Reagents and antibodies
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco Invitrogen Corporation (Gibco, Carlsbad, CA, USA). Polyvinylidene difluoride (PVDF) membranes were purchased from Immobilon®-PSQ (Millipore, CA, USA). An enhanced chemiluminescence substrate for the detection of Horseradish peroxidase (HRP) and a LightShift® chemiluminescent electrophoretic mobility shift assay (EMSA) kit were obtained from Thermo Fisher Scientific, Inc. (Rockford, USA). Biotin-labeled NF-κB consensus oligonucleotide, pNF-κB-luciferase plasmid and pRL-TK plasmid were acquired from Beyotime (Haimen, China). Antibodies against p65, Caveolin 1, FN, ICAM-1, IL-6 and MCP-1 (diluted 1: 500, Proteintech Group, Chicago, IL, USA), phosphorylation of p65, TGF-β1 (diluted 1: 1000) and β-actin (diluted 1: 5000, Cell Signaling Technology, Danvers, USA), Lamin B, ANF and BNP (diluted 1: 1000, Abcam, Cambridge, UK), TNF-α (diluted 1: 1000, Abclone, Baltimore Avenue, USA), rabbit IgG (Beyotime, Haimen, China) were purchased from commercial sources. HRP-conjugated secondary antibodies were acquired from Beyotime (Haimen, China).
Rat H9C2 cardiomyocytes were purchased from GuangZhou Jennio Biotech Co., Ltd (Jennio, Guangzhou, China). They were maintained in low glucose’ DMEM in the presence of 10% fetal bovine serum (CellMax, Beijing, China) at 37 °C. Cells culture at 80% confluence were rendered quiescent by incubation for 12 h in serum-free medium before treating with HG’ DMEM.
Plasmids, si-RNA, transient transfection
PcDNA3-Caveolin 1 was provided by Yusheng Cong (Institute of Aging Research, School of Medicine, Hangzhou Normal University); pcDNA3 vector was preserved in our laboratory. The si-RNA targeting Caveolin 1 was purchased from GenePharma (GenePharma, shanghai, China). The special sequences of Caveolin 1-siRNA were displayed as: 5’-GACGUGGUCAAGAUUGACUTT-3’, 3’-AGUCAAUCUUGACCACGUCTT-5’. We plated H9C2 cardiomyocytes in 60 mm plates 24 h prior to transfection. Dilute 5 μL Lipofectamine ® RNAiMAX Reagent in 125 μL Opti-MEM ® Medium, and dilute 5 μL siRNA in another 125 μL Opti-MEM ® Medium. Then add the diluted Caveolin 1 siRNA to diluted Lipofectamine ® RNAiMax Reagent (1: 1 ratio) and incubate 5 min. At last, the siRNA-reagent complex was added to H9C2 cells. After incubation 36 h, the transfection medium was abandoned and 2 mL low glucose’ DMEM without serum was added to recover cell for 12 h. After specified treatment, the cells were collected for assays.
Western blot assays
Western blotting was carried out as previously described  to detect the protein levels of Caveolin 1, FN, ICAM-1, IL-6, MCP-1, TGF-β1, TNF-α, ANF, BNP phosphorylation and total NF-κB p65. The signals were visualized with a GE ImageQuant 350 instrument, and were quantified by densitometry using a Gel Doc XR System (Bio-Rad, USA); the signals were analyzed using Quantity One Protein Analysis Software (Bio-Rad).
Confocal laser scanning fluorescence microscopy
Immunofluorescence assay was carried out as previously described . The images were collected using a DMi8 laser confocal fluorescence microscope (Nikon, Tokyo, Japan).
Nuclear protein extraction
The nuclear proteins were extracted with a Nuclear Extract Kit (BestBio, Shanghai, China). Cells were collected after centrifuging at 500 g for 3 minutes at 4 °C. PBS was used to wash the cells two times and the 200 μL cold Buffer A was added with 2μL protease inhibitor cocktail. Subsequently, the cells were vortexes with maximal velocity for 15 s and placed in the ice for 10 min. After being vortexes with maximal velocity for 5 s, the cells were centrifuged at 16,000g for 5 min at 4 °C and the supernatants (cytoplasm protein) were collected, besides, the sediments were added with 200 μL cold Buffer B with 2μL protease inhibitor cocktail. Afterwards, the sediments were vortexed with maximal velocity for 15 s and placed in the ice for 40 min. The supernatants (nuclear protein) were collected after centrifuged at 16,000g for 10 min at 4 °C.
Electrophoretic mobility shift assay (EMSA)
The EMSA assay was carried out as previously described . Activation of NF-κB signaling was determined based on EMSA using nuclear extracts, the sequence of the biotin-labeled oligonucleotide probes of nf-κb was as follows: 5’-AGTTGAGGGGACTTTCCCAGGC-3’, 3’-TCAACTCCCCTGAAAGGGTCCG -5’ containing the acknowledged NF-κB binding site. The procedures were performed following the instructions of the manufacturer (Light Shift Chemiluminescent EMSA Kit, Pierce, USA). The images were captured and quantified using Image Quant 350 (GE Healthcare, USA).
Dual luciferase reporter assay
The dual luciferase reporter assays were carried out as previously described . H9C2 cardiomyocytes were seeded in 96-well culture plates and cotransfected with 0.1 μg of pNF-κB -luciferase (Beyotime, Haimen, China) and 0.02 μg of pRL-TK (Beyotime, Haimen, China) in the presence or absence of 0.05 μg of Caveolin 1 plasmid or Caveolin 1 si-RNA. After various treatments, cells were lysed and the luciferase activity was determined using the Dual-Luciferase reporter assay system kit (Solarbio, Beijing, China) to analyze the effect of Caveolin 1 on the activation of NF-κB pathway. Luciferase activity was normalized to the renilla luciferase activity.
Values were expressed as mean ± SEM. All data were assessed by the Graphpad Prism 5.0 software. Unpaired Student’s t test was used for comparison between two groups. For multiple comparisons, data were analyzed by one-way ANOVA with Bonferroni post hoc test multiple comparisons. Independent experiments were performed at least thrice with similar results. P < 0.05 was considered statistically significant.