Figure 1. C. violaceum infection induces... | Rockefeller University Press

Figure 1.

$C. violaceum infection induces mitophagy in vivo and in vitro. (A) Immunoblot analysis of LC3B lipidation (LC3B-II) in the livers of C57BL/6N mice (n = 3) intraperitoneally injected with PBS or 12 × 106 CFU C. violaceum WT strain for 10 h. Liver homogenates were immunoblotted with anti-LC3B antibody. C.v. WT, C. violaceum WT strain. The abbreviations and symbols used in this article have consistent meanings throughout. (B) Immunofluorescence analysis of LC3B puncta in the livers of C57BL/6N mice (n = 3). Mice were intraperitoneally injected with PBS or C.v. WT-expressing RFP (magenta) at 12 × 106 CFU for 10 h. Livers were subjected to immunofluorescence by anti-LC3B (cyan). Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. (C) Quantification of LC3B puncta as shown in B. Mean ± SEM, n = 35 cells in each group. (D) The level of Tom20 in the livers of C57BL/6N mice (n = 2) treated as in A. Livers were homogenized and lysed. The level of Tom20 was analyzed by immunoblotting with anti-Tom20 antibody. (E) Immunoblotting of LC3B-II and p62 in 293T cells infected with C.v. WT at a MOI of 5 for indicated time points. (F and G) Immunofluorescence of LC3B puncta in 293T cells or HeLa cells. 293T cells were infected with C.v. WT-expressing RFP (magenta) at a MOI of 5 for 3 h. HeLa cells were infected with C.v. WT-expressing RFP (magenta) at a MOI of 10 for 4 h. LC3B (cyan) was stained with anti-LC3B antibody. Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of LC3B puncta in mock and C.v. WT-infected cells (G). Data are shown as mean ± SEM (n = 25 cells in each group). (H and I) Immunofluorescence of GFP-LC3B puncta in 293T cells stably expressing GFP-LC3B or HeLa cells stably expressing GFP-LC3B. 293T cells stably expressing GFP-LC3B (cyan) were assayed at a MOI of 5 for 3 h. HeLa cells stably expressing GFP-LC3B (cyan) were infected with C.v. WT at a MOI of 10 for 4 h. C. violaceum (magenta) was labeled with anti–C. violaceum antibody. Scale bar, 10 µm. Quantification of GFP-LC3B puncta in mock and C.v. WT-infected cells (I). Data are shown as mean ± SEM (n = 25 cells in each group). (J and K) Immunofluorescence of p62 in HeLa GSDMD−/− cells. Cells were infected with C.v. WT-expressing GFP (cyan) at a MOI of 10 for 5 h. Then p62 (magenta) were stained with anti-p62 antibody. The white dashed line in the figure shows the outline of the cell. Unless otherwise specified, the white dashed lines used in this article have consistent meanings throughout. Scale bar, 10 µm. Quantification of p62 puncta (K). Data are shown as mean ± SEM (n = 35 cells in each group). (L)C.v. WT infection induces mitochondrial localization of LC3B-II. 293T cells were infected with C.v. WT (MOI = 5) for 2 h. As a positive control, Parkin-transfected 293T cells were treated with DMSO or CCCP (10 µM, 2 h). Subcellular fractionation was performed to isolate cytosolic (Cyto) and mitochondrial (Mito) fractions, followed by immunoblotting analysis of LC3B-II. Tubulin and Tom20 served as markers for cytosolic and mitochondrial fractions, respectively. (M and N)C. v. WT infection promotes the fusion of mitophagosome with lysosome. 293T cells were transfected with pmRFP-GFP-Mito for 12 h. Then cells were infected with C.v. WT (MOI = 5) for 5 h in the presence of DMSO or 50 nM bafilomycin A1 and analyzed by immunofluorescence. Scale bar, 10 µm. mRFP-Mito was labeled with magenta. mRFP-Mito was labeled with cyan. C. violaceum (yellow) was labeled with anti–C. violaceum antibody. Quantification of mRFP and mRFP+GFP+ puncta (N). Mean ± SEM, n = 40 cells in each group. P values were determined by two-way ANOVA. (O)C. violaceum infection triggers mitophagy in KCs. Primary KCs were infected with C.v. WT (MOI = 2, 3 h). Whole-cell lysates were subjected to immunoblotting using antibodies against LC3B, p62, and mitochondrial markers (Tom20/HSP60). (P) Immunoblots showing Tom20 and LC3B-II levels in 293T cells treated as in E. (Q and R) Immunoblotting showing levels of Tim23 and HSP60 in 293T cells infected with C.v. WT (MOI = 5) for indicated time. Cells were lysed and immunoblotted with indicated antibodies (Q). Quantification of normalized Tim23 and HSP60 (R). Data are represented as mean ± SEM (n = 3, three independent experiments). P values were determined by one-way ANOVA with Dunnett’s test. (S and T)ATG5 deficiency blocks mitophagy. WT and ATG5−/− MEF cells were infected with C.v. WT (MOI = 5) for 4 h and subsequently subjected to immunoblot using the indicated antibody (S). Quantification of normalized Tim23 and HSP60 (T). Data are represented as mean ± SEM (n = 3, three independent experiments, two-way ANOVA) All data are representative of three independent experiments. Unpaired two-sided Student’s t tests were used to measure significance (C, G, I, and K). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F1.$

