Figure 6 ( A ) Wild - type cells transformed with a plasmid containing GFP - Atg8 were grown for 4 h in synthetic media with no drug , with UPR - inducing conditions ( + DTT and + TM ) , or under nitrogen starvation conditions ( N starv ) , and then harvested for protein preparation . Protein extracts were analyzed by Western blotting using antibodies against GFP ( top panel ) or Hac1 ( bottom panel ) . Total protein concentration was measured by BCA protein assay . Same concentration of protein was loaded in each lane , and transfer efficiency was checked by Ponceau staining . The identities of the different bands are indicated . ( B ) Wild - type cells expressing GFP - Atg8 grown under the conditions described above were visualized by fluorescence microscopy . ( C ) GFP - Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i ( + DOC ) by Western blotting using antibodies against GFP . ( D ) Western blot using antibodies against GFP of extracts from hac1Δ , ire1Δ , or vps4Δ pep4Δ cells expressing GFP - Atg8 . Mutant cells were grown under regular conditions , UPR - inducing conditions ( + DTT ) , or nitrogen starvation conditions ( N starv ) .

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Figure 6(A) Wild-type cells transformed with a plasmid containing GFP-Atg8 were grown for 4 h in synthetic media with no drug, with UPR-inducing conditions (+DTT and +TM), or under nitrogen starvation conditions (N starv), and then harvested for protein preparation. Protein extracts were analyzed by Western blotting using antibodies against GFP (top panel) or Hac1 (bottom panel). Total protein concentration was measured by BCA protein assay. Same concentration of protein was loaded in each lane, and transfer efficiency was checked by Ponceau staining. The identities of the different bands are indicated.(B) Wild-type cells expressing GFP-Atg8 grown under the conditions described above were visualized by fluorescence microscopy.(C) GFP-Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i (+DOC) by Western blotting using antibodies against GFP.(D) Western blot using antibodies against GFP of extracts from hac1Δ, ire1Δ, or vps4Δ pep4Δ cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).


Figure 7Localization of GFP - Atg8 during UPR InductionSome of the DTT - treated cells shown in Figure 4B expressing GFP - Atg8 and Sec61 - cherry ( as an ER marker ) were visualized using fluorescence microscopy . GFP - Atg8 localizes in close proximity to the ERAs detected by the ER marker .

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Figure 7Localization of GFP-Atg8 during UPR InductionSome of the DTT-treated cells shown in Figure 4B expressing GFP-Atg8 and Sec61-cherry (as an ER marker) were visualized using fluorescence microscopy. GFP-Atg8 localizes in close proximity to the ERAs detected by the ER marker.


Figure 8Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild - type , hac1Δ , atg1Δ , atg8Δ , atg9Δ , atg16Δ , and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich - media plates with no drug ( YPD ) or with different concentrations of tunicamycin ( TM ; 0 . 2 or 1 . 0 μg / ml ) . atg19Δ gave an identical result to the other autophagy genes shown here ( unpublished data ) .

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Figure 8Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild-type, hac1Δ, atg1Δ, atg8Δ, atg9Δ, atg16Δ, and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich-media plates with no drug (YPD) or with different concentrations of tunicamycin (TM; 0.2 or 1.0 μg/ml). atg19Δ gave an identical result to the other autophagy genes shown here (unpublished data).


Figure 1 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.


Figure 2 ( 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 + / - ) , 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 ) . ( 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 . Western blot analysis performed with lysates of ( G ) control and stable IRGM shRNA knockdown HT29 cells ( H ) IRGM + / + and IRGM + / - HT29 cellsWestern 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 cellsWestern 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 cellsWestern 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 . 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 B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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+/-),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).(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.Western blot analysis performed with lysates of (G) control and stable IRGM shRNA knockdown HT29 cells (H) IRGM+/+ and IRGM+/- HT29 cellsWestern 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 cellsWestern 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 cellsWestern 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.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.


Figure 3 ( 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 . ( 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 . ( 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 . Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated . Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated . 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 . ( 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 .

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Figure 3(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.(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.(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.Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated.Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated.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.(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.


Figure 4 ( 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 . ( 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 . 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 . 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 . ( 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 ) 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 .

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Figure 4(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.(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.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.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.(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) 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.


Figure 5 ( 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 . ( 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 . ( 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 . ( 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 . ( 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 ) ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.(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.(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)(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.(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.(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).


Figure 6 ( 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 . ( 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 . ( 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 . ( 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 ) . ( 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 . ( 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 . 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 .

