( F ) Comparison of proteomic data with RNA - seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples . Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA - seq values of polyribosome associated mRNA with total mRNA ( y axis ) . Green dots represent mRNAs encoding proteins upregulated upon stimulation ( identified in MS experiments ) while orange dots represent mRNAs encoding mitochondrial proteins . Density curves for transcripts encoding mitochondrial proteins ( orange ) and upregulated proteins ( green ) detected by the mass spectrometry are present on the right side of the plot . Please note that both classes represent high abundant mRNAs with good ribosome association values . Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red .

O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

(F) Comparison of proteomic data with RNA-seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples. Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA-seq values of polyribosome associated mRNA with total mRNA (y axis). Green dots represent mRNAs encoding proteins upregulated upon stimulation (identified in MS experiments) while orange dots represent mRNAs encoding mitochondrial proteins. Density curves for transcripts encoding mitochondrial proteins (orange) and upregulated proteins (green) detected by the mass spectrometry are present on the right side of the plot. Please note that both classes represent high abundant mRNAs with good ribosome association values. Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red.


( A ) Respiration activity of permeabilized synaptoneurosomes ( upper panel ) as compared to permeabilized HEK293T ( lower panel ) . Oxygen concentration ( blue line ) and oxygen consumption ( red line ) measured using O2k oxygraph are shown .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(A) Respiration activity of permeabilized synaptoneurosomes (upper panel) as compared to permeabilized HEK293T (lower panel). Oxygen concentration (blue line) and oxygen consumption (red line) measured using O2k oxygraph are shown.


( B ) Blue native gel electrophoresis ( BN - PAGE ) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-CELL_LINE B-SUBCELLULAR O

(B) Blue native gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria.


( C ) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN - PAGE followed by Western blot using antibodies specific for : complex I ( Ndufb8 ) , complex II ( Sdha ) , complex III ( Uqcrc2 ) , complex IV ( Cox6a ) and complex V ( Atp5a ) . Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-CELL_LINE B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O

(C) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN-PAGE followed by Western blot using antibodies specific for: complex I (Ndufb8), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox6a) and complex V (Atp5a). Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria.


( D ) Selected bands ( 1 - 8 ) visible in blue - native gel were excised and analyzed using mass spectrometry ( LC - MS ) . Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands .

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O

(D) Selected bands (1-8) visible in blue-native gel were excised and analyzed using mass spectrometry (LC-MS). Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands.


( A ) Synaptoneurosomes were NMDAR - stimulated and incubated with radioactive 35S - methionine / cysteine mix for 1h . Samples were separated using blue - native electrophoresis ( BN - PAGE ) . Protein complexes were visualized by Coomassie staining ( left ) and autoradiography ( right ) . Autoradiography of the BN - PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes .

O O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

(A) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 1h. Samples were separated using blue-native electrophoresis (BN-PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN-PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.


To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m - chlorophenyl hydrazone ( 10 μM CCCP ) ( B ) Protein complexes were separated on BN - PAGE and visualized by Coomassie staining and autoradiography . 1 hour after the stimulation , significant increase in 35S - labelled proteins was observed ( B , n = 4 biological replicates , * , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in the presence of CCCP ( B ; n = 4 biological replicates , * p = 0 . 02 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test )

O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * , ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test)


To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture ( containing 1 μM valinomycin , 20 μM oligomycin , 8 μM antimycin ) ( C ) . Additionaly , in order to block protein synthesis , SN were pretreated with puromycin ( PURO , 3mM ) ( C ) . Protein complexes were separated on BN - PAGE and visualized by Coomassie staining and autoradiography . 1 hour after the stimulation , significant increase in 35S - labelled proteins was observed C , n = 5 biological replicates , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in the presence of VOA ( C ; n = 5 biological replicates , * * p = 0 . 0044 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Also , significant inhibition of 35S - methionine / cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin ( C , n = 5 biological replicates , * p = 0 . 0236 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Error bars indicate SEM .

O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) (C). Additionaly, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3mM) (C). Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed C, n=5 biological replicates, ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of VOA (C; n=5 biological replicates, ** p=0.0044, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S-methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n=5 biological replicates, * p=0.0236, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( B ) Equal amounts of synaptoneurosomal samples isolated from mouse brains ( Fmr1 KO and wild - type littermates ) were permeabilized by saponin and assayed on MitoPlates for 2 hours . Results are presented as the average rate / min / µg of protein , + / - SEM ( n = 3 per genotype ; two - way ANOVA , post - hoc Sidak ' s multiple comparisons test ; * * * p < 0 . 0001 ) .

O O O O O O B-SUBCELLULAR O O O B-ORGANISM B-TISSUE O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Equal amounts of synaptoneurosomal samples isolated from mouse brains (Fmr1 KO and wild-type littermates) were permeabilized by saponin and assayed on MitoPlates for 2 hours. Results are presented as the average rate/min/µg of protein, +/- SEM (n=3 per genotype; two-way ANOVA, post-hoc Sidak's multiple comparisons test; *** p< 0.0001).


