P c myc s62

Antibodies against PIM1, p-Smad2 (S467), p-Smad3 (S423 and S425) and p-c-Myc (S62) were purchased from Abcam (Cambridge, MA, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, ZEB1, ZEB2, Snail1, Snail2 (Slug), Twist, MMP2, MMP9, Smad2, Smad3, c-Myc, PCNA, Ki67 and GAPDH were purchased from Proteintech Group (Chicago, USA). Wright-Giemsa was purchased from Solarbio (Beijing, China). The inhibitors 10058-F4 and SGI-1776 were purchased from MedChem Express (New Jersey, USA). TGF-β was purchased from PeproTech (New Jersey, USA). The Alexa Fluor 488- and 594-conjugated secondary antibodies were purchased from Invitrogen (CA, USA).

Cells were harvested, washed, and lysed. Equal quantities of protein extract were electrophoresed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary IgG antibody. The signals were visualized using the ECL detection system (Thermo Fisher, USA). Antibodies against PARP1 (#9532), caspase 3 (#9662) and 9 (#9502), p-ERK1/2 (T202/Y204, #4370), ERK1/2 (#4695), p-AKT (S473, #4060), AKT (#4691), p-mTOR (S2448, #5536), mTOR (#2983), p-STAT3 (Y705, #9145), STAT3 (#4904), p-p65 (S536, #3033), p65 (#8242), MAX (#4239), and β-actin (#4970) were purchased from Cell Signaling Technology (Beverly, MA, USA); c-Myc (#ab32072), p-c-Myc (T58, #ab28842), and p-c-Myc (S62, #ab51156) was from Abcam (Cambridge, UK).