Itgβ1

EVTs were isolated from the gels using cold PBS-EDTA. For dissolving the Matrigel/Collagen layer, Collagenase treatment was performed after the initial wash. Each well was incubated with Collagenase Type I (1,000 U/ml, Calbiochem #234153) for 30 min at 37 °C. Total RNA was isolated by TRIzol (Thermo Fisher Scientific) according to manufacturer’s protocol. RT-PCR assays were performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific) and normalized to the L32 gene. Cell lysates were prepared according to the RIPA buffer protocol (CS9806), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed with antibodies indicated. Antibodies were obtained from the following companies: Zeb1(sc-25388), N-cadherin (BD610921), E-cadherin (BD610182), p-Src Y416 (CS2101), Src (CS2108), p-Cortactin Y421 anti-mouse (Invitrogen44854), p-Cortactin Y421 anti-human (NBP1-60806), Cortactin (MP05-180), p-FAK Y397 (Invitrogen44624G), p-FAK Y576/577 (CS3281), p-FAK Y861(Invitrogen44-626G), p-FAK Y925 (CS3284), FAK (InvitrogenAHO0502), p-Paxillin Y118 (Abcam4833), Paxillin (Abcam2264), Itgβ1 (CS4706), p-p130Cas Y410 (CS4011), p130Cas (MP06-500), CRKL (MP05-414).

Cell lysates were prepared according to the RIPA buffer protocol (CS9806). Antibodies were obtained from the following companies: Zeb1 (sc-25388), N-cadherin (BD610921), E-cadherin (BD610182), Vimentin (CS3932), p-Src Y416 (CS2101), Src (CS2108), p-FAK Y861 (Invitrogen 44-626G), FAK (Invitrogen AHO0502), p-Paxillin Y118 (Abcam4833), Paxillin (Abcam2264), Itgβ1 (CS4706), p-p130Cas Y410 (CS4011), p130Cas (MP06-500), p-CRKL Y207 (CS3181), CRKL (MP05-414).

Cells were stained with antibodies against CVH, to assess the changes in PGC-related gene expression. To assess the level of SSC-related gene expression, cells were stained with ITGβ1 (all from Abcam, Cambridge, UK). Samples were analyzed by BD FACS Aria flow cytometer (BD Biosciences, provided by the testing center of Yangzhou University).