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Figure 6(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.(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.(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.(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).(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.(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.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.


Figure 7 ( 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 . ( 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 . ( 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 .

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Figure 7(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.(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.(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.


Figure 4 Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant , visualized by anti - HA staining ( green ) . Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus , respectively . A novel MS - microscopy approach was used to further define the molecular context of the proteins . This analysis identified possible chromosomal , endosomal and membrane localization of the proteins . The possible endosomal localization is in agreement with the anti - HA immunofluorescence microscopy results . ( key : the scale bar for immunofluorescence images is 10 µm , and the color gradient on the MS - microscopy indicates the localization scores calculated by the MS - microscopy tool . The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control . The error bars designate the standard deviation ( SD )

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

Figure 4 Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant, visualized by anti-HA staining (green). Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus, respectively. A novel MS-microscopy approach was used to further define the molecular context of the proteins. This analysis identified possible chromosomal, endosomal and membrane localization of the proteins. The possible endosomal localization is in agreement with the anti-HA immunofluorescence microscopy results. (key: the scale bar for immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-microscopy tool.  The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control. The error bars designate the standard deviation (SD)


Figure 5 Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization . Both the wild type and the R105W mutant display nuclear , cytoplasmic and plasma membrane localization . However , the MS - microscopy analysis suggests a more prominent endosomal localization with the R105W mutant . ( key : the scale bar for the immunofluorescence images is 10 µm , and the color gradient on the MS - microscopy indicates the localization scores calculated by the MS - Microscopy tool . The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L , whereas the R105W mutant only slightly increased the number of colonies . The error bars designate the standard deviation ( SD )

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

Figure 5 Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization. Both the wild type and the R105W mutant display nuclear, cytoplasmic and plasma membrane localization. However, the MS-microscopy analysis suggests a more prominent endosomal localization with the R105W mutant. (key: the scale bar for the immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-Microscopy tool.  The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L, whereas the R105W mutant only slightly increased the number of colonies. The error bars designate the standard deviation (SD)


Figure 1Examples of CT scan images of several metastases ( M032R1 , M032R2 , and M032R5 ) . Volumetric measurements based on the PET and CT scans could be generated from M032 , M032R1 , and M032R5 . Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment , but regressed upon vemurafenib treatment . M032R5 showed progressive disease after 4 months , whereas M032R1 still displayed stable disease . M032 was excised and no recurrence was observed .

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Figure 1Examples of CT scan images of several metastases (M032R1, M032R2, and M032R5).Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.


Figure 2Hematoxylin - eosin ( H & amp ; amp ; E ) and p - ERK stainings on FFPE material of all metastases showed that all vemurafenib - resistant tumors had reactivation of the MAPK pathway . Scale bar represents 100 μmqPCR was performed on gDNA retrieved from each of the metastases , using primers for BRAF and CRAF and normalized on LINE levels . Bars represent the mean of three replicates , error bars indicate standard deviation . The results confirmed that BRAF was amplified in M032R1 , M032R2 , and M032R5 . Staining for BRAFV600E with a mutant epitope - specific antibody confirmed the upregulation of BRAFV600E in R1 , R2 , and R5 . Scale bar represents 100 μm .

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Figure 2Hematoxylin-eosin (H&amp;E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μmqPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.Staining for BRAFV600E with a mutant epitope-specific antibody confirmed the upregulation of BRAFV600E in R1, R2, and R5. Scale bar represents 100 μm.


Figure 3Immunoblotting for MAPK pathway components confirmed reactivation of p - ERK and p - MEK in all resistant metastases . Immunoblotting for M032 patient ' s samples using an antibody directed against either the mutant BRAFV600E epitope or an N - terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3 , of approximately 80 kDa . This 80 - kDa band could not be detected with the N - terminus - recognizing antibody .

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Figure 3Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases.Immunoblotting for M032 patient's samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.


Figure 4Validation of T55delinsRT was done by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used . Transfection of pQXCIP - GFP , MEK1 - WT , or MEK1 - T55delinsRT into HEK293T cells showed that this mutation induces p - ERK and p - RSK . Validation of expression of pQXCIP - GFP , MEK1WT , or MEK1T55delinsRT in A375 melanoma cells by immunoblotting . Dose - response curves for A375 melanoma cells expressing GFP , MEK1WT , or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib , ERK inhibitor SCH772984 , or MEK inhibitor trametinib . Error bars indicate standard deviation . Colony formation assays with A375 melanoma cells , infected with GFP , MEK1WT , or MEK1T55delinsRT - encoding lentivirus , and treated with indicated doses and inhibitors . Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO ( − ) , 250 nM dabrafenib ( D ) , 10 nM trametinib ( T ) , or a combination ( D + T ) .