( C ) Mitochondrial O2 consumption rates in SN were measured by high - resolution respirometry in the presence of following respiratory substrates : malate , pyruvate , and glutamate for Complexes I - IV , succinate for Complexes II - IV , glycerol 3 - phosphate for mGpDH - IV and ascorbate and TMPD for complex IV , and normalized to protein content . Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD ( * p = 0 . 0483 and * p = 0 . 0360 respectively , n = 9 per genotype , paired two - tailed t - test ) . Data are presented as a box - and - whiskers graph ( the box extends from 25th to 75th percentiles , central horizontal line is plotted at the median , whiskers show 5th - 95th percentile ) .

O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Mitochondrial O2 consumption rates in SN were measured by high-resolution respirometry in the presence of following respiratory substrates: malate, pyruvate, and glutamate for Complexes I-IV, succinate for Complexes II-IV, glycerol 3-phosphate for mGpDH-IV and ascorbate and TMPD for complex IV, and normalized to protein content. Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD (*p=0.0483 and *p=0.0360 respectively, n=9 per genotype, paired two-tailed t-test). Data are presented as a box-and-whiskers graph (the box extends from 25th to 75th percentiles, central horizontal line is plotted at the median, whiskers show 5th - 95th percentile).


( D ) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild - types . Mitochondria are indicated by arrows ; presynaptic part in blue , postsynaptic part in green . 3 mice per genotype were analyzed . Scale bars , 0 . 5 µm .

O O O O O B-GENEPROD O B-TISSUE O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-ORGANISM O O O O O O O O O O O O O

(D) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild-types. Mitochondria are indicated by arrows; presynaptic part in blue, postsynaptic part in green. 3 mice per genotype were analyzed. Scale bars, 0.5 µm.


( A ) The bar graph represents top 10 biological pathways upregulated in gene ontology ( GO ) based Reactome pathway analysis using a set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald Test ) , 3 biological replicates ) in RNA - seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where the gene expression matrix was transformed into z - score . The heat map was generated from the common genes present in the three GO terms indicated by the three black lines . The numbers on the bars indicate the p - value of that particular GO term .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The bar graph represents top 10 biological pathways upregulated in gene ontology (GO) based Reactome pathway analysis using a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in RNA-seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( D ) The qRT - PCR validation of RNA - seq data in control and IRGM KD HT29 cells .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-CELL_LINE O O

(D) The qRT-PCR validation of RNA- seq data in control and IRGM KD HT29 cells.


( E ) The bar graph represents the top pathways upregulated in GO - based Reactome pathway analysis using set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald Test ) , 3 mice each group ) in the brain of Irgm1 - / - mice compared to Irgm1 + / + wild type mice . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where the gene expression matrix was transformed into z - score . The heat map was generated from the common genes present in the three GO terms indicated by the three black lines . The numbers on the bars indicate the p - value of that particular GO term .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The bar graph represents the top pathways upregulated in GO-based Reactome pathway analysis using set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 mice each group) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( H ) The qRT - PCR validation of RNA - seq data in Irgm1 + / + and Irgm1 - / - mouse brain .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O

(H) The qRT-PCR validation of RNA- seq data in Irgm1+/+ and Irgm1-/- mouse brain.


( A ) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1 - / - mouse brain and BMDMs .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O O O B-ORGANISM B-TISSUE O B-CELL_TYPE O

(A) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1-/- mouse brain and BMDMs.


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( B ) HT29 control ( henceforth IRGM + / + ) and single allele CRISPR knockout IRGM cells ( henceforth IRGM + / - ) ,

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O

Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (B) HT29 control (henceforth IRGM+/+) and single allele CRISPR knockout IRGM cells (henceforth IRGM+/-),


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( C ) HT29 cells stably expressing control shRNA or IRGM shRNA ,

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O

Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (C) HT29 cells stably expressing control shRNA or IRGM shRNA,


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( D ) Irgm1 + / + and Irgm1 - / - mouse brain ( n = 3 mice ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O B-ORGANISM O O

Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (D) Irgm1+/+ and Irgm1-/- mouse brain (n=3 mice).


( E ) Western blot analysis to determine levels of adaptor proteins in control and IRGM + / - HT29 cells .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O

(E) Western blot analysis to determine levels of adaptor proteins in control and IRGM+/- HT29 cells.


( F ) SDD - AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells . Western blot analysis with cytoplasmic fraction was also performed .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O

(F) SDD-AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells. Western blot analysis with cytoplasmic fraction was also performed.


Western blot analysis performed with lysates of ( G ) control and stable IRGM shRNA knockdown HT29 cells ( H ) IRGM + / + and IRGM + / - HT29

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE

Western blot analysis performed with lysates of (G) control and stable IRGM shRNA knockdown HT29 cells (H) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( I ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of TBK1 protein .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD O O

Western blot analysis performed with lysates of (I) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of TBK1 protein.


Western blot analysis performed with lysates of ( J ) IRGM + / + and IRGM + / - HT29

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE

Western blot analysis performed with lysates of (J) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( K - L ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of IRF proteins .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O

Western blot analysis performed with lysates of (K-L) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of IRF proteins.


Western blot analysis performed with lysates of ( M ) IRGM + / + and IRGM + / - HT29

Western blot analysis performed with lysates of (M) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( N ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of STAT proteins ( n = 3 mice ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O

Western blot analysis performed with lysates of (N) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of STAT proteins (n=3 mice).


