Bradford assay dye reagent

HUVECs and HAECs were washed twice with PBS, and lysis with 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma) contained 1X cell lysis buffer (9803, Cell signaling). Quantitative analysis of samples were determined with a Bradford assay dye reagent (500-0006, Bio-Rad). Fifteen μg of sample protein was mixed with 1X loading dye and boiled for 5 min, and electrophoresis on 10% SDS-polyacrylamediume gel. After transferred to the polyvinylidene fluoride membrane (ISEQ00010, Millipore), membranes were blocked in 5% skimmilk contained 1X TBST (WH400028806, 3M) for 1 hr. The membranes were incubated with anti-p-FGFR1 (1:1000, ab173305, abcam), anti-FGFR1 (1:1000, 9740, cell signaling) and anti-GAPDH (1:10000, G8795, Sigma) antibody at 4 °C for overnight. Next, the membranes were washed three times in TBST and incubated with a horseradish peroxidase-conjugated secondary anti-mouse HRP antibody (1:7000, SC-2005, Santa Cruz Biotechnology) and anti-rabbit HRP antibody (1:7000, SC-2030, Santa Cruz Biotechnology) in TBST at room temperature for 90 min. Chemiluminescence were visualized using Amersham ECL Prime Westerm Blotting Reagent (RPN2232SK, GE Healthcare Life Sciences) and exposed to X-ray film. Quantification of blotting intensity were performed using Quantity One (Bio-Rad) program.

The hESC-COs were washed twice with PBS and lysed with a 1× cell lysis buffer (9803, Cell Signaling Technology, Danvers, MA, USA) plus 1 mM of phenylmethylsulfonyl fluoride (P7626, Sigma–Aldrich). Then, the protein concentrations of the samples were determined using a Bradford assay dye reagent (500-0006, Bio-Rad Laboratories). The sample protein (10 μg) was boiled in 1× loading dye for 8 min, electrophoresed on a 10% sodium dodecyl sulfate–polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane (10600023, Thermo Fisher Scientific). The membranes were blocked with 5% bovine serum albumin (A0100-010, GenDEPOT) containing 1× TBST (a mixture of Tris-buffered saline and Tween 20; WH400028806, 3M) at RT for 1 h, incubated with the corresponding antibodies (Table S3), washed thrice with TBST, and further incubated with a horseradish peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology) at RT for 1 h. Chemiluminescence was visualized using ECL Plus reagents (32132, Thermo Fisher Scientific) and a ChemiDoc™ Touch Imaging System (1708370, Bio-Rad Laboratories). Protein levels were normalized to GAPDH (G8795, Sigma-Aldrich).

Tissue samples were lysed in lysis buffer containing 1 % Triton X-100, 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄ and 75 U of aprotinin and allowed to stand for 20 min at 4 °C. The tissue suspension was mechanically disrupted by Dounce homogenization (ten strokes). The lysate was centrifuged for 5 min at 1300×g to remove nuclei and large cellular debris. After evaluation of the protein concentration by Bradford dye reagent assay (Bio-Rad, 500-0006), the lysate was subjected to 8 % (for AMBRA1) or 15 % (for other antigens) sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, 162-0177). Membranes were blocked with 5 % defatted dried milk in TBS, containing 0.05 % Tween-20 and probed with rabbit polyclonal anti-LC3 antibody (MBL Int Corporation, PD014), with rabbit polyclonal anti-AMBRA1 (NOVUS, NBP1-07124), with rabbit polyclonal anti-p62 SQSTM1/sequestosome antibody (Sigma, P0067) or with anti-alpha-tubulin Mab (Sigma, T6199) Bound antibodies were visualized with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma, A1949) or anti-mouse IgG (Sigma, A9044) and immunoreactivity assessed by chemiluminescence reaction, using the ECL Western detection system (Amersham, RPN2106).

ASCs untreated or treated with 20 ng/ml KGF for 24 h were lysed in lysis buffer containing 1% Triton X-100, 10 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄, and 75 U of aprotinin and allowed to stand for 20 min at 4°C. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes). Then, lysate was centrifuged for 5 min at 1300 ×g to remove nuclei and large cellular debris. After evaluation of the protein concentration by Bradford Dye Reagent assay (Bio-Rad, 500-0006), the lysate was subjected to lipid analysis. Neutral lipid extracts were separated by high-performance thin layer chromatography (HPTLC) using a solvent system of hexane/diethyl ether/acetic acid (70 : 30 : 1, v/v/v) and were detected by staining with 2% copper acetate solution in 8% phosphoric acid and subsequent heating at 120°C for 15 min. After about 3 min, free cholesterol, cholesterol esters, and triglycerides yielded red spots on a white background, which were converted into pink-brown spots after 10 min. Quantitative analysis was carried out using NIH Image1.62 as software (Mac OS X, Apple Computer International).

hDPSCs were lysed in lysis buffer containing 0.1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄ and 75 U of aprotinin and allowed to stand for 20 min at 4 °C. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes). The lysate was centrifuged for 5 min at 1300× g to eliminate nuclei and large cellular debris. After protein concentration analysis by Bradford Dye Reagent assay (Bio-Rad, Milano, Italia), the lysate was tested with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were electrophoretically transferred to PVDF membranes (Bio-Rad, Milan, Italia) which were blocked with 5% nonfat dried milk (Bio-Rad, Milan, Italia), or Bovine Serum Albumine (BSA) (Sigma-Aldrich, Milan, Italy), in TBS (Bio-Rad, Milan, Italia), containing 0.05% Tween 20 (Bio-Rad, Milan, Italia), and probed with rabbit anti-p-ERK1/2 pAb, rabbit anti-total ERK1/2 pAb, rabbit anti-p-Akt pAb, rabbit anti-total Akt pAb. Antibodies were visualized with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology Danvers, MA, USA) and immunoreactivity assessed by chemiluminescence reaction, using the ECL detection system (ThermoFisher Scientific, Rockford, IL, USA). Densitometric scanning analysis was accomplished with NIH Image 1.62 software by Mac OS X (Apple Computer International).