Fig 1: DAPA targets SGLT2 to attenuate the calcification of VSMCs. (a) Murine aortic SGLT2 levels were analyzed by Western immunoblotting (n = 6). (b) SGLT2 levels in murine aorta sections were analyzed with immunofluorescence staining. Scale bars: 100 μm. (c) SGLT2 levels in HASMCs were analyzed via Western immunoblotting (n = 6). (d) SGLT2 levels in HASMCs were analyzed with immunofluorescence staining. Scale bars: 50 μm. (e) Relative SGLT2 protein levels were analyzed in HASMCs following SGLT2 plasmid transfection (n = 6). (f) Alizarin Red S staining was used to analyze calcium deposition in HASMCs overexpressing SGLT2 (n = 6). (g) RUNX2, BMP2, α-SMA, and SM22α levels in HASMCs overexpressing SGLT2 were analyzed via Western immunoblotting (n = 6). Data were analyzed with two-tailed t-tests (a, c, and e) and one-way ANOVAs, and are presented as means ± SEM
Fig 2: DAPA promotes caloric restriction-mediated SIRT1 expression to protect against vascular calcification. (a, b) The NAD+/NADH ratio was measured in (a) murine aortas (n = 6) and (b) HASMCs (n = 6). (c) SIRTs family mRNA levels were analyzed in DAPA-treated HASMCs (n = 3). (d, e) Western immunoblotting was used to detect SIRT1 protein levels in (d) murine aortas (n = 6) and (e) following the overexpression of SGLT2 (n = 6). (f) Alizarin Red S staining analyses of HASMCs following SIRT1 knockdown (n = 6). (g) Western immunoblotting was used to analyze the expression of SIRT1, RUNX2, BMP2, α-SMA, and SM22α following SIRT1 knockdown (n = 6). (h) Alizarin Red S staining of EX527-treated HASMCs (n = 6). (i) Western immunoblotting was used to analyze the expression of SIRT1, RUNX2, BMP2, α-SMA, and SM22α in EX527-treated HASMCs (n = 6). (j) Von Kossa staining of EX527-treated rat aortic rings (n = 6). Scale bars: 200 μm. Data were analyzed with one-way ANOVAs, and all are presented as means ± SEM
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