Western blot analysis performed with lysates of ( O ) IRGM + / + and IRGM + / - HT29 cells . 2 biological replicates are shown .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O

Western blot analysis performed with lysates of (O) IRGM+/+ and IRGM+/- HT29 cells. 2 biological replicates are shown.


Western blot analysis performed with lysates of ( P ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of ISG proteins ( n = 2 mice ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O O O O O B-ORGANISM O O

Western blot analysis performed with lysates of (P) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of ISG proteins (n=2 mice).


( Q ) Representative confocal image of immunofluorescence assay performed with IRGM + / + and IRGM + / - HT29 cells stained with MX1 antibodies . Scale bar , 10 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O

(Q) Representative confocal image of immunofluorescence assay performed with IRGM+/+ and IRGM+/- HT29 cells stained with MX1 antibodies. Scale bar, 10 µm.


( A ) Immunoprecipitation ( IP ) analysis of the interaction between endogenous IRGM and endogenous RIG - I , cGAS , MDA5 , TLR3 , TLR4 , TLR7 , NLRC4 , and AIM2 in THP - 1 cell lysates . IgG ( H ) , IgG heavy chain .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O

(A) Immunoprecipitation (IP) analysis of the interaction between endogenous IRGM and endogenous RIG-I, cGAS, MDA5, TLR3, TLR4, TLR7, NLRC4, and AIM2 in THP-1 cell lysates. IgG (H), IgG heavy chain.


( B - D ) Co - immunoprecipitation ( Co - IP ) analysis of the interaction between ( B ) GFP - IRGM and Flag - RIG - I , ( C ) GFP - IRGM , and Flag - cGAS , ( D ) GFP - IRGM and Flag - TLR3 in HEK293T cell lysates . L . E , Long exposure of input blot . IgG ( L ) , IgG Light chain ; IgG ( H ) , IgG heavy chain .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(B-D) Co-immunoprecipitation (Co-IP) analysis of the interaction between (B) GFP-IRGM and Flag-RIG-I, (C) GFP-IRGM, and Flag-cGAS, (D) GFP-IRGM and Flag-TLR3 in HEK293T cell lysates. L.E, Long exposure of input blot. IgG (L), IgG Light chain; IgG (H), IgG heavy chain.


( E ) GST pull - down assay of in - vitro translated and radiolabelled myc - tagged cGAS , RIG - I , and TLR3 with GST or GST - tagged IRGM .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O

(E) GST pull-down assay of in-vitro translated and radiolabelled myc-tagged cGAS, RIG-I, and TLR3 with GST or GST-tagged IRGM.


( F ) Western blot analysis with the lysates of HT - 29 cells stably transfected with control or Flag - IRGM plasmid and probed with the indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

(F) Western blot analysis with the lysates of HT-29 cells stably transfected with control or Flag-IRGM plasmid and probed with the indicated antibodies.


( G - I ) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP - IRGM and ( G ) Flag - RIG - I or ( H ) Flag - cGAS or ( I ) or Flag - TLR3 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O

(G-I) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP-IRGM and (G) Flag-RIG-I or (H) Flag-cGAS or (I) or Flag-TLR3.


( J - K ) The qRT - PCR analysis with RNA isolated from ( J ) control and Flag - IRGM stable HT - 29 cells ( K ) THP - 1 cells transiently transfected with control or Flag - IRGM plasmid .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O

(J-K) The qRT-PCR analysis with RNA isolated from (J) control and Flag-IRGM stable HT-29 cells (K) THP-1 cells transiently transfected with control or Flag-IRGM plasmid.


Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O

Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated.


( O ) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 ( 300 nM ; 4 h and 8 h ) treated Flag - IRGM expressing HT29 stable cell lines .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O

(O) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 (300 nM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.


( P ) Western blotting analysis with lysates of control and untreated or chloroquine ( 50 µM ; 4 h and 8 h ) treated Flag - IRGM expressing HT29 stable cell lines .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O

(P) Western blotting analysis with lysates of control and untreated or chloroquine (50 µM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.


( Q ) The qRT - PCR analysis with RNA isolated from untreated or Bafilomycin A1 ( 300 nM ; 4 h , 8 h , and 12 h ) treated control or Flag - IRGM expressing HT - 29 stable cells as indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

(Q) The qRT-PCR analysis with RNA isolated from untreated or Bafilomycin A1 (300 nM; 4 h, 8 h, and 12 h) treated control or Flag-IRGM expressing HT-29 stable cells as indicated.


( A ) The cell lysates of control or BECLIN1 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM were subjected to immunoblotting with indicated antibodies .

O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O

(A) The cell lysates of control or BECLIN1 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.


( B ) The cell lysates of control or ATG5 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM were subjected to immunoblotting with indicated antibodies .

(B) The cell lysates of control or ATG5 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.


( C - D ) qRT - PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

(C-D) qRT-PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


qRT - PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

qRT-PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


( G - H ) qRT - PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

(G-H) qRT-PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


Co - IP analysis of the interaction between GFP IRGM with ( I ) Flag - p62 or Flag - NDP52 or Flag - NBR1 in HEK293T cell lysates .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

Co-IP analysis of the interaction between GFP IRGM with (I) Flag-p62 or Flag-NDP52 or Flag-NBR1 in HEK293T cell lysates.