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Figure 4Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used.Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.Validation of expression of pQXCIP-GFP, MEK1WT, or MEK1T55delinsRT in A375 melanoma cells by immunoblotting.Dose-response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.Colony formation assays with A375 melanoma cells, infected with GFP, MEK1WT, or MEK1T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).


Figure 5A375 melanoma cells expressing GFP , MEK1WT , or MEK1T55delinsRT were injected into immune - deficient mice ( n = 8 per group ) , and after the tumor size of ˜ 100 mm3 was reached , mice were treated with 30 mg / kg dabrafenib or vehicle . Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment . Error bars indicate standard error of the mean . Average tumor weight of all groups at the end of the experiment described in ( A ) . Error bars indicate standard deviation . Unpaired t - test was used to calculate statistical significance . P - values are indicated in the graph . Immunoblotting for MAPK pathway components performed on tumors of all groups . Immunohistochemistry for Sirius Red ( staining for collagen ) and H & amp ; amp ; E on tumors from all groups , to identify grade of demarcation and local invasion . Scale bar represents 500 ( upper row ) and 100 ( lower row ) μm .

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Figure 5A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.Immunoblotting for MAPK pathway components performed on tumors of all groups.Immunohistochemistry for Sirius Red (staining for collagen) and H&amp;E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.


Figure 6gDNA was isolated from another fragment of the tumor resection , and the PCR with the insertion - specific primers was repeated . Primers amplifying exon 2 of MEK1WT were used as an input control . Source data are available online for this figure .

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Figure 6gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.Source data are available online for this figure.


Figure 7Staining for hematoxylin - eosin ( H & amp ; amp ; E ) and p - ERK on the PDX samples M032 . X1 , M032R1 . X1 , M032R2 . X1 , M032R4 . X1 , and M032R5 . X1 . Stainings showed that p - ERK is higher in the PDX derived from the resistant metastases . Scale bar represents 100 μm . Immunoblotting for components of the MAPK pathway confirmed reactivation of p - ERK in the PDX derived from the vemurafenib - resistant metastases , although the p - ERK signal is heterogeneous in the pre - treatment PDX . Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX , using primers for BRAF and CRAF and normalized on LINE expression . Bars represent the mean of three replicates , error bars indicate standard deviation . The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5 , but not from M032R1 . Presence of the MEK1T55delinsRT was analyzed by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used . The insertion was found in M032R1 . X1 and M032R4 . X1 .

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Figure 7Staining for hematoxylin-eosin (H&amp;E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm.Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1.Presence of the MEK1T55delinsRT was analyzed by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used. The insertion was found in M032R1.X1 and M032R4.X1.


Figure 1ATAC - seq peaks more accessible in GPR56low ( violet ) or GPR56high ( turquoise ) against the background ( grey ) are enriched for enhancers associated with specific hematopoietic cell types . P - values and odds ratios are given for a pairwise , two - sided Fisher ' s Exact test comparing each category ( GPR56low / high ) against the background . Enhancer annotations are taken from EnhancerAtlas 2 . 0 . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 . Knockdown efficiency of two shRNAs against GPR56 ( shGPR56weak and shGPR56strong ) versus shLuc as negative control measured on protein level by flow cytometry in CD34 + CB cells . Shown are representative FACS plots ( left ) and the percentage of GPR56 + cells of transduced GFP + cells on day 5 ( right panel ) . Biological N = 4 , unpaired t - test , bars and error bars represent mean and standard deviation . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 . Integrative Genome Viewer ( IGV ) snapshot showing ATAC - seq peaks along and upstream of the VWF gene in GPR56high vs . low samples ( top , average peak size of 10 GPR56high ( turquoise ) and 15 GPR56low samples ( violet ) ) , RNA - seq reads of the same location in AML samples with high ( n = 9 ) versus low ( n = 11 ) GPR56 expression ( two middle tracks ) , and RNA - seq reads of shLuc versus GPR56 knockdown CD34 + cells ( 3 bottom tracks , one of two replicates shown for each condition ) . Track height was group - scaled . Dashed vertical lines indicate binding sites for the annotated TFs . TFBS : transcription factor binding site derived from the HOCOMOCO v10 database ; TSS : transcription start site . TFs in blue bind to differentially accessible chromatin regions . Volcano plot of differential transcription factor ( TF ) motif accessibility ( activity ) in GPR56high ( turquoise ) vs GPR56low ( violet ) samples and their corresponding adjusted p - values determined with diffTF . Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA - seq dataset .

