P21cip1 waf1

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), the Propidium Iodide (PI) Cell Cycle Assay Kit and the Annexin V-FITC/PI Apoptosis Detection Kit were purchased from Beyotime (Jiangsu, China). The All-in-One™ First-Strand cDNA Synthesis Kit, the All-in-One™ miRNA qRT-PCR Detection Kit and miRNA primers were purchased from Genecopoeia (Rockville, MD, United States). DMEM and fetal bovine serum were obtained from Thermo Fisher Scientific (Waltham, MA, United States). All primary antibodies including p21Cip1/Waf1, E-cadherin, β-catenin, vimentin and snail, mTOR, p-mTOR (Ser2448), p-4E-BP1 (Thr37/46), LC3 and β-actin were purchased from Cell Signaling Technologies (Danvers, MA, United States). All other common chemicals and buffers were from Boster (Wuhan, China).

For western blot analysis, cell extracts (MDA-MB231, MB231_SC-4 d, MB231_SC-7 d) were prepared. Each protein's concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). The proteins (30 μg per sample) were separated on 4–12% gradient gels (Thermo Fisher Scientific) and transferred to nitrocellulose membrane (Thermo Fisher Scientific) by iBlot 2 Dry Blotting System (Thermo Fisher Scientific). Blots were blocked with blocking buffer (Bio-Rad Laboratories, Hercules, CA) for 30 min and incubated at 4 °C overnight with one of the following primary antibodies: p21^Cip1/Waf1 (1 : 1000 dilution, Cell Signaling Technology, #2947), phospho-Rb (Ser807/811, 1 : 1000 dilution, Cell Signaling Technology, #9308) Lamin B1 (1 : 1000 dilution, Cell Signaling Technology, #13435) and beta-actin (1 : 1000 dilution, Cell Signaling Technology, #4967). The next day, blots were washed 3 times (5 min each) with PBS (0.1% Tween-20). The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit IgG secondary antibody (1 : 2000 dilution, Cell Signaling Technology) for 1 h at RT. The immunoreactive proteins on the membranes were visualized using Supersignal™ West Femto substrate (Thermo Fisher Scientific). Immunoblot signals were developed with a C400 image machine (Azure Biosystems, Dubin, CA) and quantified by ImageJ.

TOFA was obtained from SANTA CRUZ (California, USA), Fetal bovine serum (FBS), DMEM medium and penicillin/streptomycin were obtained from Hyclone (Logan, UT, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) Annexin V-FITC/PI Apoptosis Detection Kit and Cell Cycle Assay Kit were purchased from Beyotime (Jiangsu, China). Bovine serum albumin (BSA), Dimethyl sulfoxide (DMSO), Ribonuclease (RNase A) and LY294002 were purchased from Sigma-Aldrich (St.Louis,MO,USA). RIPA Lysis Buffer, Pierce™ BCA protein assay kit, protease inhibitor cocktail, the polyvinylidene difluoride (PVDF) membrane and Super Signal West Pico Chemiluminescent Substrate detection kit was purchased from Thermo Fisher Scientific (Waltham, MA, United States). The antibodies such as p21Cip1/Waf1, CDK1, Cyclin B1, Bax, Bcl-2, Cleaved caspase 3, p-AKT (Ser473), p-mTOR (Ser2448), p-p70S6K (Ser371) and GAPDH were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA).