Fig. 4: Fn-OMV enhances the expression levels of PANoptosis effector proteins.

a 4T1 cells were infected with different bacterial titers (MOI = 0, 1, 10, 20, 50, 100) for 24 h, cellular protein was analyzed by western blotting to detect the expression level of GSDMD, GSDME, and MLKL (upper) and corresponding quantitative analyses (below). Western blotting was done thrice independently with similar results. n.s. not significant. b The protein expression of GSDMD, GSDME, and MLKL was evaluated using western blotting in 4T1 (left) and 4MOSC1 (right) cells treated with different bacterial species. Western blotting was done thrice independently with similar results. n.s. not significant. c Schematic illustration of outer membrane vesicle (OMV) isolation and transmission electron microscopy image of Fn-OMV. Scale bars = 50 μm. Data were repeated thrice independently with similar results. d Confocal Laser Scanning Microscope shows the phagocytosis of Fn-OMV by 4T1 cells and 4MOSC1 cells. Scale bars = 10 μm. Data were repeated thrice independently with similar results. e Hydrodynamic diameter (Dh) of Fn-OMV in PBS at pH 7.4. Data were repeated thrice independently with similar results. f Effect of Fn and its secreted OMV on protein expression of GSDMD, GSDME, and MLKL was evaluated using western blotting in 4T1 cells. Western blotting was done thrice independently with similar results. n.s. not significant. Western blotting analysis was performed to evaluate the protein stability in 4T1 cells of GSDMD (g), GSDME (h), and MLKL (i) upon treatment with Fn-OMV (1 μg ml⁻¹) and MG132 (10 μM) in the presence of 50 μg ml⁻¹ cycloheximide (CHX). Immunoprecipitation (IP) and western blotting analysis were performed to examine the ubiquitination and expression of GSDMD (j), GSDME (k), and MLKL (l) in Control and Gsdmd, Gsdme, or Mlkl-depleted 4T1 cells after 24 h of Fn-OMV (1 μg ml⁻¹) treatment. Western blotting was done thrice independently with similar results. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test in (a). Data represent the mean ± s.e.m. Source data are provided in the Source Data file.