Gel documentation 2000 system

The cells were harvested using radioimmunoprecipitation assay (RIPA) buffer (Millipore, Billerica, MA, USA) supplemented with a protein inhibitor cocktail (Sigma, St. Louis, MO). Then, the cell lysates were sonicated at 4 °C and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatants were collected and quantified using a Pierce™ bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Then, 40 μg of total protein was diluted with Laemmli’s sodium dodecyl sulfate sample buffer (Boston Bioproducts, Ashland, MA, USA) and denatured at 95 °C for 10 min. The proteins were loaded and separated on 4–15% Criterion™ Tris-HCl Precast Gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA), followed by blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline-0.1% Tween 20 (TBST) buffer for 1 h at room temperature. The membrane was sequentially incubated with specific primary antibodies and HRP-conjugated secondary antibodies. The protein bands were visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) with a Gel Documentation 2000 system (Bio-Rad, Hercules, CA, USA).

The treated cells were harvested using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protein inhibitor cocktail (Sigma, St. Louis, MO). The protein concentrations of the cleared lysates were determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL), and 20 mg of the total protein was resolved by 4–15% SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were electro-transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was then blocked with 5% BSA in phosphate-buffered saline-0.1% Tween 20 (PBST) and then sequentially incubated with specific primary antibodies and HRP-conjugated secondary antibodies. The blots were visualized with the Super Signal enhanced chemiluminescence (ECL) detection system and documented using the Gel Documentation 2000 system (Bio-Rad, Hercules, CA). The antibodies were purchased from the following sources: anti-NRF2 from Epitomics Inc. (Burlingame, CA); anti-NQO1, anti- HO1 and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA); anti-DNTM1, anti-DNMT3a and anti-DNMT3b from IMGENEX (San Diego, CA); and anti-HDAC 1–6 from Cell Signaling Technology (Danvers, MA).

TRAMP-C1 cells were seeded at a density of 1 × 10⁵ cells in 10-cm dishes with 10% FBS/DMEM. After incubation for 24 h, the cells were treated with 0.1% DMSO as a control and FN1 (50 nM, 100 nM and 250 nM) in DMEM containing 1% FBS. Following treatment for 3 days, the cells were washed with ice-cold PBS and harvested in ice-cold 1× RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protein inhibitor cocktail (Sigma). The protein concentrations of the cell lysates were measured using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA). Identical concentrations of protein (20 µg) were subjected to 4 to 15% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA) electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA and sequentially incubated with specific primary antibodies and HRP-conjugated secondary antibodies. The antibody-bound proteins were visualized with SuperSignal West Femtov Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) and measured with a Gel Documentation 2000 system (Bio-Rad).

HCT116 cells (1x10⁶) treated with vehicle control and various concentrations of NaB for 24h and cells were harvested using RIPA buffer supplemented with protein inhibitor cocktail (Sigma, St. Louis, MO). Protein concentrations of each cleared lysates were measured using the BCA method (Pierce, Rockford, IL). Total 45μg proteins of each sample were separated by 4-15% SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). Then the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) followed by blocking with 5% BSA in Tris-buffered saline-0.1% Tween 20 (TBST) buffer. Then the membrane was sequentially incubated with specific primary antibodies and then HRP-conjugated secondary antibodies. The blots were visualized by SuperSignal enhanced chemiluminiscence (ECL) detection system and recorded using a Gel Documentation 2000 system (Bio-Rad, Hercules, CA). Primary antibodies were purchased from different resources: anti-DNA Methyltransferase 1 (DNMT1) and anti-GAPDH were purchased from Cell Signaling (Boston, MA); anti-NRF2, anti-KEAP1, anti-Heme Oxygenase 1 (HO1), anti-MYCL proto-oncogene (c-MYC), and anti-NAD(P)H dehydrogenase [quinone] 1 (NQO1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

The treated cells from above were harvested using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protein inhibitor cocktail (Sigma, St. Louis, MO). The clear supernatants from whole cell lysates were loaded (20 μg/lane) and separated by 4–15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). Then, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) followed by blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline-0.1% Tween 20 (TBST) buffer. Primary antibodies against MMP2 and MMP9 (Abcam, Cambridge, MA) and β-Actin (Santa Cruz Biotechnology, Santa Cruz, CA) were used. The blots were visualized by using a SuperSignal enhanced chemiluminiscence (ECL) detection system and recorded using a Gel Documentation 2000 system (Bio-Rad, Hercules, CA). Densitometry of the bands was analyzed using ImageJ (Version 1.48d, NIH).