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Figure 1ATAC-seq peaks more accessible in GPR56low (violet) or GPR56high (turquoise) against the background (grey) are enriched for enhancers associated with specific hematopoietic cell types. P-values and odds ratios are given for a pairwise, two-sided Fisher's Exact test comparing each category (GPR56low / high) against the background. Enhancer annotations are taken from EnhancerAtlas 2.0. *** p<0.0005, ** p<0.005, * p<0.05.Knockdown efficiency of two shRNAs against GPR56 (shGPR56weak and shGPR56strong) versus shLuc as negative control measured on protein level by flow cytometry in CD34+ CB cells. Shown are representative FACS plots (left) and the percentage of GPR56+ cells of transduced GFP+ cells on day 5 (right panel). Biological N=4, unpaired t-test, bars and error bars represent mean and standard deviation. *** p<0.0005, ** p<0.005, * p<0.05.Integrative Genome Viewer (IGV) snapshot showing ATAC-seq peaks along and upstream of the VWF gene in GPR56high vs. low samples (top, average peak size of 10 GPR56high (turquoise) and 15 GPR56low samples (violet)), RNA-seq reads of the same location in AML samples with high (n=9) versus low (n=11) GPR56 expression (two middle tracks), and RNA-seq reads of shLuc versus GPR56 knockdown CD34+ cells (3 bottom tracks, one of two replicates shown for each condition). Track height was group-scaled. Dashed vertical lines indicate binding sites for the annotated TFs. TFBS: transcription factor binding site derived from the HOCOMOCO v10 database; TSS: transcription start site. TFs in blue bind to differentially accessible chromatin regions.Volcano plot of differential transcription factor (TF) motif accessibility (activity) in GPR56high (turquoise) vs GPR56low (violet) samples and their corresponding adjusted p-values determined with diffTF. Highlighted are TFs whose RNA expression was also positively or negatively affected by GPR56 KD in the RNA-seq dataset.


Figure 2Violin plots showing geometric mean ( horizontal line ) and individual values ( circles ) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34 + cells following overnight - infection with shGPR56strong ( turqouise ) and shGPR56weak ( gray ) versus shLuc control ( violet ) . Shown are percentages of indicated populations , which are also co - positive for human CD45 and GFP , relative to all harvested bone marrow cells . HSPC : CD34 + SSClowCD33 - CD19 - , B precursor : CD34 + CD19 + CD33 - , myeloid progenitor ( myelo . prog . ) : CD34 + CD33 + CD19 - , mature B cells : CD19 + CD34 - , mature myeloid cells : CD33 + CD19 - CD34 - . Multiple unpaired t - tests of log - transformed values , p - values were Benjamini and Hochberg corrected ( padj ) , * padj < 0 . 05 , * * padj < 0 . 005 , * * * padj < 0 . 0005 . Adhesion of control and GPR56 KD K562 cells on fibronectin - functionalized substrates . Left : Overlay of phase contrast and microinterferometry images of control ( above ) and GPR56 KD cells ( below ) . The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet , respectively . Right : Comparison of adhesion areas extracted by microinterferometry , reduction of adhesion area by factor 1 . 9 in GPR56 KD cells ( p = 1 . 0 x 10 - 10 , two - sided Mann - Whitney test , box plots showing medians , quartiles , and outliers according to the Tukey method ) . Technical replicates Ncontrol = 71 , Technical replicates NKD = 104 , scale bar 10 µm .

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Figure 2Violin plots showing geometric mean (horizontal line) and individual values (circles) of the engraftment levels of different hematopoietic cell types in the bone marrow of NSG mice 20 weeks after transplantation of CB CD34+ cells following overnight-infection with shGPR56strong (turqouise) and shGPR56weak (gray) versus shLuc control (violet). Shown are percentages of indicated populations, which are also co-positive for human CD45 and GFP, relative to all harvested bone marrow cells. HSPC: CD34+SSClowCD33-CD19-, B precursor: CD34+CD19+CD33-, myeloid progenitor (myelo. prog.): CD34+CD33+CD19-, mature B cells: CD19+CD34-, mature myeloid cells: CD33+CD19-CD34-. Multiple unpaired t-tests of log-transformed values, p-values were Benjamini and Hochberg corrected (padj), * padj<0.05, ** padj<0.005, *** padj<0.0005.Adhesion of control and GPR56 KD K562 cells on fibronectin-functionalized substrates. Left: Overlay of phase contrast and microinterferometry images of control (above) and GPR56 KD cells (below). The area of tight adhesion extracted by microinterferometry is highlighted in turquoise and violet, respectively. Right: Comparison of adhesion areas extracted by microinterferometry, reduction of adhesion area by factor 1.9 in GPR56 KD cells (p = 1.0 x 10-10, two-sided Mann-Whitney test, box plots showing medians, quartiles, and outliers according to the Tukey method). Technical replicates Ncontrol = 71, Technical replicates NKD = 104, scale bar 10 µm.


