Fig. 6: TSG-101 knock down does not affect pyroptosis susceptibility, whereas dynamin inhibition increases pyroptosis susceptibility and blocks membrane repair.

A Immunoblot of control and shRNA-TSG101#2 and #4 knockdown THP-1 cells. B Flow cytometry detection of control and shRNA TSG101#4 cells at 1 h and 21 h after treatment with 40 µM nigericin with and without wash-out at 1 h. The y-axis shows the percentage of cells with high PI fluorescence intensity (PI uptake). N = 4. Significance was tested with one-way ANOVA, ns=non-significant; p**** ≤ 0.0001. C Flow cytometry detection of control and 80 µM dynasore pretreated (−30 min) cells at 1 h and 21 h after treatment with 40 µM nigericin with and without wash-out at 1 h. The y-axis shows the percentage of cells with high PI fluorescence intensity (PI uptake). n = 4. Significance was tested with one-way ANOVA, ns = non-significant; p**** ≤ 0.0001. D Immunoblot of control and 80 µM dynasore pretreated (-30 min) THP-1 cells at 1 h and 21 h post treatment with 40 µM nigericin. Nigericin was washed out at 1 h. Actin was used as internal loading control. The N-GSDMD signal intensities were quantified by using Image J software. The mean intensity of the bands was normalized to the mean intensity of the actin bands. The background intensity was subtracted from the values. E Representative confocal microscopy images showing THP-1 cells 1 h after treatment with 20 µM nigericin and 40 min after 20 µM nigericin (1 h) wash out. EEA1-Alexa-555 (magenta), N-GSDMD-Alexa-488 (green) are shown. White demonstrates colocalization of EEA1 and N-GSDMD. A single z-plane is presented. In Alexa 488 channels, minimum threshold set to 7000; in Alexa 555, minimum threshold set to 3000, maximum threshold set to 30,000. Scale bar = 10 µM.