Figure 7.

SIRT6 gene silencing blocks the SGLT2i protective effects against hyperglycemia injury. (A) Cytotoxicity, assessed by LDH Assay Kit-WST, and (B, C) cytokine levels in endothelial cells measured after SIRT6 silencing and hGluc stress condition for 48 h, preceded or not by SGLT2i pre-treatment for 8 h. (D, E) Intracellular ROS content detected by flow cytometry analysis using DCF probe. EC after SIRT6-siRNA were subjected or not subjected to 8 h SGLT2i pre-treatment before starting 48 h of incubation with hGluc. (F) Representative images of confocal laser scanning analyses of mitochondrial ROS generation detected by MitoSOX probe and (G) mitochondrial superoxide levels detected by flow cytometry analysis. Results are expressed as median fluorescence intensity (MFI). Scale bars = 10 μm. The cytoskeleton is marked with Phalloidin 488 (green), while DAPI was used for the nuclei counterstain (blue). (H) Representative confocal images of NF-$\text{B}(\text{NF}-\text{κB})$ (red) and vimentin (green) and (I) fluorescence intensity analysis, performed using ImageJ software, expressed as arbitrary fluorescence units. (J, K) SIRT6 protein levels, assessed by Western blot analysis. Endothelial cells, after SGLT2 silencing, were subjected or not subjected to 8 h of pre-treatment with SGLT2i before starting 48 h of hGluc stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc, lane 7 = scramble siRNA, lane 8 = SGLT2-siRNA, lane 9 = SGLT2-siRNA+hGluc, lane 10 = SGLT2-siRNA+SGLT2i+hGluc. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). α-Tubulin was used as an internal control. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc.