C myc

Flag (1:1000), c-Myc (Santacruz-SC-40; 1:750 and sc-764; 1:1000), p62 (BD; 1:2000), Actin (Abcam; 1:5000), IRGM (Abcam; 1:500), HA (CST; 1:1000), IL-1β (CST; 1:1000), Caspase-1(Adipogen; 1:1000), NLRP3 (Adipogen; 1:1000), GFP (Abcam; 1:5000), ASC (Santacruz; 1:1000), ATG-5 (CST, 1:1000), ATG7 (CST, 1:1000), NLRC4 (CST; 1:1000) and AIM2 (CST; 1:1000), IRGM Antibody Rodent specific (CST; 1:1000), c-Myc (Santa Cruz; 1:750). Phsopho-p38 MAPK (CST, 1:1000), p-38α (Santa cruz, 1:1000), Anti-pro Caspase1+p10+p12 Antibody (Abcam; 1:1000), NF-kb p65 (Santa cruz; 1:1000), p- NF-kb p65 (Santa cruz; 1:1000).
HRP conjugated secondary antibodies were purchased from Santa cruz (1:2000) or Promega (1:5000) or Abcam (1:10000).

Grape seed extract (GSE, ORAC value 9000–13,000 μmole Trolox equivalents/g, total phenolic content > 85 % gallic acid equivalents) was a generous gift from San Joaquin Valley Concentrates (Fresno, CA). We had previously characterized the GSE used in this study using UPLC-MS and we detected presence of (+)-catechin and (−)-epicatechin monomers and their oligomers, and their gallate derivatives similar to other published papers [24 (link), 25 (link)]. The GSE used in this study lacks resveratrol (RSV) as described earlier [10 (link)]. BrdU Cell Proliferation Assay Kit was obtained from Cell Signaling Technology (Danvers, MA). Antibodies for PARP and cleaved PARP, p53, pGSK3β, Bax, Bcl-2, β-actin, β-catenin, cyclin D1, c-Myc, COX-2 and topoisomerase-2β (Topo ii b) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cytochrome C was obtained from Cell Signaling Technology (Beverly, MA). All other chemicals including RSV were obtained from Sigma (St. Louis, MO).

Western blot analysis of the inhibitor-treated cells was performed using a standardized protocol [35 (link)]. The primary human antibodies used in these analyses included c-MYC, SMO, and β-Actin (Santacruz, CA), AKT, phospho-AKT, S6K, phospho-S6K, GLI1 and SOX2 (Cell Signaling Technology, MA) and, cyclin D1, Bcl-2 and CD133 (BD Biosciences, CA). Immunoreactivity was detected using appropriate peroxidase-conjugated secondary antibodies (Santacruz, CA) and visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, IL).

Immunohistochemical staining was performed as previously described [62 (link)]. EZH2 staining was classified into 2 groups (negative and strong expression) based on the percentage of positive cells and staining intensity [63 (link)]. The percentage of positive cells was divided into the following 5 score ranges: <10% (0), 10% to 25% (1), 25% to 50% (2), 50% to 75% (3), and >75% (4). The intensity of staining was classified into the following 4 groups: no staining (0), light brown (1), brown (2), and dark brown (3). EZH2 staining positivity was determined using the following formula: immunoreactivity score (IRS) = intensity score × quantity score. An overall score of ≤3 was defined as negative, >3 as weak positive, and >6 as strong positive. All specimens were evaluated by two pathologists in a blinded manner.
For the immunocytochemistry experiments, cells were cultured on coverslips, fixed with 4% paraformaldehyde for 30 minutes at room temperature, and permeabilized with 0.2% Triton X-100 for 15 minutes at room temperature, and they were then incubated with the primary antibodies described above. An antibody against EZH2 was obtained from Cell Signaling Technology (CST, Beverly, MA, USA), and additional antibodies against Ki67, β-catenin, cyclin D1 and c-myc were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The various primary antibodies, unless otherwise mentioned, were purchased from Sigma-Aldrich (USA), Cell Signalling Technology (USA), Abcam (UK) or Bangalore Genei (India). The HRP-conjugated anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bangalore Genei (India). The catalog # of antibodies used in this study are as follows: RNAPII (Rpb1 CTD, Cell Signaling Technology, Cat #2629; 1: 6500 dilution), c-MYC (Santa Cruz, sc-40; 1: 2500), H3 (Cell Signaling Technology, Cat #3638), RPA (Cell Signaling Technology, Cat #2208), BRG1 (Sigma Aldrich, Cat #B8184: 1: 5000), and β-actin (Sigma Aldrich, Cat #A1978: 1: 10,000 dilution). SMARCAL1 antibody (1: 1800 dilution) was raised against the N-terminus HARP domain as discussed previously35 (link).

Cells and tissue homogenates (20 μg protein) were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for antibody probing. After washing with TBST (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), membranes were blocked with 5% skim milk for 2 h and then incubated with primary antibodies specific to HIF-1α, PrP^C, janus kinase 2 (JAK2), phosphorylated-JAK2, signal transducer and activator of transcription 3 (STAT3), p-STAT3, cyclin D1, c-Myc, p-NF-κB, cleaved caspase-3, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After incubation of the membranes with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands were detected using enhanced chemiluminescence reagents (Amersham Biosciences, Little Chalfont, UK) in a dark room.