Cell culture
The mBMSCs were obtained as previously described [16]. Cultured mBM-MSCs between passages 3 and 5 were used for the following experiments.
Adenovirus infection
The adenovirus harboring Mst1 shRNA (Ad-sh-Mst1) and the control vector for Mst1 shRNA (Ad-NC-Mst1) were purchased from WZ Biosciences (China). Vector details have been described previously [16]. The shRNA sequence targeting mouse Mst1 was GCCCTCACGTA GTCAAGTATT.
Nude mouse tumorigenicity
A total of 10 female nude mice (4 weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (No. 110011211101205428) and maintained in a specific pathogen-free environment. Then, 5.0×106 of the mBMSCs, mBMSC/NC-Mst1, and mBMSC/sh-Mst1 were injected into the right flank near the hind leg of each nude mouse. The tumors were measured with a caliper every 4 days. At 60 days after cell inoculation, all mice were terminated with ether anesthesia, and the tissues were harvested. All animal procedures were approved by the Animal Care and Use Committee of Shandong First Medical University (No. 2021-177).
Differentiation capacity assay
For the osteogenic differentiation, mBMSCs, mBMSC/NC-Mst1, and mBMSC/sh-Mst1 were cultured for 21 days in a complete α-MEM medium containing 10−7 M dexamethasone, 10 mM β-glycerol phosphate, and 50 µM ascorbate-2-phosphate. The media were changed every 3 days. Calcium deposits were detected by alkaline phosphatase staining.
For the adipogenic differentiation, the cells were incubated for 21 days in a complete α-MEM medium containing 10−6 M dexamethasone, 0.5 µM isobutylmethylxanthine, and 10 ng/ml insulin. The adipogenic induction medium was changed every 3 days. The lipoid substances were stained with Oil Red O. Both alkaline and Oil Red O staining were visualized by light microscopy.
siRNA transfection
The siRNAs were obtained from GenePharma (China). The sequences of the sense and antisense strands of the siRNA were as follows: mouse siRNA-ITGα5, 5ʹ-GCAGGGAGAUGAAGAUCUACCʹ (sense) and 5ʹ-UAGAUCUUCAUCUCCCUGCAGʹ (antisense); mouse siRNA-ITGβ1, 5ʹ-GGAGAACCACAGAAGUUUACA-3ʹ(sense) and 5ʹ-UAAACUUCUGUGGUUCUCCUG-3ʹ (antisense); and siRNA-negative control (NC), 5ʹ-UUCUCCGAACGUGUCACGUTTʹ (sense) and 5ʹ-ACGUGACACGUUCGGAGAATT-3ʹ (antisense). Then, 24 h after infecting Ad-sh-Mst1, the mBMSCs at 75% confluence were transfected with ITGα5 or ITGβ1 or NC siRNA (50 nM) by using the Lipofectamine RNAi MAX (Invitrogen) according to the manufacturer’s instructions. The level of ITGα5 or ITGβ1 expression was significantly blocked by the transfected siRNA.
Cell treatment
Dishes coated with poly-hydroxyethyl methacrylate (Poly-HEMA; Sigma, USA) were used to prevent the adhesion of cells to the tissue culture plates. Briefly, poly-HEMA stock were dissolved in 95% ethanol at a concentration of 12 mg/ml, and 1 ml of the 12 mg/ml poly-HEMA was added to each well of a 6-well plate, which was dried overnight in a clean bench. Cells were transfected as previously described. Cells (5 × 105) were plated in each well coated with 12.5 mg/ml Poly-HEMA for a certain time.
Cell viability assay
The CCK-8 assay (Beyotime Institute of Biotechnology, China) was then employed to assess the cell viability. After being cultured in poly-HEMA-coated dishes, the cells were collected and seeded (3×103/well) in 96-well plates. Then, 10 µl/well CCK‑8 at 10% dilution was added to each well, and the plate was incubated for 1 h in an incubator. Spectrophotometric readings were normalized using cell-free medium as a blank. Absorbance was measured at 450 nm by using a Multiskan MK3 microplate reader, and the experiments were repeated thrice. Cell viability was calculated as the ratio of control.
