Anti vimentin

Anti-vimentin, anti-Snail, and anti-Slug antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NUCB2, anti-Twist1, and anti-Ki-67 antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-E-cadherin, anti-p21^waf1, anti-BCL2, and anti-cyclin D1 antibodies were from Dako (Copenhagen, Denmark). Anti-p27^kip1, anti-Rb, anti-X-linked inhibitor of apoptosis (XIAP), anti-BAX, and anti-N-cadherin antibodies were from BD Biosciences (San Jose, CA, USA). Anti-ZEB1 and anti-β-actin antibodies and adriamycin (ADR: D1515) were from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-phospho (p) Rb at Ser807/811, anti-cleaved caspase-3, and anti-poly (ADP-ribose) polymerase 1 (PARP1) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cyclin A2 and anti-cyclin B1 antibodies were from Novocastra (Newcastle, UK) and Santa Cruz Biotech (Santa Cruz, CA, USA), respectively.

Western blots were performed as previously described [11] (link). Briefly, as soon as the harvested cells were lysed and the supernatant was collected, 60 µg protein was loaded onto SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). Membranes were blocked with 5% skimmed milk for 1 hour and incubated overnight with one of the following primary antibodies: anti-PSMD10 (diluted at 1∶1,000; Sigma, St. Louis, MO, USA), anti-Twist2 (diluted at 1∶200; Abcam, Cambridge, UK), anti-Vimentin (diluted at 1∶500; Cell Signaling Technology, Beverley, MA, USA), anti-β-catenin (diluted at 1∶500; Cell Signaling Technology), anti-E-cadherin (diluted at 1∶500; Cell Signaling Technology) and anti-cyclin D1(diluted at 1∶500; Cell Signaling Technology) rabbit monoclonal antibody, followed by 1 hour of incubation with the appropriate secondary antibody (1∶5,000). The anti-GAPDH (Epitomics) rabbit monoclonal antibody was diluted to 1∶1,000 for use as a sample loading control.

B16-F10 cells (2 × 10⁶ cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).

Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.

We prepared histones as described previously^{49 (link)}. Briefly, we lysed harvested HCT116, HT29 and DLD-1 cells after treatment of DMSO, 2 µM JIB-04, or 10 µM salinomycin for 24 h by mechanical shearing on a rotator in hypotonic lysis buffer at 4 °C. We extracted histones from the cell lysates with H₂SO₄ and precipitated them in TCA. After washing the precipitated histones with acetone, we dissolved the yields in distilled water.
We used the following antibodies for western blotting: anti-CD133 (Novus Biologicals, #NB120-16518), anti-E-cadherin (Cell signaling, #3195), anti-EpCAM (Cell signaling, #2929), anti-Vimentin (Cell signaling, #5741), anti-LGR5 (Novus Biologicals, #NBP1-28904), anti-CD44 (Santa Cruz Biotechnology, #sc-7297), anti-GAPDH (AbClon, #AbC-2003), anti-H3 (Abcam, #ab1791), anti-H3K9me2 (Abcam, #ab1220), anti-H3K9me3 (Millipore, #CS207324), anti-H3K36me3 (Abcam, #Ab9050), anti-H3K4me2 (Milipore, #07-030), and anti-H3K27me3 (Abcam, #Ab6002).

This method was performed as described previously^{17 (link)}. The cells grown on plates were trypsinized, and detached cells were collected by centrifugation. The cell pellet was lysed with cold lysis buffer supplemented with protease inhibitors. Protein samples (30 µg) from each cell lysate were equivalently loaded on a precast gel and used for electrophoresis. Gels were subsequently blotted onto nitrocellulose membranes (0.45 μM; Bio-Rad, Hercules, CA), followed by blocking nonspecific binding with a solution containing 1 × PBS, 0.1% Tween-20, and 5% nonfat dry milk powder at room temperature for 1 h. Membranes were incubated with the primary antibodies anti-E-cadherin, anti-Snail, anti-Vimentin, anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) and anti-Sema4C (Abcam, Cambridge, MA, USA) at 4 °C overnight. After extensive washing with TBST, horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) were incubated for 1 h at room temperature. Bands were detected with an enhanced chemiluminescence kit (Super Signal West Pico Substrate; Pierce, Rockford, IL, USA). Quantification of signal intensities was performed by densitometry on a Xerox scanner using NIH ImageJ software (ImageJ, Bethesda, MD).