One step tunel

PD (purity>99%, Fig. S1) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). LTA from S. aureus was obtained from Sigma‐Aldrich Chemical Co. (Saint Louis, Missouri, USA). The indicated antibodies, including the NF‐κB Pathway Sampler Kit and Cleaved Caspase Antibody Sampler Kit, were obtained from Cell Signaling Technology (Beverly, MA, USA). 2′,7′‐Dichlorofluorescein diacetate (2′,7′‐DCFH‐DA), One Step TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling), Apoptosis Assay Kit and FITC Annexin V Apoptosis Detection Kit with PI (propidium iodide), BAY‐11‐7082 (an inhibitor of NF‐κB) and N‐acetyl‐L‐cysteine (NAC) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Foetal bovine serum (FBS) was purchased from Sigma‐Aldrich Chemical Co. (Saint Louis, Missouri, USA). All of the other chemicals and reagents were of the highest commercial grade available.

Cell proliferation was examined using Cell Counting Kit 8 (Invitrogen, Carlsbad, CA). That is, 100 μL of cell suspension (2,000 cells • well-1) were seeded into a 96-well plate. Next, after 0, 24, 48, and 72 hours (h) of culture, we added 10 μL of Cell Counting Kit 8 reagent and incubated for 1 h prior to making detections. Cell viabilities were examined by measuring optical density at 450 nm with a specrophotometric plate reader (BioTek, VT, USA). Each experiment was repeated in triplicate.
For apoptosis assays, HO8910 or A2780 cells were harvested after transfection and double stained using fluoresce in isothiocyanate (FITC)-conjugated Annexin V and propodium iodide (PI). Next, the percentage of early apoptotic cells was analyzed on a flow cytometer (Becon Dickinson FACSCalibur, NY, USA). Apoptosis was examined by One Step TUNEL (TdT-mediated dUTP Nick-End Labeling) Apoptosis Assay Kit (Beyotime, Shanghai, China) in accordance with manufacturer protocols. HO8910 or A2780 cells were fixed with 4 % paraform for about 30 minutes (min), then stained with Hoechst 33342 (Beyotime, Shanghai, China) for 20 min and photographed under light fluorescence microscopy (Leica, Wetzlar, Germany). We performed three independent replicated experiments for these analyses.

According to the manufacturer’s protocol, a one-step TUNEL (TdT-mediated dUTP gap end labelling) apoptosis detection kit (Beyotime, C1090) was used to examine the apoptosis of HCT116 and DLD-1 cells after the knockdown and overexpression of SCRIB, respectively. The cells were photographed under an Olympus FSX100 microscope (Tokyo, Japan).