Figure 3 β - catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a ( representative Western Blot ( above ) and quantification after normalization to GAPDH ( below ) . Shown is fold - change compared to shLuc in control media , bars and error bars represent mean and standard deviation of three biological replicates , unpaired t - test ) . Left : Representative immunofluorescence ( IF ) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc ( above ) or shGPR56strong ( below ) . The non - motile primary cilium is visualized using antibodies against γ - tubulin , which stains the basal body , and the ciliary membrane marker ARL13B . DAPI indicates the nucleus . Images on the right show selected cells at 3 x magnification . Brightness was increased in all images to enhance visibility of cilia . Right : Percentage of RPE - 1 cells with primary cilium in shLuc vs . shGPR56strong RPE - 1 cells at different time points after the start of serum starvation . Unpaired t - test , bars and error bars represent mean and stdev of three biological replicate wells . * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

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Figure 3  β-catenin protein expression in K562 cells infected with shGPR56strong or shLuc negative control in presence and absence of Wnt3a (representative Western Blot (above) and quantification after normalization to GAPDH (below). Shown is fold-change compared to shLuc in control media, bars and error bars represent mean and standard deviation of three biological replicates, unpaired t-test).  Left: Representative immunofluorescence (IF) images showing primary cilium formation in RPE cells upon starvation after infection with shLuc (above) or shGPR56strong (below). The non-motile primary cilium is visualized using antibodies against γ-tubulin, which stains the basal body, and the ciliary membrane marker ARL13B. DAPI indicates the nucleus. Images on the right show selected cells at 3 x magnification. Brightness was increased in all images to enhance visibility of cilia. Right: Percentage of RPE-1 cells with primary cilium in shLuc vs. shGPR56strong RPE-1 cells at different time points after the start of serum starvation. Unpaired t-test, bars and error bars represent mean and stdev of three biological replicate wells. **** p<0.0001, *** p<0.0005, ** p<0.005, * p<0.05.


Figure 1B Relative ( left ) and absolute ( right ) amount of 13C - Lys - labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75 - 125 d ( n = 1 ) , 75 - 175 d ( n = 4 ) , 50 - 100 d ( n = 5 ) , 50 - 125 d ( n = 2 ) , 50 - 150 ( n = 4 ) or 50 - 175 d ( n = 9 ) of age . Error bars show mean ± SD . C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17 - 28 ( 663 . 3 m / z ) and 13C - Lys Aβ17 - 28 ( 666 . 3 m / z ) in an APPtg brain after labeling from 75 - 175 d . % - ion intensity , m / z - mass - to - charge . Data were acquired in data - independent analysis mode ( MSE ) . D Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in an APPtg brain after labeling from 50 - 175 d . The intensities of 4 transitions ( y10 , y9 , y8 , and y7 ) were assessed . Data were acquired by MRM in nanoflow mode . F Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in a non - APPtg brain after labeling from 90 - 100 d . Data were acquired by MRM in microflow mode .

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Figure 1B Relative (left) and absolute (right) amount of 13C-Lys-labeled and unlabeled Aβ in APPtg mouse brains after labeling from 75-125 d (n = 1), 75-175 d (n = 4), 50-100 d (n = 5), 50-125 d (n = 2), 50-150 (n = 4) or 50-175 d (n = 9) of age. Error bars show mean ± SD.C Representative chromatogram and mass spectra of doubly charged ions of unlabeled Aβ17-28 (663.3 m/z) and 13C-Lys Aβ17-28 (666.3 m/z) in an APPtg brain after labeling from 75-175 d. %- ion intensity, m/z- mass-to-charge. Data were acquired in data-independent analysis mode (MSE). D Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in an APPtg brain after labeling from 50-175 d. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed. Data were acquired by MRM in nanoflow mode.F Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in a non-APPtg brain after labeling from 90-100 d. Data were acquired by MRM in microflow mode.


