Panel A shows a schematic of 293T cells transfected with LINGO4 constructs containing FLAG and 6xHis tags, along with flow cytometry histograms depicting surface and intracellular LINGO4 expression levels. Panel B shows representative immunofluorescence images of FLAG-LINGO4 expressing 293T cells, including DAPI, FLAG signal, and merged images. Panel C presents pathway enrichment analysis of proteins immunoprecipitated using anti-FLAG, highlighting biological processes associated with LINGO4. Panel D shows pathway enrichment analysis of differentially expressed genes from RNA sequencing data. Panel E shows representative transmission electron microscopy images of intestinal lymphoid cells from wild type and Lingo4 knockout mice. Panel F shows flow cytometry histograms and scatter bar plots of Mitotracker Green and Mitotracker Red fluorescence intensity in wild type and Lingo4 knockout ILC3s. Panel G shows flow cytometry plots and scatter bar plots of mitoSOX positive ILC3 frequencies. Panel H shows flow cytometry plots and scatter bar plots of lipid peroxidation measured using BODIPY 581 slash 591 C11, including frequency and mean fluorescence intensity. Panel I shows flow cytometry plots and scatter bar plots of phosphorylated STAT3 positive ILC3s, including frequency and mean fluorescence intensity. Panel J shows flow cytometry plots and scatter bar plots of Annexin V and 7AAD staining indicating apoptosis in ILC3s. Panel K shows flow cytometry plots and scatter bar plots of Ki67 positive proliferating ILC3s.
Lingo4 is required to sustain mitochondrial function in ILC3s. (A) Scheme: 293T cell were stably transfected with Lingo4 having FLAG-tag was fused to its N-terminal part and 6xHis-tag to its C-terminal part. Flow cytometry histograms of surface and intracellular LINGO4 expression on 293T cells by anti-FLAG staining. Representative of three experiments. (B) Representative immunofluorescence image of FLAG-positive 293T-Lingo4 cells. Scale bar: 5 μm. Representative of three experiments. (C) Pathway analysis of proteins immunoprecipitated with anti-FLAG antibody from 293T-Lingo4 cells but not from 293T control cells (n = 100; peptide coverage ≥10%). (D) Pathway analysis of DEGs obtained by RNA-seq analysis of sort purified siLP ILC3 from WT and Lingo4−/− mice. n = 4; padj < 0.01. (E) Representative TEM images of sort purified siLP ILC3 from WT and Lingo4−/− mice. n = 4. Scale bars, 500 nm. (F) Representative flow cytometry plots and MFI of MitoTracker Green and MitoTracker Red staining in WT and Lingo4−/− siLP ILC3 upon ex vivo stimulation. Mean ± SEM, n = 3; representative of two experiments. (G) Representative flow cytometry plots and frequency of mitoSOX+ siLP ILC3s from WT and Lingo4−/− mice. Mean ± SEM, n = 6; two experiments combined. (H) Representative flow cytometry plots, frequency, and MFI of lipids staining by BODIPY 581/591 C11 in WT and Lingo4−/− siLP ILC3s. Mean ± SEM, n = 4; representative of two experiments. (I) Representative flow cytometry plots, frequency, and MFI of pSTAT3+ siLP ILC3. Mean ± SEM, n = 5; representative of two experiments. (J) Representative flow cytometry plots and frequency of Annexin-V and 7AAD staining of siLP ILC3. Mean ± SEM, n = 9; three experiments combined. (K) Representative flow cytometry plots and frequency of Ki67 staining siLP ILC3. Mean ± SEM, n = 4; representative of two experiments. (F–K) Statistical significance was determined using unpaired two-tailed t test or one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.