Ripa cell lysis buffer

To quantitatively determine the total protein contents on growth and adipogenic ASCs secreted ECM before and after decellularization, samples prepared in 60 mm diameter plates were analyzed using BCA assay kit (Thermo Fisher Scientific). After rinsing three times with PBS, the samples were solubilized in RIPA cell lysis buffer (Sigma-Aldrich) with EDTA-free protease inhibitor cocktail (Thermo Fisher Scientific) and then collected in the 1.5 ml centrifuge tubes. Next, the samples were suspended and rocked gently for 30 min at 4°C and centrifuged at 12,000 g for 5 min. The supernatants were transferred into a new tube and subjected to BCA assay immediately. The total protein amount of each sample was normalized to the surface area of culture dishes, and thus, the concentration was denoted as mg/cm².

Cells were collected by centrifugation upon dissociation with 0.25% Trypsin‐EDTA solution. Whole‐cell lysates were prepared using RIPA cell lysis buffer (Sigma–Aldrich, R0278) containing 1:100 phosphatase inhibitor cocktail 2 (Sigma–Aldrich, P5726), phosphatase inhibitor cocktail 3 (Sigma–Aldrich, P0044), and protease inhibitor (Sigma–Aldrich, P8340). Samples were rotated for at least 30 min at 4 °C and spun at maximum speed for 10 min at 4 °C. The supernatants of the samples were collected, and protein concentration was determined by the Bradford assay (Bio‐Rad, 23246). Equal amounts per sample were combined with Laemmli buffer, denatured for 5 min at 95 °C, and subjected to SDS/PAGE separation, followed by immunoblotting. The antibodies used in this study are listed in Table S1 (Supporting Information). β‐Actin was used as an internal loading control. Protein quantification was performed with ImageJ software from n = 3 independent biological replicates. Uncropped scans of performed western blots were included in Supporting information.

Cells were first lysed by RIPA cell lysis buffer (R0278; Sigma‐Aldrich, St. Louis, MO, USA). Protein samples were isolated by SDS/PAGE and then transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% fat‐free milk in Tris‐buffered saline with Tween 20 and incubated with the primary antibodies for the detection of KIF3A, Ki67, MMP2, MMP9 and β‐actin at room temperature for 2 h. Then polyvinylidene fluoride membranes were washed with Tris‐buffered saline with Tween 20 four times and incubated with HRP‐conjugated secondary antibodies for 45 min. After washing, signals were detected using an enhanced chemiluminescence kit.

To extract whole-cell proteins, BMDCs were lysed in RIPA Cell Lysis Buffer (Sigma, V900854) containing protease and phosphatase cocktail inhibitor (Sigma, PPC1010). The lysates were sonicated and then centrifuged at 12,000 g for 15 min at 4 °C to collect whole-cell proteins in the supernatant. The lysosomal fraction was isolated using the Lysosome Isolation Kit (Sigma, LYSISO1) according to the manufacturer’s instructions.
Protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 23,225). Thirty micrograms of protein per sample was separated by 10% SDS-PAGE and transferred to 0.2-μm PVDF membranes (Millipore, ISEQ00010). Membranes were blocked with 5% BSA (Sigma, V900933) or 5% non-fat milk for 1 h and then incubated with primary antibodies overnight at 4 °C. On the second day of the experiment, the membranes were washed and then exposed to the respective secondary antibodies diluted 1:2000–5000 for 2 h at room temperature. Blots were covered with the ECL (Tanon, 180–506) and visualized using an Electrophoresis Gel Imaging Analysis System (Azure Biosystems, USA, C500).
For the whole-cell protein samples, band densities were normalized to β-actin as a loading control. For the lysosomal fraction, LAMP2 was used as a loading control. Details of the primary antibodies used for western blotting are shown in Table S1.