C. violaceum infection induces mitophagy in vivo and in vitro. (A) Immunoblot analysis of LC3B lipidation (LC3B-II) in the livers of C57BL/6N mice (n = 3) intraperitoneally injected with PBS or 12 × 10⁶ CFU C. violaceum WT strain for 10 h. Liver homogenates were immunoblotted with anti-LC3B antibody. C.v. WT, C. violaceum WT strain. The abbreviations and symbols used in this article have consistent meanings throughout. (B) Immunofluorescence analysis of LC3B puncta in the livers of C57BL/6N mice (n = 3). Mice were intraperitoneally injected with PBS or C.v. WT-expressing RFP (magenta) at 12 × 10⁶ CFU for 10 h. Livers were subjected to immunofluorescence by anti-LC3B (cyan). Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. (C) Quantification of LC3B puncta as shown in B. Mean ± SEM, n = 35 cells in each group. (D) The level of Tom20 in the livers of C57BL/6N mice (n = 2) treated as in A. Livers were homogenized and lysed. The level of Tom20 was analyzed by immunoblotting with anti-Tom20 antibody. (E) Immunoblotting of LC3B-II and p62 in 293T cells infected with C.v. WT at a MOI of 5 for indicated time points. (F and G) Immunofluorescence of LC3B puncta in 293T cells or HeLa cells. 293T cells were infected with C.v. WT-expressing RFP (magenta) at a MOI of 5 for 3 h. HeLa cells were infected with C.v. WT-expressing RFP (magenta) at a MOI of 10 for 4 h. LC3B (cyan) was stained with anti-LC3B antibody. Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. Quantification of LC3B puncta in mock and C.v. WT-infected cells (G). Data are shown as mean ± SEM (n = 25 cells in each group). (H and I) Immunofluorescence of GFP-LC3B puncta in 293T cells stably expressing GFP-LC3B or HeLa cells stably expressing GFP-LC3B. 293T cells stably expressing GFP-LC3B (cyan) were assayed at a MOI of 5 for 3 h. HeLa cells stably expressing GFP-LC3B (cyan) were infected with C.v. WT at a MOI of 10 for 4 h. C. violaceum (magenta) was labeled with anti–C. violaceum antibody. Scale bar, 10 µm. Quantification of GFP-LC3B puncta in mock and C.v. WT-infected cells (I). Data are shown as mean ± SEM (n = 25 cells in each group). (J and K) Immunofluorescence of p62 in HeLa GSDMD^−/− cells. Cells were infected with C.v. WT-expressing GFP (cyan) at a MOI of 10 for 5 h. Then p62 (magenta) were stained with anti-p62 antibody. The white dashed line in the figure shows the outline of the cell. Unless otherwise specified, the white dashed lines used in this article have consistent meanings throughout. Scale bar, 10 µm. Quantification of p62 puncta (K). Data are shown as mean ± SEM (n = 35 cells in each group). (L)C.v. WT infection induces mitochondrial localization of LC3B-II. 293T cells were infected with C.v. WT (MOI = 5) for 2 h. As a positive control, Parkin-transfected 293T cells were treated with DMSO or CCCP (10 µM, 2 h). Subcellular fractionation was performed to isolate cytosolic (Cyto) and mitochondrial (Mito) fractions, followed by immunoblotting analysis of LC3B-II. Tubulin and Tom20 served as markers for cytosolic and mitochondrial fractions, respectively. (M and N)C. v. WT infection promotes the fusion of mitophagosome with lysosome. 293T cells were transfected with pmRFP-GFP-Mito for 12 h. Then cells were infected with C.v. WT (MOI = 5) for 5 h in the presence of DMSO or 50 nM bafilomycin A1 and analyzed by immunofluorescence. Scale bar, 10 µm. mRFP-Mito was labeled with magenta. mRFP-Mito was labeled with cyan. C. violaceum (yellow) was labeled with anti–C. violaceum antibody. Quantification of mRFP and mRFP⁺GFP⁺ puncta (N). Mean ± SEM, n = 40 cells in each group. P values were determined by two-way ANOVA. (O)C. violaceum infection triggers mitophagy in KCs. Primary KCs were infected with C.v. WT (MOI = 2, 3 h). Whole-cell lysates were subjected to immunoblotting using antibodies against LC3B, p62, and mitochondrial markers (Tom20/HSP60). (P) Immunoblots showing Tom20 and LC3B-II levels in 293T cells treated as in E. (Q and R) Immunoblotting showing levels of Tim23 and HSP60 in 293T cells infected with C.v. WT (MOI = 5) for indicated time. Cells were lysed and immunoblotted with indicated antibodies (Q). Quantification of normalized Tim23 and HSP60 (R). Data are represented as mean ± SEM (n = 3, three independent experiments). P values were determined by one-way ANOVA with Dunnett’s test. (S and T)ATG5 deficiency blocks mitophagy. WT and ATG5^−/− MEF cells were infected with C.v. WT (MOI = 5) for 4 h and subsequently subjected to immunoblot using the indicated antibody (S). Quantification of normalized Tim23 and HSP60 (T). Data are represented as mean ± SEM (n = 3, three independent experiments, two-way ANOVA) All data are representative of three independent experiments. Unpaired two-sided Student’s t tests were used to measure significance (C, G, I, and K). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F1.