Figure 2A MALDI - TOF mass spectrum of trypsin - digested , synthetic Aβ1 - 42 ( 10 pmol ) . The Aβ17 - 28 [ MH ] + fragment was detected with the highest intensity . Measurements in reflectron positive mode . The sequence of Aβ1 - 42 with the tryptic cleavage sites is indicated at the topB MS / MS spectrum of unlabeled Aβ17 - 28 and 13C15N - Lys Aβ17 - 28 synthetic peptides in the 50 fmol range . m / z of precursors ions ( m / z 663 . 34 for Aβ17 - 28 [ MH ] 2 + , m / z 667 . 36 for 13C15N - Lys Aβ17 - 28 [ MH ] 2 + ) and their derived fragments are indicated as a series of y ions ( y4 - y10 ) . The peptides were analyzed in data independent acquisition MSE modeC Decreasing quantities of synthetic 13C15N - Lys Aβ1 - 42 were trypsin - digested and analyzed by MRM . The intensities of 4 transitions ( y10 , y9 , y8 , and y7 ) were assessed for 13C15N - Lys Aβ17 - 28 , showing a detection limit level of 25 amol . Measurements in technical triplicates , normalization to total ion current . Error bars show mean ± SDD Decreasing quantities of synthetic 13C15N - Lys Aβ1 - 42 were spiked into the insoluble fraction of APPtg brains , immunoprecipitated , trypsin - digested , and analyzed by MRM . Assessment of the intensities of y10 , y9 , y8 and y7 showed a detection limit level of 50 amol for 13C15N - Lys Aβ17 - 28 and detected an excess of brain - derived unlabeled Aβ17 - 28 . Measurements in technical triplicates , normalization to total ion current . Error bars show mean ±

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Figure 2A MALDI-TOF mass spectrum of trypsin-digested, synthetic Aβ1-42 (10 pmol). The Aβ17-28 [MH]+ fragment was detected with the highest intensity. Measurements in reflectron positive mode. The sequence of Aβ1-42 with the tryptic cleavage sites is indicated at the topB MS/MS spectrum of unlabeled Aβ17-28 and 13C15N-Lys Aβ17-28 synthetic peptides in the 50 fmol range. m/z of precursors ions (m/z 663.34 for Aβ17-28 [MH]2+, m/z 667.36 for 13C15N-Lys Aβ17-28 [MH]2+) and their derived fragments are indicated as a series of y ions (y4-y10). The peptides were analyzed in data independent acquisition MSE modeC Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were trypsin-digested and analyzed by MRM. The intensities of 4 transitions (y10, y9, y8, and y7) were assessed for 13C15N-Lys Aβ17-28, showing a detection limit level of 25 amol. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SDD Decreasing quantities of synthetic 13C15N-Lys Aβ1-42 were spiked into the insoluble fraction of APPtg brains, immunoprecipitated, trypsin-digested, and analyzed by MRM. Assessment of the intensities of y10, y9, y8 and y7 showed a detection limit level of 50 amol for 13C15N-Lys Aβ17-28 and detected an excess of brain-derived unlabeled Aβ17-28. Measurements in technical triplicates, normalization to total ion current. Error bars show mean ± SD


Figure 3B Temporal MRM analysis of 13C - Lys Aβ17 - 28 and unlabeled Aβ17 - 28 in the liver , spleen , mesenteric lymph nodes , and peritoneal cells after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice per time point and tissue at 1 - 10 d , except for n = 2 mice for peritoneal cells at 3 d ; n = 4 mice per time point and tissue at 25 - 100 d , except for n = 3 mice for spleen at 100 d ) . Measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and additionally to organ weight ( mg ) for lymph nodes . Error bars show median ± interquartile range . C Temporal 4G8 immunoassay quantification of Aβ42 in the liver , spleen , mesenteric lymph nodes , and peritoneal cells after unlabeled APPtg ( n = 8 mice at 5 h - 7 d , except for n = 5 mice for peritoneal lavage , n = 5 mice at 10 - 14 d , n = 4 mice at 21 d ) or non - tg brain extract injection ( n = 2 mice at 5 h and 3 d , n = 3 mice at 1 d , n = 1 mouse at 5 and 7 d , except for peritoneal cells where n = 1 mouse at 5 h and 3 d , and n = 2 mice at 1 d ) . Error bars show median ± interquartile range . D Normalization of APPtg extract group data presented in ( C ) to organ weight ( mg ) and injected Aβ amount ( range 1 , 027 - 1 , 426 ng ) . Error bars show median ± interquartile range .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B Temporal MRM analysis of 13C-Lys Aβ17-28 and unlabeled Aβ17-28 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice per time point and tissue at 1-10 d, except for n = 2 mice for peritoneal cells at 3 d; n = 4 mice per time point and tissue at 25-100 d, except for n = 3 mice for spleen at 100 d). Measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and additionally to organ weight (mg) for lymph nodes. Error bars show median ± interquartile range.C Temporal 4G8 immunoassay quantification of Aβ42 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after unlabeled APPtg (n = 8 mice at 5 h - 7 d, except for n = 5 mice for peritoneal lavage, n = 5 mice at 10-14 d, n = 4 mice at 21 d) or non-tg brain extract injection (n = 2 mice at 5 h and 3 d, n = 3 mice at 1 d, n = 1 mouse at 5 and 7 d, except for peritoneal cells where n = 1 mouse at 5 h and 3 d, and n = 2 mice at 1 d). Error bars show median ± interquartile range. D Normalization of APPtg extract group data presented in (C) to organ weight (mg) and injected Aβ amount (range 1,027-1,426 ng). Error bars show median ± interquartile range.


