Figure 7.

KCs PM rupture and fragmentation during Lm infection due to the loss of TMEM16F in vivo. A) Intravital microscopy images (left) of the liver infected with the GFP‐expressing Lm (5×10⁷ bacteria) at 12 h post‐infection. Scale bar, 500 µm in tile scan and 50 µm in zoom. B) Quantification of the surface coverage of GFP‐expressing Lm in WT and TMEM16F KO mice shown in (A) (n = 4). C) Quantification of the KCs number per field of view (FOV) after GFP‐expressing Lm infection. Mice were infected with 5×10⁷ CFU GFP⁺ Lm intravenously, and images were taken 12 h post‐infection. n = 15. D) Intravital microscopy images of Kupffer cell lysis (F4/80⁺TIM‐4⁺) after GFP‐expressing Lm infection in vivo. Mice were infected with 5×10⁷ CFU GFP⁺ Lm intravenously, and images were taken 12 h post‐infection. Bright green dots were GFP⁺ Listeria and white arrows indicated the Lm‐containing KCs. The white lines represent the cell membrane boundaries in WT and KO KCs, and the yellow lines represent cell fragmentation in KO KCs. Scale bar, 10 µm. E) Quantification of the lytic KCs in WT and TMEM16F KO mice shown in (D) (n = 14‐17). F) Detection of KCs (TIM‐4⁺) plasma membrane rupture elicited by GFP⁺ Lm infection in vivo. Mice were infected with 1×10⁸ CFU GFP⁺ Lm intravenously, and nucleic acid staining dye PI was injected 1 h post‐infection. Images were taken right after PI injection. Plasma membrane rupture was monitored by red fluorescence of PI, when the plasma membrane integrity was lost. White arrows represent the location where the bacteria escape from the KCs. The white lines represent the PM boundaries in WT and KO KCs, while the yellow lines represent PM components derived from cell rupture in KO KCs. Scale bar, 10 µm. G) Quantification of the ruptured KCs in WT and TMEM16F KO mice shown in (F) (n = 21‐46). Data are from at least two independent experiments and presented as mean ± SEM. Two‐tailed Mann–Whitney test for panels B, C, E and G. ^* p<0.05, ^** p<0.01, ^*** p<0.001. See related Supplemental Videos S1 and S2 (Supporting Information).