Cell adhesion
After being cultured in poly-HEMA-coated dishes, the collected cells were resuspended in the complete α-MEM medium and then plated in triplicates (5× 104 cells/well) onto the well coated with fibronectin (10 g/ml), which was previously blocked for 1 h with 1% BSA. After 6 h, the cells were washed by PBS and stained with crystal violet. Before 10% acetic acid was used, the unbound dye was removed by PBS. The absorbance was read at 630 nm by using a Multiskan MK3 microplate reader. The experiment was repeated thrice. Cell viability was calculated as the ratio of control.
Anoikis assay
Anoikis assay was performed using the Anoikis Assay Kit (ab211153, Abcam, USA) according to the manufacturer’s instructions. After transfection, the cells were added to each well of the 96-well Anchorage Resistant Plate for 36 h and then treated with s AM/EthD-1. Quantitative measurement was performed using a microplate reader at Ex/Em = 485/515 nm (Calcein AM) and Ex/Em = 525/590 nm (EthD-1).
Flow cytometry
After 36 h of incubation in poly-HEMA-coated dishes, the cells were collected after centrifugation at 300 ×g for 5 min and incubation in the antibody (ITGα4 [1/500 dilution, 553157, BD], ITGα5 [1/500 dilution, 557447, BD], ITGαv [1/300 dilution, 740946, BD], ITGβ1 [1/500 dilution, 561796, BD], ITGβ3 [1/100 dilution, 740677, BD]) for 1 h, which was performed according to the operation manual.
Anoikis was also analyzed using in situ Direct DNA Fragmentation (TUNEL) Assay Kit (ab66108, Abcam). After being cultured in poly-HEMA-coated plate, the cells were collected and added to 70% ice ethanol for 30 min. Then, ethanol was removed, and the cells were resuspended by a wash buffer. Then, the cells were stained with a staining solution for 60 min. Prior to the addition of the PI/ RNase A solution, the cells were treated using a rinse buffer twice. Quantification was analyzed by the BD FACSDiva software (Ex/Em = 488/520 nm for FITC, and 488/623 nm for PI).
Quantitative PCR (qPCR)
The qPCR was performed as previously reported [16]. The total mRNA from the mBMSCs underwent differentiation via 21-day exposure to osteogenic or adipogenic condition, or the cells transfected with siRNA were isolated using Trizol Reagent (Thermo Fisher Scientific, Inc.). The RNA was subsequently reverse transcribed to cDNA, and amplified using SYBR® Premix Ex TaqTM Ⅱ kit (Takara, Dalian, China) on ABI 7500 real-time PCR system (Applied Biosystems). Each experiment was repeated thrice. Data were normalized using the housekeeping gene GAPDH by the 2−ΔΔCT method. The primer sequences are shown in the Supporting Information Table 1.
Western blot analysis
To determine the protein expression, Western blot analysis was performed. After being cultured in poly-HEMA-coated plate, the whole-cell protein extracts were prepared in RIPA lysis buffer, subjected to SDS-PAGE, and transferred onto PVDF membranes. The membranes were then blocked for 1 h with 5% skimmed milk or BSA in TBST and incubated overnight at 4 ℃ with the following primary antibodies (diluted by Western Primary Antibody Buffer, Beyotime): Mst1 (1:1000, ab51134, Abcam), ITGα5 (1:1000, ab150361, Abcam), ITGβ1(1:1000, ab179471, Abcam), Phospho-FAK (Tyr397) (1:500, 3283S, CST), FAK (1:1,000, 3285S, CST), activate caspase 3 (1:1000, ab214430, Abcam), and caspase 3 (1:1000, ab18297, Abcam). GAPDH (1:1000, 5174S, CST) served as the loading control. The anti-rabbit IgG and HRP-linked antibody (1:1000, 7074S, CST) were used. The relative protein expression levels were compared with those of GAPDH by using ImageJ software.
Statistical analysis
All results were expressed as means ± SD. The data were analyzed by one-way ANOVA. P value less than 0.05 was considered significant.