Figure 4A Temporal MRM analysis of unlabeled Aβ17 - 28 and 13C - Lys Aβ17 - 28 in the soluble brain fraction after 13C - Lys APPtg brain extract i . p . injection ( n = 3 mice per time point at 1 - 10 d , n = 4 mice per time point at 25 - 100 d ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD . Comparative MRM analysis of 13C - Lys Aβ17 - 28 ( B ) in the insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) and 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 , 50 , 100 d , n = 3 mice in the APPtg extract group and n = 4 in the non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - tailed t - test * p < 0 . 05 ( P = 0 . 0129 at 3 d , P = 0 . 0156 at 10 d , P = 0 . 0109 at 25 d ) , * * p < 0 . 01 ( P = 0 . 0091 at 1 d , P = 0 . 0011 at 5 d ) , * * * p < 0 . 001 ( P = 0 . 0006 at 7 d ) , 13C - Lys APPtg extract - injected vs 13C - Lys non - tg extract - injected for each time point . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD . C Comparative MRM analysis of unlabeled Aβ17 - 28 ( C ) in the insoluble brain fraction after 13C - Lys - APPtg brain extract ( donors labeled for 50 - 125 d ) and 13C - Lys - non - tg brain extract ( donors labeled for 10 d ) i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 , 50 , 100 d , n = 3 mice in the APPtg extract group and n = 4 in the non - tg extract group at 75 d ) , right side zoom for time points 1 - 10 d . Unpaired two - tailed t - test * p < 0 . 05 ( P = 0 . 0129 at 3 d , P = 0 . 0156 at 10 d , P = 0 . 0109 at 25 d ) , * * p < 0 . 01 ( P = 0 . 0091 at 1 d , P = 0 . 0011 at 5 d ) , * * * p < 0 . 001 ( P = 0 . 0006 at 7 d ) , 13C - Lys APPtg extract - injected vs 13C - Lys non - tg extract - injected for each time point . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD . D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C - Lys APPtg or 13C - Lys non - tg brain extract i . p . injection ( n = 3 mice per group and time point at 1 - 10 d , n = 4 mice per group and time point at 25 - 100 d ) , right side zoom for time points 1 - 10 d . Normalization to protein concentration . E Representative chromatograms of unlabeled Aβ17 - 28 ( left ) and 13C - Lys Aβ17 - 28 ( right ) in the brain of an unlabeled 50 - day - old APPtg mouse not injected with labeled brain extract . Note the absence of 13C - Lys Aβ17 - 28 . F MRM analysis of unlabeled and 13C - Lys - labeled Aβ17 - 28 in APPtg mouse brains after 13C - Lys - labeled diet for 1 or 3 d ( n = 3 mice per time point ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD . G Comparative MRM analysis of 13C - Lys Aβ17 - 28 in the insoluble brain fraction after 13C - Lys APPtg and 13C - Lys non - tg brain extract ( all donors labeled for 100 d ) i . p . injection ( n = 6 mice per group ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD . H MRM analysis of unlabeled and 13C - Lys - labeled Aβ17 - 28 in non - APPtg mouse brains after 13C - Lys - labeled diet for 1 d ( n = 3 mice ) or at 1 d after 13C - Lys - APPtg brain extract i . p . injection ( n = 3 mice ) . Data information : measurements in technical triplicates , normalization to 13C15N - Lys Aβ17 - 28 intensity as a spiked internal standard and brain weight ( mg ) . All error bars show mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Temporal MRM analysis of unlabeled Aβ17-28 and 13C-Lys Aβ17-28 in the soluble brain fraction after 13C-Lys APPtg brain extract i.p. injection (n = 3 mice per time point at 1-10 d, n = 4 mice per time point at 25-100 d). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.Comparative MRM analysis of 13C-Lys Aβ17-28 (B) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.C Comparative MRM analysis of unlabeled Aβ17-28 (C) in the insoluble brain fraction after 13C-Lys-APPtg brain extract (donors labeled for 50-125 d) and 13C-Lys-non-tg brain extract (donors labeled for 10 d) i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25, 50, 100 d, n = 3 mice in the APPtg extract group and n = 4 in the non-tg extract group at 75 d), right side zoom for time points 1-10 d. Unpaired two-tailed t-test * p<0.05 (P=0.0129 at 3 d, P=0.0156 at 10 d, P=0.0109 at 25 d), ** p<0.01 (P=0.0091 at 1 d, P=0.0011 at 5 d), *** p<0.001 (P=0.0006 at 7 d), 13C-Lys APPtg extract-injected vs 13C-Lys non-tg extract-injected for each time point. Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.D Comparative 4G8 immunoassay quantification of Aβ42 in the insoluble brain fraction after 13C-Lys APPtg or 13C-Lys non-tg brain extract i.p. injection (n = 3 mice per group and time point at 1-10 d, n = 4 mice per group and time point at 25-100 d), right side zoom for time points 1-10 d. Normalization to protein concentration.E Representative chromatograms of unlabeled Aβ17-28 (left) and 13C-Lys Aβ17-28 (right) in the brain of an unlabeled 50-day-old APPtg mouse not injected with labeled brain extract. Note the absence of 13C-Lys Aβ17-28.F MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in APPtg mouse brains after 13C-Lys-labeled diet for 1 or 3 d (n = 3 mice per time point). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.G Comparative MRM analysis of 13C-Lys Aβ17-28 in the insoluble brain fraction after 13C-Lys APPtg and 13C-Lys non-tg brain extract (all donors labeled for 100 d) i.p. injection (n = 6 mice per group). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.H MRM analysis of unlabeled and 13C-Lys-labeled Aβ17-28 in non-APPtg mouse brains after 13C-Lys-labeled diet for 1 d (n = 3 mice) or at 1 d after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice). Data information: measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and brain weight (mg). All error bars show mean ± SD.


