Fig. 8: MFN2 interacts with Rab7 for the activation of xenophagy.

a Immunoprecipation analysis from RAW264.7 cells transfected with MFN2-RFP or Rab7-GFP. Representative immunoblots for the indicated protein expression. b–d Confocal microscopy images of BMDMs infected with Mtb (MOI 5) and stained with MFN2 (green), Rab7 (red), and LAMP1 (magenta). Representative images for MFN2 co-localization with Rab7 or LAMP1. Scale bars, 8 μm and 0.5 μm (b). c, d Quantitative data of co-localization of MFN2 and Rab7 (c) or LAMP1 (d). Fifty cells in six (c) or ten (d) fields were counted in each group from two different experiments. e BMDMs from Mfn2 WT and Mfn2 CKO mice transduced with a control lentivirus, virus expressing a mouse WT-Rab7 or constitutively active (CA)-Rab7 plasmid for 48 h, were infected with Mtb-ERFP (MOI 5) for 6 h and then stained with LAMP1 and DAPI (for nuclei). Quantitative data of co-localization of Mtb-ERFP and LAMP1 are shown. f, g BMDMs from Mfn2 WT and Mfn2 CKO were infected with Mtb (MOI 5) for 18 h. Representative electron microscopy image of mitochondria (M) and lysosome (L) contact (yellow arrows) (f). Scale bars, 1 μm and 500 nm. Quantitative data of percentage of lysosomes contacting mitochondria (for <15 nm; g). Data are presented as mean ± SEM. Two-tailed Student’s t test is used to calculate the significance (c–e, g). WCL, whole cell lysate; CA, constitutively active; Un, uninfected; M, mitochondria; L, lysosome.