Lysis buffer

Western blotting of five proteins was conducted as described in our previous study (24 (link)). Briefly, cells and xenografts were washed in phosphate-buffered saline (PBS) and incubated in lysis buffer (Sangon Biotech, Co., Ltd., Shanghai, China). Protein was separated by 12% SDS-PAGE and transferred to a PVDF membrane (Sangon Biotech). Non-specific binding sites were blocked by incubation with TBST containing 5% (w/v) non-fat dried milk. Next, the membrane was incubated with rabbit anti-human Akt, p-Akt, KLF-4, ERK or p-ERK (1:1,000 dilution) primary antibodies at 37°C for 1 h. Subsequently, membranes were incubated overnight with an HRP-conjugated anti-rabbit IgG secondary antibody (1:1,000 dilution) at 4°C. Signals were visualized by an ECL chemiluminescence detection kit and semi-quantitated using ImageJ software. Equal protein loading was assessed by the expression of GAPDH.

Cells were first lysed in lysis buffer (Sangon Biotech Co., Ltd., Shanghai, China) and then total protein was extracted. Followed by protein concentration was measured by the bicinchoninic acid method. Equal aliquots (20 µg protein per lane) of protein lysate were separated by 8–12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Subsequent to blocking with 5% skimmed milk at 37°C for 1 h, Western blots were probed with primary antibodies against nuclear factor (NF)-κB (1:1,000; cat no. ab32360; Abcam, Cambridge, MA, USA), Akt (1:1,000; cat no. ab126811; Abcam), cyclin B (1:1,000; cat no. ab18221; Abcam), P53 (1:1,000; cat no. ab21985; Abcam), growth arrest and DNA damage inducible β (GADD45B) (1:1,000; cat no. ab128920; Abcam) and β-actin (1:1,000; cat no. ab6276; Abcam) overnight at 4°C. Three consecutive washes were performed for 10 min in PBS-Tween, followed by incubation with the alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; cat no. ab6722; Abcam) diluted in 5% skimmed milk at room temperature for 1 h. The immunoblots were visualized with enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA) and autoradiography. The experiments were repeated three times, and the results were detected using the Image Lab software (version 4.1; Bio-Rad Laboratories, Inc.) on ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.).

To create the cell sheet, the released cells were seeded at 4×10⁴ cells/cm² into flasks cultured in 10 ml of MEM-α supplemented with 10% FBS (Gibco, Thermo Fisher Scientific Inc.) for a minimum of 1 week. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂, and the medium was replaced daily. After 1 week, the layered BMSCs were formed. The cells were then rinsed with phosphate-buffered saline (Gibco) twice, and then lifted as a cell sheet using a scraper (Fig. 1) (12 (link)). Cell proliferation was determined by DNA content assay using a fluorescent dye Hochest 33258 (Sangon Biotech Co., Ltd., Shanghai, China). The MSC sheets were harvested and stored at −20°C until the assay was performed. For the DNA content assay, the cells were thawed at room temperature and homogenized in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.6, 0.1% v/v Triton X-100; Sangon Biotech Co., Ltd.). The lysate was sonicated on ice for 30 sec. After centrifugation, the supernatant was collected for measurement. Cells (~4.8×10⁶) were contained within a single cell sheet, as determined by DNA quantification using an PerkinElmer LS 55 Fluorescence(PerkinElmer, Inc., Waltham, MA, USA) and the fluorescent dye, Hoechst 33258 (13 (link)).