Anti bcl 2

Apigenin (HPLC>98%) was purchased from the Nanjing TCM Institute of Chinese Materia Medica and was dissolved in sodium carbonate (20 mM). Purified mouse anti-Bax and anti-Bcl-2 antibodies were purchased from Biolegend (USA). Mouse monoclonal anti-phospho-ERK1/2, anti-caspase-3, anti-caspase-8, anti-phospho-p38, rabbit monoclonal anti-ERK1/2, anti-p38, mouse monoclonal anti-β-actin, rabbit/goat anti-mouse IgG and goat anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology (USA). Mouse monoclonal anti-phospho-JNK and anti-JNK antibodies were purchased from Cell Signaling Technology (USA). Apoptosis, DNA Ladder Extraction Kit with Spin Column, DNA Molecular Weight Marker was purchased from Beyotime Institute of Biotechnology (Shanghai, China). In Situ Cell Death Kit, Fluorescein was purchased from Roche Applied Science, Mannheim, Germany.

Colons were flushed with PBS and frozen at –70 °C. Colon tissues were mechanically disrupted using the Bullet Blender® (Next Advance) at 4 °C and after lysis with RIPA buffer with Phosphatase Inhibitor Cocktail (PhosStop EASYpack, Roche) and Protease Inhibitor Cocktail (complete Tablets EASYpack, Roche). Tissues were centrifuged for 15 min at 4 °C and 16,000 g. Supernatants were collected, run on SDS gels, and transferred onto membranes. The membranes were blocked and probed with anti-Bcl-2 (Biolegend, San Diego, CA, USA) and anti-β-actin (Biolegend, San Diego, CA, USA). ImageJ was used for densitometry.

Cells were lysed with radioimmunoprecipitation assay buffer (Fujifilm Wako Pure Chemical) containing protease/phosphatase inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were transferred onto polyvinylidene difluoride membranes. After blocking, the blots were incubated with the following antibodies: anti‐p21^Cip1/Waf1 (#2947; Cell Signaling Technology [CST]), anti‐p16^Ink4a (SPC‐1280; StressMarq Biosciences), anti‐γH2AX(Ser¹³⁹) (#9718; CST), anti‐Bcl‐2 (#658701; BioLegend), anti‐Bcl‐xL (#2764; CST), anti‐Mcl‐1 (#54535; CST), anti‐survivin (#71G4B7; CST), anti‐cFLIP (ALX‐804–428; Enzo Life Sciences), anti‐TATA‐binding protein (TBP; #22006‐I‐AP; Proteintech), anti‐PARP (#46D11; CST), anti‐caspase‐3 (#9668; CST), anti‐c‐Myc (1472–1; EPT), anti‐GAPDH (#015‐25473; Fujifilm Wako Pure Chemical), and anti‐β‐actin (#622102; BioLegend). Nuclear and cytoplasmic proteins were prepared using the LysoPure™ Nuclear and Cytoplasmic Extraction Kit (Fujifilm Wako Pure Chemical). After washing, membranes were incubated with goat anti‐rabbit or horse anti‐mouse horseradish peroxidase‐conjugated secondary antibody (#7074 and #7076; CST). Protein bands were visualized using an Amersham ImageQuant™ 800 Biomolecular Imager (General Electric Company).

Total cell lysates (20 μg) from drug-sensitive and -resistant cells were resolved on 6% SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked in milk in phosphate buffered saline (PBS) at 5% (w/v) and probed with P-gp specific mAb (0.1 μg/ml of C219 mAb) or anti-Bcl-2 (0.5 μg/ml; BioLegend, San Diego, USA; anti-Bcl-2 (D55G8) rabbit mAb, 1:1000 v/v (human specific), Cell Signaling Technology, Massachusetts, USA; or anti-β-Actin mAb, 0.5 μg/ml, Sigma-Aldrich, Toronto, CA) in 5% milk/PBS, followed by several washes in PBS and incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 v/v, BioRad, Ont., CA). The reactive signals were visualized using Western Breeze Chemiluminescent Kit and captured using ECL-imager from Thermo-Fisher Scientific. Tubulin expression was detected on the same PVDF membrane using anti-α-tubulin specific monoclonal antibody (1 μg/ml Sigma-Aldrich, Ont., CA), followed by HRP-conjugated goat anti-mouse IgG (1:5000 (v/v), BioRad, Ont., CA).

Organoids were digested into single cells with trypsin. Then, cells were fixed with a Fixation/Permeabilization Solution Kit (BD, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, 100 µL of Fixation/Permeabilization solution was mixed with resuspended cells and incubated for 20 min at 4 °C. The cells were washed twice with 1× BD Perm/Wash buffer. Then, 5 µL of Alexa Fluor 488 conjugated anti-Bcl-2 (Biolegend, San Diego, CA, USA) or anti-Bcl-XL (CST, Danvers, MA, USA) antibodies were added into the tube and incubated for 30 min at 4 °C. Stained cells were analyzed using a BD Aria SORP Flow Cytometer (San Jose, CA, USA). Results were plotted using FlowJo (LLC, Ashland, OR, USA).