Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Canagliflozin stimulates AMPK in human endothelial cells. ( a ) HUVECs were stimulated with A769662 (100 μmol/l, 30 min) or the indicated concentrations of canagliflozin, dapagliflozin or empagliflozin for 15 min and lysates prepared. ( b ) HAECs were stimulated with the indicated concentrations of canagliflozin, empagliflozin or A769662 for 30 min and lysates prepared. AMPK was immunoprecipitated from lysates and assayed for AMPK activity. Data shown represents AMPK activity (% vehicle) from three independent experiments. **p < 0.01; ***p < 0.001 vs vehicle (two-tail t-test).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: Immunoprecipitation, Activity Assay

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Canagliflozin inhibits 2-deoxyglucose uptake in HUVECs without influencing cell proliferation. ( a ) HUVECs were preincubated in the presence or absence of canagliflozin (10 μmol/l) for the times indicated or dapagliflozin (10 μmol/l, 30 min) and 2-deoxyglucose uptake assessed. Data shown represents the mean 2-deoxyglucose uptake relative to vehicle from three independent experiments. **p < 0.01, ***p < 0.001 relative to vehicle (one-way ANOVA). ( b ) Cell lysates from HUVECs (2 independent lysates), HAECs (4 independent lysates) and HEK-293 cells and mouse kidney membranes were resolved by SDS-PAGE and immunoblotted with anti-SGLT2 antibodies. The immunoblot shown has been cropped, with the full-length immunoblot shown in Supplementary Figure . ( c , d ) HUVECs were incubated in the presence or absence of canagliflozin, dapagliflozin or empagliflozin and ( c ) proliferation assessed in 2.5% (v/v) or 5% (v/v) MV2 Supplement mix (serum) by BrdU incorporation or ( d ) viability assessed by MTS assay. Data shown represents the mean proliferation or viability relative to vehicle (in 5% MV2 supplement mix) from three independent experiments in each cae. ## p < 0.01 relative to vehicle (two-tailed t-test).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: SDS Page, Western Blot, Incubation, BrdU Incorporation Assay, MTS Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Expression of SGLT2 and SGLT1 mRNA in human endothelial cells. Expression of SGLT2, SGLT1 relative to TBP was assessed by qPCR in cDNA prepared from three independent cultures of HUVECs, HAECs and HEK-293 cells, using cDNA from human kidney cortex as a positive control. Data shown are the mean ± SEM Ct values and 2 −ΔΔCt in each case, from three independent samples. ND = Not detected.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: Expressing, Positive Control

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Canagliflozin inhibits IL-1β-stimulated MCP-1 and IL-6 secretion in human endothelial cells. ( a , c ) HUVECs were stimulated with IL-1β (10 ng/ml) for ( a ) 6 h or ( c ) 24 h following preincubation in the presence or absence of canagliflozin (10 μmol/l, 15 min), dapagliflozin (1 μmol/l, 15 min), empagliflozin (1 μmol/l, 15 min) or A769662 (100 μmol/l, 30 min) and conditioned medium collected. ( b ) HAECs were infected with 100 pfu/cell Ad.null or Ad.AMPK-DN for 24 h and preincubated in the presence or absence of canagliflozin (10 μmol/l, 15 min) or A769662 (100 μmol/l, 30 min) prior to stimulation with IL-1β (10 ng/ml) for 6 h and conditioned medium collected. ( a , b ) MCP-1, ( a ) IL-6 or ( c ) endothelin-1 levels were assayed in conditioned medium by ELISA. Data shown represents the % IL-1β-stimulated MCP-1, IL-6 or endothelin-1 secretion from ( a , c ) three ( b ) four (Ad.null) or five (Ad.AMPK-DN) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to IL-1β alone (ANOVA).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: Infection, Enzyme-linked Immunosorbent Assay