Fig 2: HDAC6 promotes sIL-6R release by activating the TAK1–p38–ADAM17 axis.A Western blot analysis of the total protein expression and phosphorylation of ADAM17, ERK1/2 and p38. GAPDH was used as an internal reference protein. B Schematic of the experimental procedure used to analysis of HDAC6 interacting proteins. The unique peptides and peptide-spectrum matches of TAK1 are 4, which is credible. C. Stable knockdown of TAK1 in HCT116 cells. D, E Analysis of sIL-6R levels in the cell media by ELISA D and detection of ADAM17 activity E in HCT116 cells after TAK1 knockdown. F Flow cytometry assessed CD11b, CD86 and CD206 expression of cocultured macrophages in shCtrl- and shTAK1-treated HCT116 cells. G QPCR detected the expression of M1 and M2 polarization related genes of cocultured macrophages in shCtrl- and shTAK1-treated HCT116 cells. H Treatment with the TAK1 selective inhibitor Takinib decreased TAK1 phosphorylation in HCT116 cells. I, J Analysis of sIL-6R levels in the supernatants by ELISA I and detection of ADAM17 activity J in HCT116 cells after treated with Takinib. K Western blot analysis of TAK1 total protein expression and phosphorylation in HCT116 and HT29 cells with stable HDAC6 knockdown. Data were shown as the mean ± SD of three independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Suppressive effects of prostaglandin E synthase (PTGES) siRNA in IFN-γ-treated MSCs on the phenotypic change of macrophages from M1 to M2. (a) THP-1 monocytes were stimulated by PMA for 48 h and then incubated with CM from IFN-γ-treated hMSCs transfected with negative control (NC) siRNA or PTGES siRNA. After 48 h, the cells were collected and subjected to western blot analysis of CD163 and CD68. CD163 protein levels were normalized to GAPDH and CD68 levels (n = 5 in each group). The blots are the cropped images from different parts of the same gel. Full-length gel images are provided in the supplementary file. The samples derive from the same experiment and that gels were processed in parallel. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01.

Fig 4: Expression of a PKR kinase-dead mutant results in an increase of viral proteins and virus titer. (A) RD cells were transiently expressed with PKR or the K296H mutant for 24 h, and then infected with EV-A71 at an m.o.i. of 10. Immunoblot analysis was performed for detecting the presence and phosphorylation of PKR (anti-PKR, anti-PKR-p) and eIF2a (anti-eIF2a, anti-eIF2a-p), and the expression of viral 3A and 3C proteins. GAPDH expression was used as a protein loading control. A representative result from three independent experiments is shown. (B) Stable RD cells expressing vector alone, PKR, or K296H were selected by addition of 3 µg/mL puromycin. Cells were infected with EV-A71 at an m.o.i. of 10 and then the cellular extracts were harvested at 0, 6, and 8 h post-infection. Immunoblot analysis was performed to detect the presence of PKR and viral 3A and 3C proteins. A representative result based on three independent experiments is shown. (C) RD cells stably expressing PKR or the K296H mutant were infected with EV-A71 at an m.o.i. of 10 of for 8 h. The RD cells and culture supernatant were harvested for virus titer determination by plaque assay. The results are expressed as the mean ± SD (n = 3). *p < 0.05.

Fig 5: Oxidative stress promotes EndMT and has additive effects to TGF-ß.(a) Representative dihydroethidium (DHE) microscopy images of thoracic aortic sections from tamoxifen-induced end.SclCreERT;R26RstopYfp;ApoE-/- mice after 8 versus 18 weeks HFD demonstrating in situ superoxide production. Analysis by one-way ANOVA; overall P<0.0001. (b) TUNEL assay on atherosclerotic lesions from tamoxifen-induced end.SclCreERT;R26RstopYfp;ApoE-/- mice after 8 versus 18 weeks HFD demonstrating apoptotic Yfp+ cells. For a and b: L=lumen; scale bars, 100 µm. Insets at higher magnification as indicated. Arrows indicate co-positive cells. Staining performed on at least three spatially separated thoracic aortic sections per mouse (n=5 mice per group). Control experiments from non-atherosclerotic mice shown in Supplementary Fig. 8. (c) Phase contrast microscopy of HUVECs grown in basal media (ctrl) or following exposure to TGF-ß and/or H2O2 for 5 days. Scale bars, 100 µm. (d) Heatmap showing expression of endothelial genes (top), non-endothelial genes also downregulated in EndMT/EMT, genes known to be upregulated in EndMT/EMT and mesenchymal genes (bottom) for HUVECs exposed to control conditions (basal media—unstimulated), TGF-ß or TGF-ß plus 200 µM H2O2. Because there are no gene sets for ‘EndMT' or ‘fibroblast' in databases like GO, KEGG, Reactome or MSigDB, this gene list was compiled by extensive literature search (Supplementary Table 2). (e) FAP protein expression by western blot with quantification in HUVECs exposed to control (ctrl) conditions (unstimulated) or TGF-ß, with and without H2O2 at 100 or 200 µM. FAP ladder (L) represents 100 kDa (upper) and 75 kDa (lower intense band). FAP expected molecular weight/size is 90 kDa. GAPDH ladder represents 37 kDa. GAPDH expected molecular weight/size is 37 kDa. (f) FAP RNA levels assessed by qRT-PCR under identical conditions. (g) CD31 protein by western blot with quantification. CD31 ladder (L) represents 150 kDa (upper) and 100 kDa (lower intense band). CD31 expected molecular weight/size is 130 kDa. GAPDH ladder represents 37 kDa. GAPDH expected molecular weight/size is 37 kDa. (h) CD31 RNA levels assessed by qRT-PCR is presented for HUVECs under identical conditions. Data in e–h analysed by two-way ANOVA with complete results in Supplementary Table 3. (i) Cell migration for unstimulated HUVECs versus after treatment with TGF-ß only or TGF-ß+200 µM H2O2 for 5 days. Analysis by one-way ANOVA; overall P=0.015. (j) Cell invasion for unstimulated HUVECs versus after treatment with TGF-ß only or TGF-ß+200 µM H2O2 for 5 days. Analysis by one-way ANOVA; overall P<0.0001. n=6–9 for qRT-PCR and 3–6 for western blots. Migration and invasion determined from three independent experiments with each experiment having n=3. NS, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.