Cavin-1 regulates caveolae-mediated LDL transcytosis: crosstalk in an AMPK/eNOS/ NF-κB/Sp1 loop

Figures

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  Figure 1. Cavin-1 siRNA suppressed LDL uptake and LDL transcytosis in HUVECs (A, B) Cells were transfected with scrambled siRNA or cavin-1 siRNA (20 nM) for 48 h, then incubated with L-NAME (50 μM) or Compound C (1 μM ) for 48 h. (C, D) Cells were transfected with scrambled siRNA (20 nM) or cavin-1 siRNA (20 nM) for 3 h, then co-transfected with scrambled siRNA (10 nM) or AMPKα1/2 siRNA (10 nM) for 45 h. (E-F) Cells were treated with indicated concentration of AICAR. (A, C, E) Flow cytometry images of FITC-LDL uptake in HUVECs incubated with FITC-LDL. (B, D, F) Summary bar graph showing the mean FITC-LDL fluorescent intensity in each group. (H, I, J) Quantitative summary of FITC-LDL transcytosis in HUVECs. * P <0.05 vs. scrambled siRNA or AICAR (0 mM); † P <0.05 vs. cavin-1 siRNA, n=4.

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  Figure 2. Time course for caveolin-1 and eNOS expression after cavin-1 siRNA transfection Cells were transfected with cavin-1 siRNA or scrambled siRNA (20 nM) for 24-60 h. (A) The representative western blots image shows the expressions of eNOS, caveolin-1 and cavin-1 at indicated time after siRNA transfection. (B, C) The protein levels of eNOS (B), caveolin-1 (C) and cavin-1 (C) were evaluated by immunoblotting. * P <0.05 or **P <0.01 vs. Scrambled siRNA, n=4. (D) Cavin-1 siRNA enhanced NO production in HUVECs. HUVECs were transfected with cavin-1 siRNA or scrambled siRNA for indicated time and then loaded with DAF-FM-DA dye. After washing with PBS, fluorescence was measured. * P <0.05 or **P <0.01 vs. Scrambled siRNA, n=4.

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  Figure 3. Cavin-1 knockdown decreased the expression of caveolin-1, eNOS and LDLR (A, B) Cells were transfected with scrambled siRNA or cavin-1 siRNA (20 nM) for 48 h, then incubated with L-NAME (50 μM) for 48 h. (C, D) Cells were transfected with scrambled siRNA (20 nM) or cavin-1 siRNA (20 nM) for 3 h, then cotransfected with scrambled siRNA(10 nM) or AMPKα1/2 siRNA (10 nM) for 45 h. (E, F) Cells were transfected with scrambled siRNA or caveolin-1 siRNA (20 nM) for 48 h, then incubated with L-NAME (50 μM) for 48 h. (G-H) Cells were treated with indicated concentration of AICAR. The protein level of cavolin-1, LDLR, AMPK and eNOS was evaluated by western blotting. (A, C, E, G) The representative images of western blotting. (B, C, D, F) The quantitative analysis of the expression of proteins. *P <0.01 vs. scrambled siRNA or AICAR (0 mM), **P <0.01 vs. AICAR (0 mM), n=4. † P <0.05 vs. Cavin-1 siRNA.

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  Figure 4. Cavin-1 knockdown inhibited the activity of NF-κB/Sp1, consequently reducing the mRNA expression of caveolin-1, eNOS and LDLR (A, B) Cells were transfected with scrambled siRNA or cavin-1 siRNA (20 nM) for 48 h, then incubated with L-NAME (50 μM) or Compound C (1 μM ) for 48 h. (C, D) Cells were transfected with scrambled siRNA (20 nM) or cavin-1 siRNA (20 nM) for 3 h, then cotransfected with scrambled siRNA (10 nM) or AMPKα1/2 siRNA (10 nM) for 45 h. (E, F) Cells were treated with indicated concentration of AICAR. (B, D, F) The mRNA level was evaluated by qPCR. (A, C, E) The activity of NF-κB or Sp1 was detected. * P < 0.05 vs. scrambled siRNA; † P < 0.05 vs. cavin-1 siRNA; n=4.

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  Figure 5. Schematic diagram summarized the molecular mechanism underlying the cavin-1-mediated regulation of downstream proteins