Supplementary Figure 8: ER-tagged NLRX1-dLRR is sufficient to induce ER-phagy.

a, Confocal microscopy analysis of ER marker CLIMP-63 co-localized with Flag-tagged dmt-NLRX1, ret-dmt-NLRX1, ret-dmt-NLRX1-dLRR or ret-dmt-NLRX1-dLRR-dLIR in U2OS cells. Scale bars, 5μm. b, Quantification of the Pearson’s co-localization coefficient between CLIMP-63 and the individual Flag-tag proteins as shown in (a). 20 cells were counted in each group from two different experiments. c, Oligomerization of ret-dmt-NLRX1 or ret-dmt-NLRX1-dLRR overexpressed in 293T cells. d, Western blot analysis of ER marker CLIMP-63 after overexpression of the indicated proteins as in (a). e, Densitometric quantification of CLIMP-63/Actin ratios of (d). (n=3 independent experiments). f, Confocal microscopy analysis of LC3 co-localized with Flag-tag ret-dmt-NLRX1, ret-dmt-NLRX1-dLRR or ret-dmt-NLRX1-dLRR-dLIR in U2OS cells. Representative images are shown. Scale bars, 5μm. g, Quantification of the Pearson’s co-localization coefficient between LC3 and the individual Flag-tag protein as shown in (f). Cells were counted in each group from two different experiments. (n=20 in ret-dmt-NLRX1 or ret-dmt-NLRX1-dLRR-dLIR group; n=30 in ret-dmt-NLRX1-dLRR group). h, The model for L.m-induced mitophagy through a novel mitophagy receptor NLRX1. In quiescent macrophages, NLRX1 localized on mitochondria is maintained in a monomeric autoinhibition state by its LRR associated with NACHT. L.m infection or LLO stimulation decreases mitochondria membrane potential, releases its LRR suppression and induces NLRX1 oligomerization to expose its LIR motif to associate with LC3 for mitophagy induction. Data are representative of two (a-c,f,g) or three (d,e) independent experiments and represent mean ± SEM (b,e,g). Two-sided Student’s t-test was used to measure significance.