Fig 2: The mechanism for the AT1R inhibition of anti-senescence through mitochondrial quality control. ox-LDL induced mitochondrial fission through the association of LOX-1 and AT1R and the CRAF/MEK/ERK pathway, resulting in mitochondrial dysfunction and senescence. AT1R inhibition attenuated senescence by suppression of this cascade and induced Rab9-dependent alternative autophagy through the CRAF/MEK axis and aspects of the mitochondrial quality control process in human VSMCs.

Fig 3: Effects of B19V–VP1u and its truncated fragments on the phosphorylation of ERK and P38. (A) ERK, p-ERK, P38 and p-P38 expression in left ventricle of the mice receiving different treatments (PBS, VP1u, VP1u-A, VP1u-B, VP1u-C or VP1u-D). C indicates control mice treated with PBS. (B) Relative expression levels of p-ERK and p-P38 are based on total protein expression. Similar results were obtained in three-repeated experiments. *P<0.05 vs. Control (mice treated with PBS). n=4 for each group. B19V, B19 virus; VP1u, VP1-unique region; p-, phosphorylated.

Fig 4: Inhibition of Cavin-1 expression by shRNA promotes AngII-induced VSMC proliferation via further activation of extracellular signal-regulated kinase (ERK). A, Representative Western blot and statistical analysis showing the efficiency of Cavin-1 shRNA on the inhibition of Cavin-1 expression and even on CAV-1 expression in VSMC culture (n=3–4/per group, Cavin-1 expression: *P<0.05 vs Scramble shRNA alone; CAV-1 expression: # P<0.05 vs Scramble shRNA alone). B, The histogram of VSMC number showing the effect of Cavin-1 shRNA on VSMCs proliferation in normal growth culture medium (10% FBS DMEM). C, Knockdown of Cavin-1 protein expression by Cavin-1 shRNA had no effect on VSMC phenotypic marker a-SM actin and calponin (n=6–8/per group). D, [3H]-thymidine incorporation measured as count per minute (cpm) showing the effect of Cavin-1 knockdown on VSMC proliferation in stimulation with AngII (100 nmol/L) for 24 hours with or without pretreatment with proteasome inhibitor MG132 (10 µmol/L) for 1 hour. n=9, ***P<0.001 vs Scramble shRNA alone, $ P<0.05, $$ P<0.01 vs Scramble shRNA treated with AngII and ## P<0.01 vs Cavin-1 shRNA treat with AngII. E, Western blot showing the effect of Cavin-1 knockdown on AngII-induced ERK phosphorylation (p-ERK) with or without pretreatment with MG132. p-ERK/ERK RelOD indicated the percentage of the relative optical density value of p-ERK/ERK to Scramble shRNA group. n=3, **P<0.01, ***P<0.001 vs Scramble shRNA treated with AngII, # P<0.05 vs Cavin-1 shRNA treated with AngII. Ang II indicates angiotensin II; FBS, fetal bovine serum; RelOD, relative optical density; shRNA, short hairpin RNA; VSMCs, vascular smooth muscle cells.

Fig 5: Western blot validation of proteomics data in OMM2.5 cells. (A,A’): There was no change in MITF expression levels after 4 h of 20 µM ACY-1215 treatment, while treatment for 24 h with 20 µM ACY-1215 led to a significant (**, p = 0.002) reduction in MITF expression levels. (B,B’): Relative expression levels of p-ERK to total ERK remained unchanged after 20 µM ACY-1215 treatment for 4 h. At 24 h post treatment with 20 µM ACY-1215, relative expression levels of p-ERK to total ERK were significantly (****, p < 0.0001) downregulated when compared to the 0.5% DMSO treatment. (C,C’): Expression of cleaved PARP was significantly (*, p = 0.049) upregulated after 24 h treatment with 20 µM ACY-1215 in comparison to 0.5% DMSO-treated OMM2.5 cells. Representative blots for each protein probed and densitometry analysis presented, raw blots are provided in Supplementary Figure S7. ß-actin, GAPDH or a-tubulin were used as loading controls. Student’s Unpaired T test statistical analysis was performed, and data presented as mean ± SEM.