Fig. 1: GD-NT-interacting kinases may regulate the resistance of tumor cells to GD-NT-mediated pyroptosis.

A Tetracycline on (TetOn) system uses tetracycline (or one of its analogs like doxycycline) as a regulator of gene expression. Tetracycline-dependent promoter is created by placing a TRE upstream of a minimal promoter. TRE is 7 repeats of the tetracycline operator (tetO) sequence and is recognized by a reverse tetracycline-controlled transactivator (rtTA). In the presence of tetracycline or one of its analogs like doxycycline, rtTA will bind to tetracycline and the TRE, permitting target gene transcription. B Tumor cells were transduced with lentivirus to express Dox-inducible GD-NT or empty vector (EV). Cells were treated with or without Dox (2 µg/ml). GD-NT expression was detected by immunoblot (IB) analysis at 16 hours post-Dox. β-actin served as internal control. C Tumor cells expressing Dox-inducible GD-NT or empty vector were treated with Dox (2 µg/ml). Cell death was assessed by Trypan Blue staining at different time points. The cells that were resistant to Trypan Blue uptake under light microscopy were defined as living cells. The ratio of living cells is calculated as living cells/total cells x 100%. D Similar to (C), except that cell death was assessed with LDH-based Non-Radioactive Cytotoxicity Assay kit 24 hours post-Dox. E Similar to (C), except that the morphological changes were observed using phase-contrast imaging 24 hours post-Dox. F Anti-Flag immunoprecipitation (IP) coupled with MS analysis was performed in E0771 cells expressing Dox-inducible Flag-GD-NT to identify GD-NT-interacting proteins. Shown is a schematic workflow of LC-MS analysis of GD-NT interactome. G, GO enrichment analysis of GD-NT interactome with PANTHER Overrepresentation Test (molecular function). The identified proteins were acquired from the IP/LC-MS analysis (F). The interactomes of GD-NT groups and control groups were analyzed by two-tailed Student`s t-test (means ± s.e.m). The genes with a significant difference of fold ≥100 and P < 0.05 were recruited into GO analysis. GO terms were sorted based on the P values. Displayed are the gene ontology terms with the false discovery rate (FDR) P < 0.05 (Fisher’s exact test). In C and D, error bars represent the variation range of duplicated experiments. Data are representative of at least two independent experiments.