Figure 6. CipB activates the... | Rockefeller University Press

Figure 6.

$CipB activates the mitophagy-TFEB axis to suppress immune responses. (A) Volcano plot showing the effect of mitophagy on differential gene expression. log2(fold change); ratio RFP-CipB/RFP-CipB mFXXL(V). Magenta dots indicate downregulated genes in RFP-CipB–transfected cells; black dots show upregulated genes in RFP-CipB–transfected cells. The dotted black line indicates the significance cutoff (*P < 0.05). (B) qRT-PCR analysis of the mRNA levels of immune genes and immunosuppressive genes in 293T cells. Cells were transfected with RFP, RFP-CipB, or RFP-CipB mFXXL(V), and then mRNA was collected and analyzed (n = 3 per group). (C and D)C. violaceum infection induces the activation of TFEB in 293T cells. Cells were infected with C.v. WT (MOI = 5) for 3 h or treated with DMSO/330 nM Torin1 for 4 h. Immunoblotting analysis of TFEB (C). The cell lysate was subjected to nuclear–cytoplasmic fractionation and detected by immunoblotting (D). (E) Western blot images showing levels of TFEB and p-TFEB(S211) in HeLa cells transfected with Flag-BFP and Flag-CipB or treated with DMSO/Torin1. (F and G) Representative immunofluorescence images of endogenous TFEB in HeLa cells transfected with RFP/RFP-CipB (magenta) for 21 h (F). Scale bar, 20 µm. TFEB (cyan) was stained with anti-TFEB antibody. Nuclei (gray) were stained with DAPI. Percentage of cells with TFEB nuclear localization (G). More than 100 cells were quantified per condition in each experiment. Data are presented by unpaired two-sided Student’s t tests (mean ± SEM). (H–J) RavZ inhibits CipB-induced TFEB nuclear translocation. 293T cells transfected with the GFP-tagged (cyan)/RFP-tagged (magenta) plasmid. Cell lysates were analyzed by anti–LC3B-II immunoblotting (H). TFEB (yellow) was stained with anti-TFEB and then subjected to immunofluorescence (I). Nuclei (blue) were stained with DAPI. Scale bar, 20 µm. Quantifications of the percentages of cells showing nuclear TFEB (J). The data are presented as means ± SEM. n = 35 cells. ns, not significant. P values were determined by two-way ANOVA (mean ± SEM). (K and L) RFP-CipB mFXXL(V) inhibits TFEB nuclear translocation. HeLa cells were transfected with RFP, RFP-CipB, or RFP-CipB mFXXL(V) (magenta), and then analyzed by immunofluorescence (K); TFEB (cyan) was stained with anti-TFEB antibody. Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. The white dashed line in the figure shows the outline of nucleus. The percentage of nuclear TFEB-positive cells among transfected cells was calculated (L). Mean ± SEM, n = 35 cells. (M) Western blot validation of TFEB KO effect in HeLa cell lines. Cells were lysed and immunoblotted with TFEB antibody. (N) qRT-PCR testing of the mRNA levels of immune genes in WT or TFEB−/− HeLa cells. Cells were transfected with RFP-CipB and then subjected to qRT-PCR. Data are representative of three biologically independent experiments. Data are presented by unpaired two-sided Student’s t tests (mean ± SEM). (O) qRT-PCR analysis of the transcription of immune genes in WT or TFEB−/− HeLa cells after transfection with RFP-CipB or RFP-CipB mFXXL(V). P values were determined by two-way ANOVA (mean ± SEM, n = 3). Data are representative of three biologically independent experiments. P values were determined by one-way ANOVA with Tukey’s test. (B and L). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F6.$

CipB activates the mitophagy-TFEB axis to suppress immune responses. (A) Volcano plot showing the effect of mitophagy on differential gene expression. log₂(fold change); ratio RFP-CipB/RFP-CipB mFXXL(V). Magenta dots indicate downregulated genes in RFP-CipB–transfected cells; black dots show upregulated genes in RFP-CipB–transfected cells. The dotted black line indicates the significance cutoff (*P < 0.05). (B) qRT-PCR analysis of the mRNA levels of immune genes and immunosuppressive genes in 293T cells. Cells were transfected with RFP, RFP-CipB, or RFP-CipB mFXXL(V), and then mRNA was collected and analyzed (n = 3 per group). (C and D)C. violaceum infection induces the activation of TFEB in 293T cells. Cells were infected with C.v. WT (MOI = 5) for 3 h or treated with DMSO/330 nM Torin1 for 4 h. Immunoblotting analysis of TFEB (C). The cell lysate was subjected to nuclear–cytoplasmic fractionation and detected by immunoblotting (D). (E) Western blot images showing levels of TFEB and p-TFEB(S211) in HeLa cells transfected with Flag-BFP and Flag-CipB or treated with DMSO/Torin1. (F and G) Representative immunofluorescence images of endogenous TFEB in HeLa cells transfected with RFP/RFP-CipB (magenta) for 21 h (F). Scale bar, 20 µm. TFEB (cyan) was stained with anti-TFEB antibody. Nuclei (gray) were stained with DAPI. Percentage of cells with TFEB nuclear localization (G). More than 100 cells were quantified per condition in each experiment. Data are presented by unpaired two-sided Student’s t tests (mean ± SEM). (H–J) RavZ inhibits CipB-induced TFEB nuclear translocation. 293T cells transfected with the GFP-tagged (cyan)/RFP-tagged (magenta) plasmid. Cell lysates were analyzed by anti–LC3B-II immunoblotting (H). TFEB (yellow) was stained with anti-TFEB and then subjected to immunofluorescence (I). Nuclei (blue) were stained with DAPI. Scale bar, 20 µm. Quantifications of the percentages of cells showing nuclear TFEB (J). The data are presented as means ± SEM. n = 35 cells. ns, not significant. P values were determined by two-way ANOVA (mean ± SEM). (K and L) RFP-CipB mFXXL(V) inhibits TFEB nuclear translocation. HeLa cells were transfected with RFP, RFP-CipB, or RFP-CipB mFXXL(V) (magenta), and then analyzed by immunofluorescence (K); TFEB (cyan) was stained with anti-TFEB antibody. Nuclei (gray) were stained with DAPI. Scale bar, 10 µm. The white dashed line in the figure shows the outline of nucleus. The percentage of nuclear TFEB-positive cells among transfected cells was calculated (L). Mean ± SEM, n = 35 cells. (M) Western blot validation of TFEB KO effect in HeLa cell lines. Cells were lysed and immunoblotted with TFEB antibody. (N) qRT-PCR testing of the mRNA levels of immune genes in WT or TFEB^−/− HeLa cells. Cells were transfected with RFP-CipB and then subjected to qRT-PCR. Data are representative of three biologically independent experiments. Data are presented by unpaired two-sided Student’s t tests (mean ± SEM). (O) qRT-PCR analysis of the transcription of immune genes in WT or TFEB^−/− HeLa cells after transfection with RFP-CipB or RFP-CipB mFXXL(V). P values were determined by two-way ANOVA (mean ± SEM, n = 3). Data are representative of three biologically independent experiments. P values were determined by one-way ANOVA with Tukey’s test. (B and L). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F6.