Anti α smooth muscle actin

The immunostaining was performed according to the following protocol: the VSMCs were gently washed, once, before fixation with 3.7% paraformaldehyde solution for 15 min. After quenching the fixation solution in three washing steps, the cells were permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) and incubated in 0.1% sodium-borohydride solution for 15 min. The cells were then incubated in 1% BSA (Sigma-Aldrich) containing blocking solution for 30 min, then immunolabelled using anti-α-smooth muscle actin (Sigma-Aldrich) and Alexa Fluor 488 conjugated secondary antibodies (Invitrogen). Cell nuclei were stained by DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride; Sigma-Aldrich). Photomicrographs were taken using a Leica DMI6000B inverted microscope.

Rabbit anti–murine COX-2 (catalog 160106) was from Cayman Chemical; rat anti–mouse F4/80 (catalog ab6640), rabbit anti–VEGF-A (catalog ab52917), anti–IL-1β (catalog ab9722), and anti-Ki67 (catalog ab16667) were from Abcam; rat anti–mouse CD68 (catalog MCA1957B) was from Bio-Rad; rabbit anti-LAMP2A (catalog L0668), mouse anti–β-actin (catalog A1978), and anti–α-smooth muscle actin were from Sigma-Aldrich; mouse anti–IL-6 (catalog sc-32296) and anti–TNF-α (catalog sc-133192) were from Santa Cruz Biotechnology; rabbit anti–p-AKT (Ser473) (catalog 4046, clone D9E), anti-AKT (catalog 4685), anti–perilipin-1 (catalog 9449, clone D1D8), and HMGB1 (catalog 6893S) were from CST; and rabbit anti-CD31/PECAM-1 was from Novus Biologicals (catalog NB100-2284). The HFD was from Bio-Serv (36% fat accounting for 60% of calories, F3282); water-soluble dexamethasone (catalog D2951) and palmitic acid (catalog 506345) were from MilliporeSigma. For the in vitro study, 5 mM palmitate conjugated to 2 mM FFA-free BSA in sterile water (pH 7.4) was used. ONO-4819 (Rivenprost, LS-H93, LSbio) was given at 75 μg/kg/day via Alzet model mini-pump.

They were performed as previously described.^{18 (link)} Immunofluorescence analyses of adherent cells were carried out with antimyosin heavy chains (Developmental Studies Hybridoma Bank) and anti-α smooth muscle actin (Sigma-Aldrich) antibodies. Living cells were sorted by flow cytometry according to labeling with anti-CD56-APC, anti-CD15-FITC, and CD140a-PE (PDGFRα) antibodies (BD Biosciences). We used a BD FACSARIA II sorter equipped with four lasers (Becton, Dickinson and Company, Franklin Lakes, NJ, USA, http://www.bdbiosciences.com/us/s/contactus) and the BD FACSDiva software. For CD15-FITC, fluorescence was excited with the 488 nm laser and measured with a 530/30 bandpass filter. For CD140a-PE, fluorescence was excited with the 561 nm laser and measured with a 586/15 bandpass filter. For CD56-APC, fluorescence was excited with the 633 nm laser and measured with a 670/14 bandpass filter. Fluorescence minus one (FMO) controls were performed to insure that the FITC fluorescence did not spread into the PE fluorescence (Supplementary Figure 1). The fluorescence spreads were always <1%. Gates created after compensation with the appropriate FMO controls were the same as those created with single-staining controls.

Primary antibodies against Fascin-1 [mouse monoclonal (D-10) sc-46675], RhoA [mouse monoclonal (26C4) sc-418], GAPDH [rabbit polyclonal (FL-335) sc-25778], Actin [goat polyclonal (C-11) sc-1615], GLUT1 [mouse monoclonal (A-4) sc-377228], GLUT4 [rabbit polyclonal (H-61) sc-7938] and SDF-1/CXCL12 [rabbit polyclonal (FL-93) sc-28876] were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-phospho-p44/42 ERK (Thr202/Tyr204) [mouse monoclonal, #9106S] and p44/42 ERK1/2 [rabbit polyclonal, #9102] from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-FAK [rabbit polyclonal, GTX132141] was from GeneTex, Inc. (Irvine, CA, USA); anti-MEK1[rabbit monoclonal, #04-376] was from EMD-Millipore (Burlington, MA, USA); anti-α-smooth muscle Actin (mouse monoclonal (1A4), #A2547) was from Sigma-Aldrich (Saint Louis, MO, USA). Rabbit (#A0545) and goat (#A4174) peroxidase-conjugated secondary antibodies, media and sera for cell cultures were purchased from Sigma-Aldrich; mouse peroxidase-conjugated secondary antibody (#sc-2005) was from Santa Cruz Biotechnology, Inc. Plasticware was obtained from Corning Inc. (Corning, NY, USA). 2-deoxy-[3H]-D-glucose was provided by Perkin Elmer (Waltham, MA, USA). Other reagents for cell culture and microscopy were obtained from Sigma-Aldrich, except where otherwise indicated.

At the end of each experiment, fibroblasts were harvested and whole-cell lysates subjected to western analysis as described [12 (link)]. The following antibodies were used: anti-phospho-Smad2 (Cell Signaling Technology, Boston, MA, USA), anti-type I collagen (Southern Biotech, Birmingham, AL, USA), A20 (Santa Cruz, Dallas, TX, USA), anti-α smooth muscle-actin (αSMA), beta actin and tubulin (all from Sigma, St. Louis, MO, USA). Proteins were visualized using ECL reagents (Pierce, Rockford, IL, USA) and levels were quantitated by determining band intensities normalized to loading controls in each lane using ImageJ software.