Co - IP analysis of the interaction between GFP IRGM with ( J ) Flag - Optineurin or Flag - TAX1BP1 in HEK293T cell lysates .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

Co-IP analysis of the interaction between GFP IRGM with (J) Flag-Optineurin or Flag-TAX1BP1 in HEK293T cell lysates.


( K - M ) Co - IP analysis of the interaction between GFP IRGM with ( K ) Flag - cGAS , ( L ) Flag - RIG - I , ( M ) Flag - TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O O

(K-M) Co-IP analysis of the interaction between GFP IRGM with (K) Flag-cGAS, (L) Flag-RIG-I, (M) Flag-TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates.


( N ) The cell lysates of control or p62 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM ( as indicate ) were subjected to immunoblotting with indicated antibodies .

O O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O

(N) The cell lysates of control or p62 siRNA transfected THP-1 cells expressing GFP or GFP- IRGM (as indicate) were subjected to immunoblotting with indicated antibodies.


( O ) The qRT - PCR analysis with RNA isolated from control or p62 transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O

(O) The qRT-PCR analysis with RNA isolated from control or p62 transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


( P - R ) Co - IP analysis of the interaction between HA - p62 and ( P ) Flag - RIG - I , ( Q ) Flag - TLR3 , ( R ) Flag - cGAS in the absence and presence of GFP or GFP - IRGM in HEK293T cell lysates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O

(P-R) Co-IP analysis of the interaction between HA-p62 and (P) Flag-RIG-I, (Q) Flag-TLR3, (R) Flag-cGAS in the absence and presence of GFP or GFP-IRGM in HEK293T cell lysates.


( A ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 antibody . Scale bar , 5 µm . Zoom panels are digital magnifications .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 antibody. Scale bar, 5 µm. Zoom panels are digital magnifications.


( B ) The graph depicts the percentage of cells with swollen / rounded mitochondria in Irgm1 + / + and Irgm1 - / - mouse BMDMs .

O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O

(B) The graph depicts the percentage of cells with swollen/rounded mitochondria in Irgm1+/+ and Irgm1-/- mouse BMDMs.


( C ) Representative confocal ( STED ) images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies . Line profile : Co - localization analysis using line intensity profiles .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O

(C) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies. Line profile: Co-localization analysis using line intensity profiles.


( D ) Representative confocal ( STED ) images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies . Line profile : Co - localization analysis using line intensity profiles .

(D) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies. Line profile: Co-localization analysis using line intensity profiles.


( E ) Representative confocal images of control siRNA and IRGM siRNA transfected THP - 1 cells processed for IF analysis with Ubiquitin ( green ) and TOM20 ( red ) antibodies . Scale bar , 10 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O

(E) Representative confocal images of control siRNA and IRGM siRNA transfected THP-1 cells processed for IF analysis with Ubiquitin (green) and TOM20 (red) antibodies. Scale bar, 10 µm.


( F ) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP - 1 cells , probed with indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O

(F) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells, probed with indicated antibodies.


( G ) Western blot analysis of mitochondrial fraction of Irgm1 + / + and Irgm1 - / - mouse BMDM cells , probed with indicated antibodies ( n = 2 mice ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O

(G) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells, probed with indicated antibodies (n=2 mice).


( H ) Western blot analysis of mitochondrial fraction of Irgm1 + / + and Irgm1 - / - mouse BMDM cells untreated or treated with Bafilomycin A1 ( 300 nM , 3 h ) , probed with indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

(H) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.


( I ) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP - 1 cells untreated or treated with Bafilomycin A1 ( 300 nM , 3 h ) , probed with indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

(I) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.


( J ) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1 + / + and Irgm1 - / - mouse BMDM cells . The experiments were performed using the XF24 extracellular flux analyzer , and the flow chart showed the measurement of oxygen consumption rate ( OCR )

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O

(J) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1+/+ and Irgm1-/- mouse BMDM cells. The experiments were performed using the XF24 extracellular flux analyzer, and the flow chart showed the measurement of oxygen consumption rate (OCR)


( K ) Representative confocal images of control and IRGM siRNA transfected THP - 1 cells stained with CMXRos red dye .

(K) Representative confocal images of control and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye.


( L ) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP - 1 cells stained with CMXRos red dye ( 10 nM , 30 mins ) . ( M ) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP - 1 cells stained with CMXRos .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O

(L) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye (10 nM, 30 mins). (M) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP-1 cells stained with CMXRos.


( N ) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP - 1 cells stained with JC - 1 dye ( 2 µM , 30 mins ) . At low mitochondrial membrane potential , JC - 1 is predominantly a monomer that yields green fluorescence , whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(N) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP-1 cells stained with JC-1 dye (2 µM, 30 mins). At low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence, whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission.


( P ) Representative flow cytometry analysis of control and IRGM siRNA transfected THP - 1 cells stained with MitoSox red dye ( 1µM , 20 min ) . The percentage of control and IRGM knockdown cells with increased red fluorescence ( mitochondrial ROS generation ) is depicted .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O

(P) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with MitoSox red dye (1µM, 20 min). The percentage of control and IRGM knockdown cells with increased red fluorescence (mitochondrial ROS generation) is depicted.


