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: Relative mRNA expression of the GTPases in young and aged pachytene spermatocytes.(A) Cdc42, (B) Rac1, (C) RhoB, (D) Rab9, and (E) Chn2. *p<0.05, **P<0.01 and ***p<0.001. n = 5.

Fig 4: Deregulation of autophagic and mitochondrial markers in M114T transfected cells. (A) Schematic representation of plasmid constructs to overexpress PFN1 fused to eGFP gene. (B) Mitochondrial respiration was measured in HEK293T expressing ePFN1WT (white), ePFN1C71G (dark grey), ePFN1M114T (black), ePFN1E117G (light grey) and ePFN1G118V (grey) plasmid constructs using the MTT assay. To facilitate data interpretation, the mitochondrial respiration of PFN1WT was adjusted to 100%. (C) Immunoblots were performed using anti-GFP, RAB9, MFN2 and GAPDH antibodies on HEK293T protein extracts. (D) Densitometry analyses of eGFP, (E) RAB9 and (F) MFN2 protein levels in HEK293T cells transfected with the 5 plasmid constructs were normalized to those of GAPDH and are presented relative to those of ePFN1WT. Results are means ± SEM for at least 9 independent experiments. (G) NSC-34 were transfected with eGFP-PFN1 constructs expressing WT or various mutant forms of PFN1 (C71G, M114T, E117G or G118V) and triple immunofluorescence was performed to detect mitochondria (using CYCS in red) and RAB9A (in far-red) in cells with and without expression of eGFP tagged PFN1 constructs (in green). Nuclei are stained with DAPI (blue). Bar is 10 µm. (H) Fluorescent signal intensities of RAB9A and (I) CYCS were measured in cells expressing the eGFP-PFN1 constructs and are presented relative to those recorded in untransfected cells. Results are means ± SEM for 3 independent experiments with 20 to 70 transfected cells recorded for each plasmid condition. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001.

Fig 5: Rab9-dependent mitochondrial autophagy was not affected by ERK inhibition but was affected by MEK inhibition. (A) Representative images and quantitative analysis of TOMM20 (green) and LAMP2 (red) immunohistochemistry in SCH772984-treated vs. control vehicle-treated cells with ox-LDL. Scale bar = 10 μm. (n = 3 per group). (B) Representative images and quantitative analysis of TOMM20 (green) and LAMP2 (red) immunohistochemistry in PD184352-treated vs. control vehicle-treated cells with ox-LDL. Scale bar = 10 μm. *P < 0.05 when compared with ox-LDL with the control (n = 3 per group). (C) Representative images and quantitative analysis of LAMP2 (green) and Rab9 (red) immunohistochemistry in PD184352-treated vs. control cells with ox-LDL. Scale bar = 10 μm. *P < 0.05 when compared with ox-LDL with the control (n = 3 per group). All data are presented as the mean ± SEM. Statistical analysis were conducted by Wilcoxon rank sum test (A-C).