Halt protease and phosphatase inhibitor cocktail 100

RIPA Lysis and Extraction Buffer and Halt Protease and Phosphatase Inhibitor Cocktail (100×) were purchased from Thermo Scientific and stored at 4 °C. BCA protein assay kit used to quantify protein concentration were purchased from Beyotime and stored at RT. DMEM (Hyclone, Logan, UT, USA), RPMI1640 (Hyclone, Logan, UT, USA), Penicillin/streptomycin and Trypsin/EDTA were purchased from Hyclone. Horse serum was purchased from Gibco (Gibco, Grand Island, NY, USA). Fetal bovine serum (FBS) was derived from Biological Industries, GW4869 (MCE, NJ, USA), PEG8000 (Sigma-Aldrich, MO, USA), X-treme GENE9 (Roche, Basel, Switzerland), Recombinant Mouse GDF15 (R & D, Minnesota, USA).

Briefly, cells from pancreatic cell lines were washed twice with PBS and lysed in Pierce RIPA Buffer (Cat#8990, Thermo) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail 100×(Cat#78440, Thermo). Protein concentrations were quantified using Pierce BCA Protein Assay Kit (Cat#23225, Thermo). Samples were denatured at 95 °C in XT Sample Buffer 4×(Cat#1610791, Bio-Rad, Hercules, CA) and loaded onto 4–12% Criterion XT Bis-Tris Protein Gels (Cat#345-0124, Bio-Rad). Proteins were transferred onto PVDF Transfer Membrane (Cat#88518, Thermo) and probed for STING (Cat#13647S, Cell Signaling Technology, Danvers, MA), IRF3 (Cat#4302S, Cell Signaling), STAT1 (Cat#9172S, Cell Signaling), p-STAT1 (Y701) (Cat#7649S, Cell Signaling) and GAPDH (Cat#2118S, Cell Signaling). HRP-conjugated goat anti-rabbit IgG (Cat#31460, Invitrogen, Carlsbad, CA) was used as a secondary antibody. Proteins were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat#34580, Thermo).

RIPA lysis buffer (Beyotime Biotechnology, China) containing 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific) was used to lyse cells. Pierce BCA Protein assay kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration. The protein (40 μg) was separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membrane was blocked in TBST containing 3% BSA and primary antibodies were incubated overnight at 4 °C. The primary antibodies were used as follows: anti-SIRT1, anti-p53, anti-Ac-p53, anti-p-p53, anti-p-Chk2, anti-p21, anti-TRAF2, anti-p-ATM, anti-Cyclin B1 (1:1000, Abcam) and anti-GAPDH (1:5000, Huabio). Subsequently, TBST was used to wash the membranes three times and the membranes were incubated at room temperature for 1 h with 1:3000 horseradish peroxidase conjugated anti-rabbit or mouse immunoglobulin G (Southern Biotechnology Associates, USA), followed by three washings in TBST. Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) were used to visualize the specific bands with a ChemiScope 3300 Mini equipment (CLINX, China). The same membrane loaded with GAPDH served as the control.

Total protein extracts were isolated from MDA-MB-231 breast CSCs using a lysis buffer TNTE (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA) supplemented with 2 µL protease inhibitor cocktail and phosphatase 100X (Halt Protease and Phosphatase Inhibitor Cocktail 100×, Thermo Scientific, Waltham, MA, USA). The proteins (60 µg) were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Afterward, the membranes were blocked for 60 min at room temperature with TBST-1X (137 mM NaCl, 20 mM Tris, 0.1% Tween-20, at pH 7.6) containing 5% BSA (Sigma-Aldrich, St. Louis, MI, USA), as previously described [30 (link)]. Then, the samples were incubated overnight at 4 °C with mouse anti-GAPDH (1:1000, Santa Cruz Sc47724, Santa Cruz, CA, USA), mouse anti-VEGFA (1:100, Santa Cruz Sc7269), or mouse anti β-catenin (1:200, Santa Cruz Sc7963) primary antibodies. Finally, the membranes were washed 3 times in TBST-1X and incubated with horseradish peroxidase-conjugated goat anti-mouse (1:6000, Jackson Immuno Research JKSN 115-035-003, Cambridge, UK). An ECL detection kit was used to develop the signals. Densitometric analyses of immunodetected bands were performed using the myImage analysis software package.

Total proteins from the cells were extracted using a RIPA Lysis and Extraction Buffer (Thermo Scientific™, Waltham, MA, USA) including Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Scientific™, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The PVDF membranes were incubated with primary antibodies at 4°C overnight, and then further incubated with secondary antibodies for 30 min on the following day. The immunoreactive signals were visualized using the chemiluminescence reagents WesternSure^® PREMIUM Chemiluminescent Substrate (LI-COR^®, Lincoln, NE, USA). Images were captured with the Odyssey^® XF Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The following antibodies were used: XIAP Antibody (Cell Signaling Technology, Danvers, MA, USA), α-Tubulin Antibody (Cell Signaling Technology, Danvers, MA, USA).