( Q ) Representative flow cytometry analysis of control and IRGM siRNA transfected THP - 1 cells stained with CellRox - green and CellRox - orange dye ( 1µM , 30 min ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O

(Q) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with CellRox-green and CellRox-orange dye (1µM, 30 min).


( A ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 ( red ) and dsDNA ( green ) antibody .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 (red) and dsDNA (green) antibody.


( B ) Representative STED microscopy images of control THP - 1 cells processed for IF analysis with cGAS ( green ) , dsDNA ( red ) , and Lamin B1 ( cyan ) antibodies . ( C ) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP - 1 cells .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

(B) Representative STED microscopy images of control THP-1 cells processed for IF analysis with cGAS (green), dsDNA (red), and Lamin B1 (cyan) antibodies. (C) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP-1 cells.


( D - E ) Representative STED microscopy images of Irgm1 - / - mouse BMDMs ( D ) transfected with mCherry - cGAS and immunostained with dsDNA ( green ) antibody . The white line depicts the periphery of the cells . Scale bar , 3 µm . ( E ) The graph depicts the percentage of cells with micronuclei in Irgm1 + / + and Irgm1 - / - mouse BMDMs .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O

(D-E) Representative STED microscopy images of Irgm1-/- mouse BMDMs (D) transfected with mCherry-cGAS and immunostained with dsDNA (green) antibody. The white line depicts the periphery of the cells. Scale bar, 3 µm. (E) The graph depicts the percentage of cells with micronuclei in Irgm1+/+ and Irgm1-/- mouse BMDMs.


( F ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O

(F) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs.


( G ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from control and IRGM siRNA transfected HT - 29 cells .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

(G) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control and IRGM siRNA transfected HT-29 cells.


( H ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT - 29 ( Rho0 ) cells .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

(H) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.


( I ) The qRT - PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT - 29 ( Rho0 ) cells .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

(I) The qRT-PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.


( J ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs transfected with DNase I ( 15 μg , 6hr ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-GENEPROD I-GENEPROD O O O O O O O

(J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr).


( K ) The qRT - PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP - 1 cells electroporated with DNase I ( 5 μg , 1hr ) as indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O

(K) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with DNase I (5 μg, 1hr) as indicated.


( L - M ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with ( L ) dsRNA ( green ) and Tom20 ( red ) antibodies . ( M ) The graph depicts the percentage of Irgm1 + / + and Irgm1 - / - mouse BMDMs with dsRNA aggregates .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O B-SMALL_MOLECULE O O

(L-M) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with (L) dsRNA (green) and Tom20 (red) antibodies. (M) The graph depicts the percentage of Irgm1+/+ and Irgm1-/- mouse BMDMs with dsRNA aggregates.


( N ) Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with RIG - I ( red ) and dsRNA ( green ) antibodies . Line profile : Co - localization analysis using line intensity profiles .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O

(N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles.


Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with ( O ) Mda5 ( red ) and dsRNA ( green ) antibodies Co - localization analysis using line intensity profiles .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O

Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies Co-localization analysis using line intensity profiles.


Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with ( P ) Mda5 ( red ) , dsRNA ( green ) and Tia1 ( blue ) antibodies ( Scale bar , 3 µm ) . Line profile : Co - localization analysis using line intensity profiles .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O

Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles.


( A ) qRT - PCR analysis with total RNA isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG - I siRNA or cGAS siRNA and RIG - I siRNA as indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated.


( B ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG - I siRNA or transfected with three siRNAs as indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated.


( C ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O

(C) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated.


( D ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O

(D) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated.


( E ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated .

(E) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated.


( F ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O

(F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated.


RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h , Ca WT or Ca YL , and Lm WT or Lm Δhly for 60 min . ( A ) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry . Data represent the mean ± SEM of three independent biological experiments . One - way ANOVA followed by Dunnett ' s test compared to WT strains . ns = non - significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O O B-ORGANISM O O O O O B-ORGANISM O O B-ORGANISM O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One-way ANOVA followed by Dunnett's test compared to WT strains. ns= non-significant; *p ≤0.05; **p ≤0.01.


( B ) Recruitment of endogenous LRRK2 and EGFP - Rab8A to M . tuberculosis ( Mtb ) was visualised by confocal microscopy . Scale bar = 5 μm .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(B) Recruitment of endogenous LRRK2 and EGFP-Rab8A to M. tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm.


( C ) C . albicans ( Ca ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative analysis of marker association to the respective pathogen . Data represents the mean ± SEM of two to four independent biological replicates .

O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(C) C. albicans (Ca) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.


( D ) L . monocytogenes ( Lm ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative analysis of marker association to the respective pathogen . Data represents the mean ± SEM of two to four independent biological replicates .

(D) L. monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.


( A ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(A) RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot.


( B ) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot .

O O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(B) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot.


( C ) Macrophages were pre - treated with either 1 μM GSK inh or 0 . 1 μM MLi - 2 and then treated with 1 mM LLOMe for 30 min . Rab8A and Rab8A pT72 levels were analysed by Western blot .

