Anti p65

Cells were treated with HQGGT, HQ, GG, SM, BS, GC, BI, BE, WO, and WS for 48 h and processed for immunoblot analysis as previously described [12 (link)]. The following primary antibodies (obtained from Cell Signaling Inc. unless noted) were used at the indicated dilutions: anti-TS, 1:2000 (#9045); anti-E2F1, 1:1000 (#3742); anti-GAPDH, 1:10,000 (#5174; #sc-47724, Santa Cruz); anti-p-NDRG1, 1:1,000 (#5482); anti-NDRG1, 1:1,000 (#9408); anti-p-P65, 1:5,000 (#3033); anti-P65, 1:5,000 (#8242); anti-PUMA, 1:1,000 (#4976); anti-p-RB, 1:1,000 (#9307); anti-RB, 1:1,000 (#9313); anti-CHK1, 1:1,000 (#2345); anti-HSP27, 1:1,000 (#2402); anti-CyclinB1, 1:1,000 (#12231); anti-STAT3, 1:5,000 (#9139); anti-MCL1, 1:1,000 (#5453); anti-CDK4, 1:1,000 (#12790); anti-CDK6, 1:1,000 (#3136); anti-CyclinD1, 1:1,000 (#2978); anti-DHFR, 1:1,000 (#610696, BD Biosciences); anti-TK1, 1:1,000 (#40688, Novus Biologicals). Proteins were detected using SuperSignal West Pico substrate (Pierce; Rockford, IL). Quantitation of signal intensities was performed by densitometry on a HP scanner using NIH IMAGEJ software.

Total cell or tissue lysates were extracted using lysis buffer, and proteins were separated in 8–12% (v/v) SDS-PAGE gels. After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-VEGF (Santa Cruz Biotechnology, sc-152, Dallas, TX, USA), anti-stromal cell-derived factor (SDF)-1 (Cell Signaling Technology, 3530S, Boston, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, sc-52012, Dallas, TX, USA), anti-IL-6 (Santa Cruz Biotechnology, sc-1265, Dallas, TX, USA), anti-tumor necrosis factor (TNF)-α (Santa Cruz Biotechnology, sc-52746, Dallas, TX, USA), anti-p-ERK (Cell Signaling, 9106S, Boston, MA, USA), anti-ERK (Cell Signaling, 9102S, Boston, MA, USA), anti-CXCR2 (Santa Cruz Biotechnology, sc-7304, Dallas, TX, USA), anti-CXCL5 (R&D, MAB254, Minneapolis, MN, USA), anti-p-p65 (Cell Signaling Technology, #3031S, Danvers, MA, USA), anti-p65 (BD, 0079008, East Rutherford, NJ), and anti-actin (Merck, 3423208, Darmstadt, Germany) at 4 overnight. After washing three times, the membranes were incubated with HRP-conjugated secondary antibodies (1:1000) for 1 h at room temperature. Finally, the membranes were visualized using the ECL kit.

Cells were washed in PBS and fixed in 4% PBS–PFA for 10–15 minutes at RT. For p65 and c-Rel immunocytochemistry cells were incubated in ice cold methanol for 10 minutes at -20°C. For HMGB1 staining slides were incubated in Citrate buffer and subjected to microwave heating at 350 W for 5 minutes, then left to reach RT for 20 minutes and washed in PBS. Slides were incubated in blocking solution containing 10% donkey serum or 10% goat serum (depending on the source of the secondary antibody) in PBS for 1 hour at RT. Subsequently, cells were incubated overnight at 4°C with the following primary antibodies: anti-CD11b 1:100 (OX-42, Millipore), anti-p65 1:30 (BD Biosciences), anti-c-Rel 1:50 (sc-70, Santa Cruz), anti-HMGB1 1:100 (ab79823, Abcam). After rinsing, slides were incubated with the appropriate secondary antibodies for 1 hour at RT. Cells were washed in PBS and mounted in mounting medium Vectashield (Vector Laboratories). Negative control slides were incubated with blocking solution instead of the primary antibodies. Staining was visualized in a fluorescence microscope (Axiophot; Carl Zeiss, Jena, Germany). Quantification of fluorescence intensity was done with ImageJ Software (NIH, USA).