Cas 3

The protein levels in the U937 cells were determined from the SDS-PAGE gels. The proteins were moved to the membranes of PVDF membranes. After diluting the primary antibodies (1:200), the obtained membranes were kept overnight at 4 °C with the diluted primary antibodies of anti-TRPV1 (Cat #PA-748; Thermo Fisher), CAS3 (Cat #14220; Cell Signaling Technology), CAS9 (Cat #9502; 1:200; Cell Signaling Technology), Bcl-2 (Cat #ab59348), Bax (Cat #ab53154) (Abcam), and β-actin (Cat #sc-47778) (Santa Cruz). After the incubation of secondary antibodies, the protein bands of the antibodies were visualized in the visible imaging system (G:Box, Syngene, Cambridge, UK). The band signal intensities of Bax, Bcl-2, CAS3, CAS9, β-actin, and TRPV1 were measured using ImageJ software (Version 1.48, NIH, USA) [37 (link)]. The internal control of the signal intensity was a housekeeping protein (β-actin antibody) band. The signal activities of bands were measured by using ImageJ software, and they were shown as relative density.

MDA-MB-231 cells grown to 65–70% confluency were treated with 10 nM OXi6196 for varying times (0–90 min). Lysates were prepared by cold application of RIPA buffer, clarified via centrifugation, and treated with LDS/5% β-mercaptoethanol. Proteins were separated by SDS-PAGE on 4–12% Bis-Tris gradient gels (Invitrogen, ThermoFisher Scientific), then transferred to 0.2 μm PVDF membranes (EMD Millipore). After blocking, blots were incubated with primary antibodies (1:1000 for Cas-3, 1:2500 for GAPDH, Cell Signaling Technology), followed by secondary HRP-labeled goat anti-rabbit IgG antibodies (Jackson Immuno Research Labs, West Grove, PA USA). Blots were developed with Amersham ECL Prime chemiluminescent substrate and imaged with an ImageQuant LAS 4000 system (GE).

The following antibodies were used for staining: TSPO (1:5,000; Abcam Cat# ab109497), cleaved caspase-3 (Cas-3; 1:500; Cell Signaling Technology Cat# 9664, RRID:AB_2070042) and phospho-histone H3 (PHH3; 1:100; Cell Signaling Technology Cat# 9701, RRID:AB_331535). The percentages of cells from ROIs with a 700-μm radius staining positively for PHH3 and Cas-3 were analysed. For TSPO, positively stained cells from the whole tumour area were analysed. All the analyses were done with QuPath [29 ]. The analysis scripts are shown in Supplementary Tables 1 and 2.