O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(C) Macrophages were pre-treated with either 1 μM GSK inh or 0.1 μM MLi-2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.


( D - F ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ( D ) Endogenous LRRK2 and ( E ) Rab8A recruitment to LAMP - 1 positive compartments was visualised by immunofluorescence . Scale bar = 10 μm . ( F ) Quantification of D - E . Data represents the mean ± SEM of three independent biological experiments .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-F) RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP-1 positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of D-E. Data represents the mean ± SEM of three independent biological experiments.


LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min . The number of LRRK2 per cell were monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . Data represent the mean ± SEM of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s test against the untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of LRRK2 per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min . The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . Data represent the mean ± SEM of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s test against the untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


( A ) RAW264 . 7 macrophages were electroporated with EGFP - Rab8A and treated with 1 mM LLOMe for 30 min . Intracellular distribution of Galectin - 3 , CHMP4B or LC3B was visualised by immunofluorescence . Scale bar = 5 μm .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O

(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.


( B ) Macrophages were electroporated with EGFP - Rab8A and RFP - Galectin - 3 . Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging . Snapshots at the indicated time points after LLOMe addition are shown . Scale bar = 5 μm .

O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.


( C ) RAW264 . 7 macrophages were treated with 1 mM of LLOMe and LRRK2 , Rab8A , CHMP4B , Galectin - 3 and LC3B foci over time was analysed by high - content imaging . Data shows the mean ± SEM of five biological replicates .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates.


RAW264 . 7 macrophages were pre - treated with 10 μM BAPTA - AM for 1 hr , and treated with 1 mM LLOMe for 30 min . ( D ) CHMP4B recruitment was monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm.


( E ) Rab8A pT72 phosphorylation was analysed by Western blot .

O O O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O

(E) Rab8A pT72 phosphorylation was analysed by Western blot.


( F ) LRRK2 and ( G ) Rab8A recruitment was monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . ( H ) Quantification of E and F .

O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (H) Quantification of E and F.


RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show the quantification of number of CHMP4B positive vesicles per cell . Mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . Galectin - 3 vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show the quantification of number of Galectin - 3 positive vesicles per cell . Mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Live cell imaging of LysoTracker positive spots in WT , LRRK2 KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in the LLOMe treatment window were estimated using linear regression .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-GENEPROD O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


Live cell imaging of LysoTracker positive spots in WT , Rab8A KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in the LLOMe treatment window were estimated using linear regression .

Live cell imaging of LysoTracker positive spots in WT, Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) and WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show quantification of number of positive vesicles per cell . Data show the mean ± SEM of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) and WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show quantification of number of positive vesicles per cell . Data show the mean ± SEM of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


( C ) Representative images of LC3B / LAMP - 1 double positive endolysosomes in control and LLOMe treated macrophages . Scale bar = 10 μm . ( D ) Analysis of the % of LC3B / LAMP - 1 double positive endolysosomes in LLOMe treated macrophages . Data shows mean ± SEM of three independent biological experiments . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 by Student ' s t - test .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of LC3B/LAMP-1 double positive endolysosomes in control and LLOMe treated macrophages. Scale bar = 10 μm. (D) Analysis of the % of LC3B/LAMP-1 double positive endolysosomes in LLOMe treated macrophages. Data shows mean ± SEM of three independent biological experiments. *p≤0.05, ***p≤0.001 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Mtb for 24 h , Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb for 24 h, Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Ca for 60 min Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and LRRK2 KO RAW264.7 macrophages were infected with Ca for 60 min Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Lm for 60 min . Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test .

WT and LRRK2 KO RAW264.7 macrophages were infected with Lm for 60 min. Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h . Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and LRRK2 KO RAW264.7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Lm WT or Lm Δhly . Growth measured for 2 h . Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and LRRK2 KO RAW264.7 macrophages were infected with Lm WT or Lm Δhly. Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Monocyte - derived macrophages from healthy controls and PD patients carrying the G2019S ( Donor 2 - 3 ) or R1441C ( Donor 1 ) LRRK2 mutation were pre - treated with 0 . 1 μM MLi - 2 and treated with 1 mM LLOMe for 30 min . ( A ) Rab8A and Rab8A pT72 levels were analysed by Western blot .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O

Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min. (A) Rab8A and Rab8A pT72 levels were analysed by Western blot.


( B ) Rab8A and Galectin - 3 positive vesicle numbers were analysed by immunofluorescence . Data show the mean ± SEM of biological replicates . Scale bar = 20 μm . ns = non - significant , * * p ≤ 0 . 01 , by One - way ANOVA followed by Sidak ' s multiple comparisons test .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.


( A ) Diauxic growth of WT E . coli ( NCM3722 ) in minimal media containing glucose and acetate , bacterial density measured as optical density ( OD600 ) . Growth on glucose is captured by the exponential fit ( blue dashed line ) , and proceeds until glucose runs out ( black dashed line ) at time = 0 h . This is followed by a period of growth - lag lasting ~ 3 . 5h before exponential growth resumes on acetate ( red dashed line ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O

(A) Diauxic growth of WT E. coli (NCM3722) in minimal media containing glucose and acetate, bacterial density measured as optical density (OD600). Growth on glucose is captured by the exponential fit (blue dashed line), and proceeds until glucose runs out (black dashed line) at time = 0 h. This is followed by a period of growth-lag lasting ~3.5h before exponential growth resumes on acetate (red dashed line).


