated antibodies. D, E HT-29 cells were transfected with an expression vector of flag-tagged MLKL fused with two AP20187-binding (FKBPv) domains. The cells were pretreated with indicated compounds for 4 h followed by treatment with AP20187 to induce MLKL oligomerization. Cell viability was determined by CellTiter-Glo after treatment with AP20187 for 2 h (D Left). Cell lysates were separated by non-reducing SDS/PAGE and analyzed by western blotting using MLKL antibody (D Right). The cell lysates were immunoprecipitated by anti-Flag resin after treatment with AP20187 for 2 h and analyzed by western blotting using indicated antibodies (E). acMLKL, oligomerizable MLKL; endo, endogenous. TNFa, 20 ng/mL; zVAD, 25 µM; SM164, 50 nM; Nec-1s, 10 µM; NSA, 4 µM; TPCA-1, 10 µM; p38i PH-797804, 10 µM; p38i LY2228820, 4 µM; 5Z-7, 500 nM; MK2i MK2-IN-1, 10 µM; AP20187, 20 µM. Data were presented as mean ± SEM. **t-test p < 0.01, n = 3. n.s., not significant.

Fig 4: p38 MAPK activity differs among glial cells with normal or malignant phenotypes. Representative western blots of protein isolated from the indicated cells lines harvested 24 h after serum shock and probed with anti-phospho-p38 (pp38), or anti-total p38 (p38) antibodies. Amido-stained membranes were used to normalize protein loading. The graph below shows the average pp38 MAPK signal normalized to total p38 MAPK levels (n = 3, ± SEM). pp38 MAPK levels were significantly lower in C6 cells compared to IM3 (**p < 0.05) cells

Fig 5: Hyperosmotic stress dampens Fus3 activation in a Hog1-dependent manner.(A) Activation kinetics of Fus3 and Hog1; wild-type cells transformed with plasmid-borne GAL1-FUS3 were grown in SC and 2% galactose followed by stimulation with 10 µM a factor, 0.75 M KCl, or co-stimulation with 10 µM a factor+0.75 M KCl. Cell lysates were resolved by 12.5% SDS-PAGE. P-Fus3 and P-Kss1 were detected with phospho-p44/p42 antibodies. P-Hog1 was detected with phospho-p38 antibodies. Total Fus3 and Hog1 were detected with Fus3 and Hog1 antibodies. G6PDH served as a loading control. All primary antibodies were recognized by fluorescently labeled secondary antibody, detected by fluorescence scanner (Typhoon Trio) and quantified by scanning densitometry (ImageJ). The panel to the right shows averaged scanning densitometry of four individual experiments. Error bars represent ± SEM. Co-stimulation dampened P-Fus3 by 47.6%±2.2% at 5 min and 47.5%±6.6% at 30 min. (B) hog1? cells transformed with GAL1-FUS3 treated as in A. Co-stimulation dampened P-Fus3 by 44.3%±7.4% at 5 min and 7.4%±10.9% at 30 min. (C) Sequential stimulation of Hog1 and Fus3. Wild-type cells grown in SC with 2% dextrose stimulated with 0.75 M KCl, and after an indicated period of stress adaptation stimulated with 10 µM a factor for an additional 15 min. Error bars represent ± SEM.