Bio imaging system

Total protein was extracted using lysis buffer (Pierce, Rockford, IL) and quantified with the Bradford method.23 A total of 50 µg total protein per sample were separated via 15%, 10%, or 8% SDS-PAGE, and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA). Membranes were incubated overnight at 4°C with the following primary antibodies: BHLHE41(1:500, Abcam, Cambridge, UK), GAPDH (1:2000, Abcam, Cambridge, UK), His-tag (1:1000, CWBio, Shanghai, China), Myc-tag (1:1000, ImmunoWay Biotechnology Company, Plano, TX), cyclinA2, cyclinB1, cyclinD1, cyclinD2, cyclinD3, cyclinE1, cyclinE2, cyclinH (1:1000, #9869, CST). ERK (1:1000, #4695, CST), phospho (p)-ERK (1:1000, #4370, CST). PI3K (1:1000, #4249, CST), phospho (p)-PI3K (1:1000, #4228 CST). AKT (1:1000, #4691, CST), phospho (p)-AKT (1:1000, #13038, CST). I-κB (1:1000, #4812, CST), NF-κB (1:1000, #8242S CST), Bax (1:1000, 50599-2-lg Proteintech), Bcl-2(1:1000, 12789-1-AP Proteintech). Membranes were washed and incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 37°C for 2 h. Bound proteins were visualized using electrochemiluminescence (Pierce, Rockford, IL) and detected with a bio-imaging system (DNR bio-imaging systems, Jerusalem, Israel).

Total protein was extracted using a lysis buffer (Pierce, Rockford, IL, USA) and quantified with the Bradford method.9 (link) Total proteins (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with the following primary antibodies: ZNF259 (1:500, Abcam), GAPDH (1:5000, Sigma), Myc-tag, FLAG, Snail, MMP2, MMP9, cyclin D1, cyclin E1, CDK4, CDK6, p-AKT-Thr308, AKT, p-p38, p38, p-JNK, JNK p-FAK-Tyr397, FAK (1:1000; Cell Signaling Technology, Danvers, MA, USA), and E-cadherin (1:1000; BD Transduction Laboratories, Lexington, KY, USA). FAK inhibitor (PF-562271) was obtained from Selleck Chemicals (Houston, TX, USA). AKT inhibitor LY294002 was obtained from Cell Signaling Technology. Membranes were washed and subsequently incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology) at 37°C for 2 h. Bound proteins were visualized using electrochemiluminescence (Pierce) and detected with a bio-imaging system (DNR bio-imaging systems, Jerusalem, Israel).

RNA isolation from SK-BR-3 and MDA-MB-231 cells was performed using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was prepared using the ReverTra Ace RT-qPCR kit (Toyobo Life Science) with 1 µg RNA per reaction. RT was performed at 37°C for 15 min followed by 98°C for 5 min to inactivate reverse transcriptase activity. qPCR was performed with the Thunderbird SYBR qPCR mix (Toyobo Life Science) and the thermocycling conditions were as previously described (22 (link)). The primers used are listed in Table SI. Relative MMP gene expression was calculated using the 2-^ΔΔCq method (23 (link)), normalized to GAPDH and compared with the MMP3 level.
For semi-qPCR, the amplification conditions were the following: Initial denaturation at 95°C for 1 min, followed by 38 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 1 min for MMPs, and 23 cycles under the same conditions for GAPDH. Taq polymerase (Takara Bio, Inc.) was used as the DNA polymerase. Each condition was run in triplicate with the primers listed in Table SI. Amplified products were resolved by 1% agarose gel electrophoresis and visualized by 4S green plus (Sangon Biotech Co., Ltd.), and then photographed with a Bio-Imaging system (DNR Bio Imaging Systems). The band intensity of each lane was analyzed by ImageJ software (version 1.52a; National Institutes of Health) as described previously (24 (link)).

Total protein was extracted using a lysis buffer (Thermo Fisher Scientific) and quantified through the Bradford method. In total, 50 μg of protein samples was separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% resolving gel) and electroblotted onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with the following primary antibodies: anti-TMEM17 (1:200; Santa Cruz Biotechnology Inc), anti-GAPDH (1:5000; Sigma-Aldrich Co), anti-Snail, anti-Myc-tag, anti-p-ERK, anti-ERK, anti-p-AKT, anti-AKT, anti-active β-catenin, anti-cyclin D1, anti-c-myc (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin, and anti-β-catenin (1:500; BD Biosciences, San Jose, CA, USA). Membranes were washed and subsequently incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology Inc) at 37°C for 2 hours. Bound proteins were visualized using electrochemiluminescence (Thermo Fisher Scientific) and detected with a bio-imaging system (DNR bio-imaging systems, Jerusalem, Israel).

Cells were seeded in 6 cm cell culture dishes (1,000 cells per dish) 24 h after transfection and were cultured for 10 days. The medium was changed every 3 days. Cells were then washed with PBS and stained with hematoxylin for 10 min at room temperature. The number of colonies with >50 cells were counted using a bioimaging system (version 5.2.1; DNR Bio-Imaging Systems, Ltd.).