( D ) Lag - times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 ( inset ) . Addition of chlorotetracycline ( cTc ) removes the tetR repression and induces aceBA expression . As the expression of aceB / aceA during the response ( inducer added at time = 0h ) is increased , lag times decrease . Bar plot shows mean lag - times ( N = 3 biological repeats ) for different inducer concentration with error bars denoting the Standard Deviations ( SD ) .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Lag-times for controlled titration of aceBA expression using an inducer construct in strain NQ1350 (inset). Addition of chlorotetracycline (cTc) removes the tetR repression and induces aceBA expression. As the expression of aceB/aceA during the response (inducer added at time = 0h) is increased, lag times decrease. Bar plot shows mean lag-times (N=3 biological repeats) for different inducer concentration with error bars denoting the Standard Deviations (SD).


( A ) A plasmid system ( inset ) is used to control the expression of the non - required gene lacZ using the strain NQ1389 . lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion ( time = 0 h ) using the indicated range of cTc concentrations . Diauxic growth conditions with glucose and acetate , same as in Fig . 1 .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

(A) A plasmid system (inset) is used to control the expression of the non-required gene lacZ using the strain NQ1389. lacZ expression was induced using cTc to varying degrees at the moment of glucose depletion (time = 0 h) using the indicated range of cTc concentrations. Diauxic growth conditions with glucose and acetate, same as in Fig. 1.


( B ) Lag - times fall reciprocally with the allocation towards required proteins ( the fraction of mRNA encoding for required proteins ) during the shift . Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows ( top ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Lag-times fall reciprocally with the allocation towards required proteins (the fraction of mRNA encoding for required proteins) during the shift. Allocation of translation activity towards different proteins are represented by the weight of red and gray arrows (top).


( B ) For the various gene categories in panel A , the temporal kinetics of expression during the diauxic shift is plotted . Time 0 indicates time when glucose runs out . The series of RNA - seq through the growth transition was performed once .

O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(B) For the various gene categories in panel A, the temporal kinetics of expression during the diauxic shift is plotted. Time 0 indicates time when glucose runs out. The series of RNA-seq through the growth transition was performed once.


( C ) Steady - state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh . Growth rate differences between WT and the mutants are not significant in glucose ( p - value > 0 . 05 ) , but are significant for growth in acetate ( p - values < 0 . 02 ) . Additional growth conditions are shown in Fig . EV6 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

(C) Steady-state growth in glucose and acetate for the WT and the motility deletion strains ΔfliC and Δflh. Growth rate differences between WT and the mutants are not significant in glucose (p-value >0.05), but are significant for growth in acetate (p-values<0.02). Additional growth conditions are shown in Fig. EV6.


( D , E ) Growth transitions for the motility deletion strains ΔfliC and ΔflhD are substantially faster than that for the WT strain , with ( D ) showing representative growth kinetics of the three strains and ( E ) showing mean lag - times and standard error of four independent biological replicates . Lag - times for WT are significantly different from that of ∆ fliC ( p - value 0 . 01 ) and ∆ flhD ( p - value 0 . 004 ) .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O

(D, E) Growth transitions for the motility deletion strains ΔfliC and ΔflhD are substantially faster than that for the WT strain, with (D) showing representative growth kinetics of the three strains and (E) showing mean lag-times and standard error of four independent biological replicates. Lag-times for WT are significantly different from that of ∆fliC (p-value 0.01) and ∆flhD (p-value 0.004).


( F ) ∆ fliC shows a 2 - fold increase ( p - value 0 . 17 ) and ∆ flhD shows a 3 - fold increase ( p - value 0 . 02 ) in aceB expression compared to WT , as measured by qPCR .

O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O

(F) ∆fliC shows a 2-fold increase (p-value 0.17) and ∆flhD shows a 3-fold increase (p-value 0.02) in aceB expression compared to WT, as measured by qPCR.


Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT ( black ) and the motility deletion strains ΔfliC and ΔflhD strain ( magenta and green ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diauxic growth kinetics for growth on glucose and a different secondary carbon sources for the WT (black) and the motility deletion strains ΔfliC and ΔflhD strain (magenta and green).


qRT - PCR analysis of Foxp1 - 4 in murine ECs freshly isolated from P11 mesentery ( n = 6 mice , individual data points shown ) . Data are presented as mean relative expression ( normalized to Gapdh ) ± SD . Transcript levels for each transcript are presented relative to levels in LECs . P , Student ' s t - test . nd , not detected .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O

qRT-PCR analysis of Foxp1-4 in murine ECs freshly isolated from P11 mesentery (n = 6 mice, individual data points shown). Data are presented as mean relative expression (normalized to Gapdh) ± SD. Transcript levels for each transcript are presented relative to levels in LECs. P, Student's t-test. nd, not detected.


Immunofluorescence ( C ) and qRT - PCR analysis ( D ) of HDLECs grown under static conditions or exposed to OSS for 48 h ( n = 3 independent experiments ) . Data are presented as mean ± SD . P , Student ' s t - test .

