Fig 1: Carboxypeptidase A4 (CPA4) regulates HGS-ETR1/2-induced pyroptosis.a, b The upregulated expression of CPA4 in HLCZ01-ETR1/2 R cells was verified by qPCR (a) and western blotting (b). c-f Lentiviral sh-CPA4 supernatant was added to HLCZ01-ETR1/2 R cell culture medium, stable cell lines were screened with puromycin, and the silencing effect was detected by qPCR (c, e) and western blotting (d, f). g, h CPA4-silenced HLCZ01-ETR1R (g) and HLCZ01-ETR2R (h) cells were treated with 2 μg/mL HGS-ETR1 or HGS-ETR2 for 10 h, and the protein expression of GSDME, caspase-3, and cleaved caspase-3 was detected by western blotting, β-actin was used as the internal reference protein. j, k Lenti-CPA4 supernatant was added to HLCZ01 cells, CPA4 was overexpressed, and stable cell lines were screened with puromycin. After 10 h of addition of HGS-ETR1/2, proteins were collected to detect the expression of CPA4 and the cleavage of GSDME. β-actin was used as the internal reference protein. i, l HGS-ETR1/2 was added to HLCZ01-ETR1/2 R cells that silenced CPA4 (i) and HLCZ01 cells that overexpressed CPA4 (l). Ten hours later, PI and Hoechst dye were added to the cell supernatant for staining, and the cell death was recorded by fluorescence photography. Scale bar, 300 μm (i) or 125 μm (l). m HGS-ETR1/2 was added to HLCZ01 cells that overexpressed CPA4, and the pyroptosis morphology of the cells was observed 10 h later by bright field photography. n HGS-ETR1/2 was added to HLCZ01 cells that overexpressed CPA4, and the supernatant was absorbed 10 h later to detect the release of LDH in the supernatant. o-t Schematic diagram of NCG mice treated with HLCZ01(lentiviral vector/CPA4) injection (o). After the back hair removal of mice, 5 million HLCZ01 cells overexpressing CPA4 or vector were injected into them. When the tumour grew out, its volume was measured and recorded every 2 days. When the tumour volume was approximately 100 mm3, the mice were randomly grouped and injected with HGS-ETR1/2 or PBS (10 mg/kg), and then injected again 4 days later. After the second injection, the mice were sacrificed 4 days later, and the tumours were removed for photographic observation (p) and weighed. According to the measured tumour volume, growth curve analysis was performed for tumours in different groups (q). The tumour was removed and weighed to analyse the difference in tumour weight between different groups of mice (r). In order to have the samples on the same PVDF membrane, three tumor tissues were randomly selected from each group to extract proteins. The expression of GSDME and CPA4 in the tumor tissues was detected by western blot (s, t). u-y Schematic diagram of NCG mice injected with HLCZ01-ETR1R(sh-control/CPA4) (u). After the back hair removal of mice, 5 million silenced CPA4 or control HLCZ01-ETR1R cells were injected, and the volume was measured every 3 days after the growth of the tumour, and the volume was measured and recorded. When the tumour volume was approximately 100 mm3, the mice were randomly grouped and injected with HGS-ETR1 (10 mg/kg) or PBS. After 3 days, the mice were injected again for a total of 5 times. After the fifth injection, the mice were sacrificed 3 days later, and the tumours were removed for photo observation (v) and weighed. Growth curve analysis was performed for different groups of tumours based on the measured tumour volume (w). Tumour weight differences between different groups of mice were analysed by weighing the removed tumours (x). Three tumor tissues were randomly selected from each group to extract proteins, and the expression of GSDME and CPA4 in the tumor tissues was detected by western blot (y). The experiment was independently repeated two (o-t) or three times(a-n). Two-sided Student’s t test was used to analyse significant differences. (*p <0.05, **p <0.01, *** p <0.001, **** p <0.0001).