Tcf8 zeb1

DMEM, fetal bovine serum, penicillin-streptomycin solution, and 0.25% trypsin were obtained from Corning; DMSO (sigma); Cell Counting Kit-8 (CCK-8) from Dojindo; Annexin V-FITC apoptosis detection kit (BD Pharmingen™). β-actin was purchased from Abgent. Cyclin D1, Cyclin D3, CDK4, p18, caspase-3, caspase-7, PARP, E-cadherin, N-cadherin, and TCF8/ZEB1 antibodies were purchased from Cell Signaling Technology. Lamin B1, NOV, and the HRP-labeled anti-mouse and anti-rabbit antibodies were from Santa Cruz Biotechnology.

Following treatment with drugs, cells were lysed and whole cell fractions were isolated as previously described [17 (link)]. Proteins (10–30 μg) were separated by 10%–12% (w/v) polyacrylamide gel electrophoresis and electroblotted to PVDF membranes. After blocking with non-fat milk for 1 h, membranes were incubated overnight with the following primary antibodies (1:1000 dilution) from Cell Signaling Technology (Danvers, MA, USA): E-Cadherin (Cat #3195), N-Cadherin (Cat #13116), TCF8/ZEB1 (Cat #3396), β-Catenin (Cat #8480), Vimentin (Cat #5741), phospho-Akt (Ser473) (Cat #4060), Akt (Cat #9272), phospho-IGF-I Receptor β (Tyr1135/1136) (Cat #3024), IGF-I Receptor β (Cat #9750), phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cat 4228), PI3 Kinase p85 (Cat 4257), phospho-mTOR (Ser2448) (Cat #5536), mTOR (Cat #2983). β-Actin (Cat #8457) was used at the same time as a loading control. After incubation with the secondary antibody (HRP-conjugated; 1:5000 dilution) for 1 h, at room temperature, the conjugates were developed and visualized using a Molecular Imager FX^TM System (BioRad; Hercules, CA, USA).

Paclitaxel (PTX) was purchased from Bristol-Myers Squibb, dissolved in Ethanol to make 7 μM stock concentration. JNK-IN-8 (Calbiochem, UK), with a working concentration of 10 µM was prepared in DMSO. MDR1/ABCB1, E-cadherin, N-cadherin, Vimentin, Snail, TCF8/ZEB1, β-catenin, Axin-1, Claudin, PI3K p110γ, p-PDK-1, Akt, p-Akt, p-c-Raf, Cyclin D1, p-GSK3β, p-p38 MAPK, p38 MAPK, p-p44/42, c-jun, p- SAPK/JNK, SAPK/JNK, p-AMPK, Wnt3a, Wnt5a/b, p-LRP6, LRP6, Dvl2, Dvl3, β-actin, GAPDH primary, and secondary antibodies were used at 1:1000 dilutions and were from Cell Signaling Technology (CST, Beverly, MA, USA).