Figure S1.
C. violaceum infection induces mitophagy independently of CopD. (A) Confocal microscopy analysis of LC3B puncta in the livers of mice (n = 3) intraperitoneally injected with PBS or 12 × 106 CFU C. violaceum WT strain (C.v. WT for 10 h). Livers were subjected to immunofluorescence by anti-LC3B (green). Nuclei (blue) were stained with DAPI. Scale bar, 20 µm; inserts, 5 µm. (B) Confocal microscopy analysis of LC3B puncta in hepatocytes of mice (n = 3) treated as in A. Hepatocytes were labeled with anti-cytokeratin 18 antibody (magenta), and LC3B (yellow) was stained with anti-LC3B antibody. Nuclei (cyan) were counterstained with DAPI. Scale bar, 40 µm; inserts, 10 µm. (C) Immunofluorescence analysis of KCs isolated from mouse livers. Freshly isolated KCs were plated for 2 h, followed by PBS washing to remove debris and non-adherent cells. Cells were stained with the macrophage marker F4/80 (green). Nuclei (blue) were counterstained with DAPI. Scale bar: 40 µm. (D–G)C. violaceum infection induces mitophagy in HSCs (D), HepG2 cells (E), Huh7 cells (F), and AML12 cells (G). Cells were infected with C.v. WT (MOI = 5) for indicated times, followed by western blot analysis of cell lysates using the specified antibodies. (H and I) Immunoblotting analysis of TUFM (H)/Tom20 (I) in 293T cells infected with C.v. WT and treated with or without Mdivi-1 (20 μM) for 5 h. (J) LC3B levels were assessed by immunoblotting in 293T cells transfected with a vector control or Cpi-1/-1a T3SS effectors. (K) The invasion ability of different C. violaceum strains was evaluated using gentamicin protection assays in HeLa GSDMD−/− cells. C.v. ΔcopD: C. violaceum mutant strain with deletion of the copD gene. (L–N)C.v. ΔcopD infection induces mitophagy. (L) 293T cells expressing GFP-LC3B (cyan) were infected with WT or C.v. ΔcopD and analyzed by fluorescence microscopy. C. violaceum (magenta) was labeled with anti–C. violaceum antibody. Scale bar: 10 µm. (M and N) Cell lysates from infected cells were immunoblotted with anti-LC3B (M) or anti-Tom20 (N). (O and P) Confocal microscopy analysis of mitochondrial morphology in HeLa cells transfected with GFP, GFP-CipB, GFP-SipB, or GFP-IpaB (cyan), respectively. (O) Mitochondria were stained with a ΔΨm-dependent MitoTracker (pseudo-colored magenta). Scale bar, 10 µm. (P) Quantifications of the percentages of cells showing fragmented mitochondria among 100 randomly selected transfection-positive cells. P values were determined by one-way ANOVA with Dunnett’s test. All data are representative of three independent experiments. Source data are available for this figure: SourceData FS1.

C. violaceum infection induces mitophagy independently of CopD. (A) Confocal microscopy analysis of LC3B puncta in the livers of mice (n = 3) intraperitoneally injected with PBS or 12 × 106 CFU C. violaceum WT strain (C.v. WT for 10 h). Livers were subjected to immunofluorescence by anti-LC3B (green). Nuclei (blue) were stained with DAPI. Scale bar, 20 µm; inserts, 5 µm. (B) Confocal microscopy analysis of LC3B puncta in hepatocytes of mice (n = 3) treated as in A. Hepatocytes were labeled with anti-cytokeratin 18 antibody (magenta), and LC3B (yellow) was stained with anti-LC3B antibody. Nuclei (cyan) were counterstained with DAPI. Scale bar, 40 µm; inserts, 10 µm. (C) Immunofluorescence analysis of KCs isolated from mouse livers. Freshly isolated KCs were plated for 2 h, followed by PBS washing to remove debris and non-adherent cells. Cells were stained with the macrophage marker F4/80 (green). Nuclei (blue) were counterstained with DAPI. Scale bar: 40 µm. (D–G)C. violaceum infection induces mitophagy in HSCs (D), HepG2 cells (E), Huh7 cells (F), and AML12 cells (G). Cells were infected with C.v. WT (MOI = 5) for indicated times, followed by western blot analysis of cell lysates using the specified antibodies. (H and I) Immunoblotting analysis of TUFM (H)/Tom20 (I) in 293T cells infected with C.v. WT and treated with or without Mdivi-1 (20 μM) for 5 h. (J) LC3B levels were assessed by immunoblotting in 293T cells transfected with a vector control or Cpi-1/-1a T3SS effectors. (K) The invasion ability of different C. violaceum strains was evaluated using gentamicin protection assays in HeLa GSDMD−/− cells. C.v. ΔcopD: C. violaceum mutant strain with deletion of the copD gene. (L–N)C.v. ΔcopD infection induces mitophagy. (L) 293T cells expressing GFP-LC3B (cyan) were infected with WT or C.v. ΔcopD and analyzed by fluorescence microscopy. C. violaceum (magenta) was labeled with anti–C. violaceum antibody. Scale bar: 10 µm. (M and N) Cell lysates from infected cells were immunoblotted with anti-LC3B (M) or anti-Tom20 (N). (O and P) Confocal microscopy analysis of mitochondrial morphology in HeLa cells transfected with GFP, GFP-CipB, GFP-SipB, or GFP-IpaB (cyan), respectively. (O) Mitochondria were stained with a ΔΨm-dependent MitoTracker (pseudo-colored magenta). Scale bar, 10 µm. (P) Quantifications of the percentages of cells showing fragmented mitochondria among 100 randomly selected transfection-positive cells. P values were determined by one-way ANOVA with Dunnett’s test. All data are representative of three independent experiments. Source data are available for this figure: SourceData FS1.

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