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. 2023 Oct 23;8(20):e166242. doi: 10.1172/jci.insight.166242

Figure 4. Elevated autophagic flux and targeting of intracellular M. tuberculosis in Mda5-deficient, IFN-γ–primed macrophages.

Figure 4

(A–F) IFN-γ–primed Mda5-KO and NTC J774.1 cells were infected with M. tuberculosis expressing GFP on an episomal plasmid (MOI of 1:10) for 6 hours. (A) Fluorescence confocal images of fixed cells immunostained for LC3 (Alexa Fluor 647, far-red) or nuclei (Hoechst 33342, blue). (B and C) Quantification of LC3 puncta per nucleus (B) and M. tuberculosis colocalization with LC3B (C). (D) Fluorescence confocal images of fixed cells immunostained for p62 (Alexa Fluor 647, far-red) or nuclei (Hoechst 33342, blue). (E and F) Quantification of p62 puncta per nucleus (E) and M. tuberculosis colocalization with p62 (F). Scale bars: 20 μm for ×63 images. Perinuclear LC3 and p62 puncta were counted in a minimum of 200 cells across different fields. Image processing and analysis were performed using Fiji and Imaris 9.7 software. Data are mean ± SD (n = 3 biological replicates from 2 independent experiments). (G) LC3-I, LC3-II, and β-actin protein levels in IFN-γ–primed Mda5-KO and NTC J774.1 cells infected with M. tuberculosis (MOI of 1:5) for 24 hours detected by immunoblot analysis. Densitometric quantification of the LC3-II/β-actin ratio presented as the mean ± SD (n = 4 biological replicates from 4 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Tukey’s posttest.