Hrp linked antibody

Western blot analysis was carried out using antibodies targeting phospho-(Y416)-Src Rabbit pAb (Cell Signaling Technology, Danvers, MA, USA, Cat# 2101, RRID:AB_331697), Src (L4A1) Mouse mAb (Cell Signaling Technology Cat# 2110, RRID:AB_10691385), and PARP (46D11) Rabbit mAb (Cell Signaling Technology Cat# 9532, RRID:AB_659884). Mouse monoclonal antibodies were used in conjunction with anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology Cat# 7076, RRID:AB_330924). Rabbit polyclonal antibodies were used in conjunction with anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology Cat# 7074, RRID:AB_2099233). Equal loading was assessed using vinculin (Abcam Cat# ab129002, RRID:AB_11144129) or β-actin (Cell Signaling Technology Cat# 3700, RRID:AB_2242334) targeting antibodies.

Cells or tumor tissues were harvested and lysed in RIPA buffer complemented with cOmplete Mini Protease Inhibitor Cocktail inhibitors and Phosphatase inhibitor cocktails (Sigma). Lysates were denatured by boiling in 1× Laemmli buffer at 95°C for 5 min and loaded on a 4–20% gradient gel (BioRad). PVDF (polyvinylidene difluoride) membranes were used for proteins wet transfer (BioRad). Membranes were blocked in Casein + 0.1% Tween-20 (Thermo), and incubated overnight at 4°C with the primary antibodies Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370 S, Cell Signaling) at 1:2000, p44/42 MAPK (Erk1/2) (137F5) (4695 S, Cell signaling) at 1:2000, β-Actin Antibody (4967 S, Cell Signaling) at 1:2000, and α-Tubulin Antibody (2144 S, Cell Signaling) at 1:2000. Secondary antibody used were Anti-rabbit IgG, HRP-linked Antibody (7074 S, Cell Signaling) at 1:5000, and Anti-mouse IgG, HRP-linked Antibody (7076 S, Cell Signaling) at 1:5,000. Signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Fisher). BioRad GelDoc XR was used for membranes imaging. The western blot analysis was performed at least three times for each experiment in three technical replicates.

ITGB2 (#73663), GluT4 (#2213), Akt (#4691), Phospho-Akt (#4060), PI3Kinase (#4292), Anti-rabbit IgG, HRP-linked Antibody (#7074), Anti-mouse IgG and HRP-linked Antibody (#7076) were obtained from Cell Signaling Technology. ITGB2 (ab53009), Phospho-PI3Kinase (ab182651) and a-SMA (ab5694) were obtained from the Abcam company. ITGB2 (10554-1-AP), HIF1a (66730-1-Ig), MCT1 (20139-1-AP), MCT4 (22787-1-AP), LDHB (14824-1-AP) and PDK4 (12949-1-AP) were obtained from the Proteintech company. ITGB2 (AF1730) was purchased from R&D. Secondary Anti-rabbit IgG Dylight 680 (#35568), Anti-rabbit IgG Dylight 488 (#35553) and Anti-mouse IgG Dylight 800 (#35521) were obtained from ThermoFisher.
The primer sequences used in this study were obtained from commercial sources and are displayed in Table S1.

TPA (20 nM) and β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human tumor necrosis factor-α (TNF-α; 10 ng/mL) was purchased from R&D Systems (Minneapolis, MN, USA). Inhibitors of AP-1 (SR 11302) and NF-κB (Bay 11-7092) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The MAPK inhibitors SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor) were purchased from Merck Millipore (Billerica, MA, USA). Rabbit antibodies against SIRT6, phosphorylated (p-)-c-Jun, c-Jun, p-c-Fos, c-Fos, p-IKKα/β, IKKα, IKKβ, p-IκBα, SAPK/JNK, p-SAPK/JNK, p38 MAPK, p-p38 MAPK, p44/42 MAPK (Erk1/2), p-p44/42 MAPK (Erk1/2), and PKCδ were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit antibodies against NF-κB p65, NF-κB p105/p50, MMP-9, PKCα, and PKCβ were purchased from Abcam (Cambridge, UK). Mouse antibodies against IκBα were purchased from Cell Signaling Technology. Mouse anti-PCNA antibodies were obtained from Santa Cruz Biotechnology. The secondary antibodies anti-rabbit IgG, HRP-linked antibody, anti-mouse IgG, and HRP-linked antibody were purchased from Cell Signaling Technology. We used both the primary and second antibodies at a 1000:1 dilution (in 5% skim milk/1X TBS).

All thiolated oligonucleotides used in this study were purchased from Integrated DNA Technologies (Coralville, IA). Graphite powder (99%, 7–11 μM) was purchased from Alfa–Aesar (Heysham, Lancashire, UK). Sulfuric acid, phosphoric acid, hydrogen peroxide, trisodium citrate, sodium phosphate tribasic, sodium phosphate dibasic, boric acid, tetrachloroauric acid, dimethyl sulfoxide, apigenin, xylene, ethanol, 3,3-diaminobenzidine tetrahydrochloride, hematoxylin, and isopropanol were purchased from Sigma–Aldrich (Milwaukee, WI). Citric acid, calcium chloride, magnesium chloride, potassium permanganate, tris(hydroxymethyl)aminomethane (Tris), and hydrochloric acid were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Alpha-MEM and fetal bovine serum were purchased from GIBCO (Campinas, Brazil). An MTT assay kit was purchased from Thermo Fisher Scientific (Waltham, MA). MUC1 (VU4H5) mouse mAb, anti-mouse IgG, and HRP-linked antibody were purchased from Cell Signaling Technology (Danvers, MA). Milli-Q ultrapure water (18 MΩ, Millipore, Billerica, MA) was used in all experiments.