B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence (C) and qRT-PCR analysis (D) of HDLECs grown under static conditions or exposed to OSS for 48 h (n = 3 independent experiments). Data are presented as mean ± SD. P, Student's t-test.


Whole - mount immunofluorescence of P7 mesenteries , showing efficient FOXP2 depletion in Foxp2flox / - ; Tie2 - Cre vessels . Dotted line indicates vessel width . Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox / - ; Tie2 - Cre and littermate controls ( n = vessels [ mice ] , as indicated ) .

O O O B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-TISSUE O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O B-TISSUE I-TISSUE B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE O B-ORGANISM O O O O O O

Whole-mount immunofluorescence of P7 mesenteries, showing efficient FOXP2 depletion in Foxp2flox/-;Tie2-Cre vessels. Dotted line indicates vessel width. Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = vessels [mice], as indicated).


Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox / - ; Tie2 - Cre mice . A spectrum of defects in the mutants ranging from shortened integrin - α9 + FN - EIIIA + valve leaflets ( middle panels ) to a complete loss of leaflets ( right panels ) are shown . Quantification of the proportion of valves with integrin - α9 + leaflets ( G ) , and leaflet length ( H ) in P7 Foxp2flox / - ; Tie2 - Cre and littermate controls ( n = valves [ mice ] , as indicated ) . Leaflet length = 0 corresponds to valves with no leaflets .

B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE O B-ORGANISM O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-TISSUE O O B-SUBCELLULAR O

Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox/-;Tie2-Cre mice. A spectrum of defects in the mutants ranging from shortened integrin-α9+FN-EIIIA+ valve leaflets (middle panels) to a complete loss of leaflets (right panels) are shown. Quantification of the proportion of valves with integrin-α9+ leaflets (G), and leaflet length (H) in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = valves [mice], as indicated). Leaflet length = 0 corresponds to valves with no leaflets.


Whole - mount immunofluorescence ( A ) and quantification of NRP2 staining intensity ( B ) in P6 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre and littermate control mice ( n = vessels [ mice ] , as indicated ) .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-TISSUE O B-ORGANISM O O O O O O

Whole-mount immunofluorescence (A) and quantification of NRP2 staining intensity (B) in P6 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).


RNAscope - based whole - mount in situ hybridization ( C ) and quantification of Efnb2 transcript levels ( D ) in P7 mesenteric lymphatic vessels of Foxp2flox / - ; Tie2 - Cre and littermate control mice ( n = vessels [ mice ] , as indicated ) .

B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-TISSUE O B-ORGANISM O O O O O O

RNAscope-based whole-mount in situ hybridization (C) and quantification of Efnb2 transcript levels (D) in P7 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).


Whole - mount immunofluorescence of P6 mesenteries , showing valve defects in the mutant vessels . Quantification of lymphatic valve numbers ( C ) , valve leaflet length ( D ) and vessel width ( E ) in Foxp2flox / flox ; Prox1 - CreERT2 and littermate control mice ( n = vessels ( C , E ) or valves ( D ) [ mice ] , as indicated ) .

O O O B-EXP_ASSAY O O B-TISSUE O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O O B-TISSUE B-SUBCELLULAR B-EXP_ASSAY O O O O B-TISSUE B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-TISSUE O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O

Whole-mount immunofluorescence of P6 mesenteries, showing valve defects in the mutant vessels. Quantification of lymphatic valve numbers (C), valve leaflet length (D) and vessel width (E) in Foxp2flox/flox;Prox1-CreERT2 and littermate control mice (n = vessels (C, E) or valves (D) [mice], as indicated).


qRT - PCR analysis of FOXP2 , FOXC2 and GJA4 expression in control ( siCTRL ) and FOXP2 or FOXC2 siRNA - treated HDLECs ( n = 6 ( FOXP2 , FOXC2 ) or n = 3 ( GJA4 ) independent experiments ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O

qRT-PCR analysis of FOXP2, FOXC2 and GJA4 expression in control (siCTRL) and FOXP2 or FOXC2 siRNA-treated HDLECs (n = 6 (FOXP2, FOXC2) or n = 3 (GJA4) independent experiments).


Top panels : whole - mount immunofluorescence of E18 Foxc2flox / flox ; Prox1 - CreERT2 and littermate control mesenteries , showing downregulation of FOXP2 in Foxc2 deficient vessels . Bottom panels : extraction of nuclear FOXP2 staining ( grey ) using IMARIS surface mask generated based on PROX1 staining ( green ) . Unmasked FOXP2 staining is shown in magenta . Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox / flox ; Prox1 - CreERT2 and control littermates based on nuclear FOXP2 signals as in ( B ) ( n = vessels [ mice ] , as indicated ) .

O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O B-TISSUE O B-ORGANISM O O O O O O

Top panels: whole-mount immunofluorescence of E18 Foxc2flox/flox;Prox1-CreERT2 and littermate control mesenteries, showing downregulation of FOXP2 in Foxc2 deficient vessels. Bottom panels: extraction of nuclear FOXP2 staining (grey) using IMARIS surface mask generated based on PROX1 staining (green). Unmasked FOXP2 staining is shown in magenta. Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox/flox;Prox1-CreERT2 and control littermates based on nuclear FOXP2 signals as in (B) (n = vessels [mice], as indicated).