Cleaved caspase 3 casp3

Western blot analysis was conducted to isolate the target proteins using RIPA buffer (Beyotime) containing protease inhibitors. The protein concentrations were determined using a BCA Kit. Subsequently, the same amounts (0.02 mg) of protein were treated with boiled water and isolated using the SDS-PAGE technique. The SDS-PAGE gels were subsequently transferred to 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA), which were exposed to TBST containing 5% fat-free milk for 60 min and cultivated with RPN2, MCL1, BCL2, BAX, BIM (Abcam, Cambridge, MA, USA), NOXA, PUMA, cleaved caspase-3 (CASP3), STAT3, p-STAT3 and β-actin (ACTB) (Cell Signaling, Danvers, MA, USA) antibodies at 4 °C overnight. After washed with TBST for 3 times, and the membranes were cultivated with 2nd antibody (Abcam) diluted at 1:1000 for 120 min at room temperature.

Flow cytometry studies aimed at determining cell cycle profile, mitosis fraction, DNA damage and/or apoptosis of CRC-SCs were performed as previously reported [26 (link)]. The following primary antibodies were used: cleaved caspase 3 (CASP3; #9661) and phospho-histone H3 (pH3; #3377) from Cell Signaling Technology (Danvers, MA, USA), and γH2AX (#05-636) from Merck Millipore. Secondary antibodies used were: Alexa Fluor^® 488-goat anti-mouse and 647-donkey anti-rabbit ((#A-21121 and #A-31573). These antibodies were provided by Thermo Scientific. Antibody dilutions and the Research Resource Identifiers (RRIDs) are in [26 (link)]. The DNA intercalant DAPI (#D1306, from Thermo Scientific) at 10 µM was employed to stain the DNA. The samples were acquired by a BD FACSCelesta^TM flow cytometer (BD Biosciences, BD, Franklin Lakes, NJ). Data and statistical analyses were performed with the FlowJo software (FlowJo LLC, BD), gating on events showing normal forward and side scatter (FSC and SSC) parameters, on singlets and (for cell cycle analysis) upon exclusion of the sub-G₁ fraction.

Tumors were fixed in formalin and embedded in paraffin block for immunohistochemistry (IHC). Tumor sections were stained using antibodies for Ki-67 (Dako, Denmark), cleaved caspase-3 (Casp3, Cell Signaling Technology, Danvers, MA, USA), CD8 (ThermoFisher, Waltham, MA, USA) or Gal-1 (R&D Systems, Minneapolis, MN, USA). Images were captured using Olympus DP70 camera on Olympus BX61 microscope. Captured images were analyzed using IHC Profiler plugin in ImageJ.