Anti puma

After each treatment with or without TPGS, cells (1 × 10⁵ cells/well) were fixed in 80% ethanol and stored at −20° C overnight. Then, cells were washed with PBS and permeabilized with 0.2% Triton X-100 plus 1.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min. Cells were washed and incubated with anti-PUMA (Abcam, cat. No. ab-9643) and caspase-3 (Rabbit, Millipore, cat. No. AB3623) primary antibodies (1 : 500, diluted in PBS containing 0.1% BSA). Subsequently, the cells were washed and incubated with (1 : 500) Dylight donkey anti-rabbit (594 nm, cat. No. DI-1094) or -mouse (488 nm, cat. No. DI-2488) secondary antibodies for 30 min at RT in the dark. After washing with PBS, the cells were suspended in 500 μL of PBS. The analysis was performed on a BD LSRFortessa II flow cytometer (BD Biosciences). Cells without primary antibodies served as a negative control. For assessment, it was acquired 10000 events and quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software.

For whole mount immunostaining, organoids were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 20 min, and blocked with 5% BSA for an hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. The following primary antibodies (and their respective dilutions) were used: anti-cleaved-caspase3 (CST, 1:200), anti-Ki67 (CST, 1:200), anti-Cytochrome-C (Abcam, 1:200), anti-PUMA (Abcam, 1:200), anti-PCNA (Abcam, 1:200), and anti-E-cadherin (CST, 1:200). The respective secondary antibodies (Molecular Probes, Invitrogen) were diluted by 1:400 in blocking buffer and incubated at room temperature for an hour. The images of organoids were taken by confocal microscopy (OLYMPUS FV3000 Japan), and the intensity of the fluorescence was quantified by ImageJ software for the corresponding comparisons. ImageJ software was used to quantify the intensity of c-Myc staining of single cells in organoids. After analyzing the intensity of c-Myc immunofluorescence staining of single cells in organoid sections, the average c-Myc fluorescence value in all sample sections was 32.66. Therefore, we set the fluorescence value of 35 as the threshold of strong positive c-Myc and used it to calculate the proportion of strong positive c-Myc expression in each organoid.