Bs 52613r

After 7 and 14 days of culture, the TSPCs in Groups N and G with (N+ and G+) or without (N- and G-) phosphoinositide 3-kinase (PI3K)/AKT pathway inhibitor (LY 294002; Bioss) were lysed in lysis buffer containing a mixture of proteinase inhibitors (Thermo Fisher Scientific). Equal amounts of protein samples (30 μg/lane) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes. Then, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-TNC (1: 1000, ab108930; Abcam, Cambridge, UK), anti-TNMD (1: 1000, ab203676; Abcam), anti-AKT (1: 1000, 9272S; Cell Signaling Technology (CST), Danvers, MA, USA), anti-phospho-AKT (pAKT473, 1: 1000, 9271S; CST), anti-phospho-AKT (Thr308) (pAKT308, 1: 1000, 9275S; CST), anti-PIK3CA (PI3K, 1: 1000, bs-2067R; Bioss), anti-phospho-PI3KCA (p-PI3K, 1 : 1000, bs-5570R; Bioss), anti-integrin α2 (1 : 1000, bs-52613R; Bioss), anti-integrin β1 (1 : 1000, bs-0486R; Bioss), and anti-GAPDH (1 : 1000, ab8245, Abcam). GAPDH was used as a loading control. Then, the membranes were incubated with secondary antibody (10285-1-AP, 1 : 2000; Proteintech, Wuhan, China) for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (GE Healthcare, Wuxi, China), and the results were analyzed with ImageJ software.

After 7 days of culture, the TSPCs in the different groups were fixed in 4% paraformaldehyde and washed three times with PBS. The fixed cells were incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody for 90 min at room temperature. Nuclei were stained with DAPI for 10 min. The following primary and secondary antibodies were used: anti-integrin α2 (1 : 1000, bs-52613R; Bioss), anti-integrin β1 (1 : 1000, bs-0486R; Bioss), and Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (1 : 100, SA00009-2; Proteintech, Wuhan, China). Samples were observed with a laser scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany). The mean fluorescence intensity was analyzed by ZEN software (blue edition 2.3; Carl Zeiss). Three different images for each group were used for statistical analyses.