Fig 1: HSP22 inhibits hyperglycemia-induced mtROS production and mitochondrial impairment in endothelial cellsin vitro. A) Mitochondrial superoxide was measured by flow cytometry. HUVECs were incubated in Hanks' balanced salt solution containing 5 μmol/l MitoSOX for 15 min. n = 3. B) Representative images of MitoTracker Red staining showing the mitochondrial morphology in each group of cells (original magnification ×63). C) Mitochondrial density was quantified. n = 3. D) Representative images of JC-1 immunochemistry staining showing the mitochondrial membrane potential in each group of cells (original magnification ×63). E) Mitochondrial membrane potential was quantitated as a percentage in the gate. n = 3. *P < 0.05 vs. NC-NG; #P < 0.05 vs. NC-HG.
Fig 2: Effect of ATV on HSP22, eNOS and p38 MAPK in HUVECs. The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot assay, respectively. (C and D) The expression levels of p-eNOS and p-p38 MAPK were examined by western blot assay in HUVECs with an HFD and/or ATV treatment. **P<0.01 compared with the control group; #P<0.05 and ##P<0.01 compared with the ox-LDL group; ??P<0.01 compared with the ATV group. ox-LDL, oxidized low-density lipoprotein; ATV, atorvastatin; HUVECs, human umbilical vein endothelial cells; HSP22, heat shock protein 22; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated kinase; HFD, high-fat diet; p-, phosphorylated; t-, total.
Fig 3: HSP22 deficiency increases diabetes-induced endothelial oxidative stressin vivo. A)Frozen aortic sections were incubated with 5 µmol/l DHE for 20 min and analyzed using fluorescence microscopy (original magnification ×40). B) Quantification of the fluorescence intensity of ROS levels in the aorta. n = 4. C) Quantification of 8-OHdG levels in the aorta. n = 4. *P < 0.05 vs. WT-control; #P < 0.05 vs. WT-diabetes.
Fig 4: Hspb8 siRNA inhibited microRNA-126a-5p inhibitor-mediated protection against H2O2-induced injury in H9C2 cells(A) Western blot analysis of Hspb8 levels in total tissue extracts from infarct zone tissue treated with microRNA-126a-5p inhibitor (LNA-miR-126a-5p) or control inhibitor (LNA-Scr). *, P < 0.05, vs. Sham group, n=6; #, P < 0.05, vs. I/R+LNA-scr group, n=6. (B) Western blot analysis of Hspb8 levels in total cell extracts prepared from H9C2 cells treated with microRNA-126a-5p mimic or inhibitor. *, P < 0.05, vs. Ctrl group, n=8; #, P < 0.05, vs. H2O2 group, n=8. (C) Analysis of Hspb8 levels total cell extracts prepared from H9C2 cells transfected with control siRNA (siCtrl) or HSPB8-specific siRNAs. siRNA #1: #195633; siRNA #2: #195635. *, P < 0.05, vs. siCtrl group, n=4. (D, E) Analysis ofcell viability (D) and caspase 3 activity (E) in H9C2 cells exposed to H2O2 for 24h after co-transfection with microRNA-126a-5p inhibitor and Hspb8 siRNA #1 for 24h. *, P < 0.05, vs. H2O2 group, n=6; #, P < 0.05, vs. inhibitor+H2O2 group, n=6.
Fig 5: HSP22-knockdown inhibits ox-LDL-induced p-eNOS decrease and p-p38 increase in HUVECs. (A) The transfection efficiency of HSP22 shRNAs in HUVECs was measured by western blot assay. (B) The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by western blot assay. (C and D) The expression of p-eNOS and p-p38 in HUVECs with an HFD and/or ATV treatment in the absence or presence of HSP22 shRNA was measured by western blot assay. *P<0.05 and **P<0.01 compared with the corresponding NC group. ox-LDL, oxidized low-density lipoprotein; ATV, atorvastatin; HUVECs, human umbilical vein endothelial cells; HSP22, heat shock protein 22; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated kinase; HFD, high-fat diet; p-, phosphorylated; t-, total; NC, negative control; shRNA, short hairpin RNA.
Supplier Page from Abcam for Anti-Hsp22/HSPB8 antibody [EPR9714]