Figure 5 ( A ) Representative 5 - day - old seedlings of wild - type Arabidopsis thaliana Col - 0 and TF mutants gata12 and wri3 grown in the dark . Scale bar , 1 mm . ( B ) Heat map indicates which TFs significantly affects hypocotyl length , root length and the composite trait of root to total length in dark - grown seedlings and whether the effects of TFs are dependent on condition . TFs are hierarchically clustered using Euclidean distance . ( C ) Heat map of the average relative effects of TF mutant alleles on hypocotyl length , root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle - dependent growth . Mutant alleles are listed in rows and traits in columns . Cells of TF mutant alleles in heat map are colored if the AGI or AGI : TCA Metabolite linear model terms for each trait are statistically significant ( P < 0 . 05 , two - way ANOVA , 16 - 20 seedlings per genotype per condition across two experiments per genotype ) . Mutant alleles are hierarchically clustered using Euclidean distance .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative 5-day-old seedlings of wild-type Arabidopsis thaliana Col-0 and TF mutants gata12 and wri3 grown in the dark. Scale bar, 1 mm.(B) Heat map indicates which TFs significantly affects hypocotyl length, root length and the composite trait of root to total length in dark-grown seedlings and whether the effects of TFs are dependent on condition. TFs are hierarchically clustered using Euclidean distance.(C) Heat map of the average relative effects of TF mutant alleles on hypocotyl length, root length and the ratio of root to total length reveal that TF lesions significantly perturbed TCA cycle-dependent growth. Mutant alleles are listed in rows and traits in columns. Cells of TF mutant alleles in heat map are colored if the AGI or AGI:TCA Metabolite linear model terms for each trait are statistically significant (P < 0.05, two-way ANOVA, 16-20 seedlings per genotype per condition across two experiments per genotype). Mutant alleles are hierarchically clustered using Euclidean distance.


Figure 6 ( A ) Heat map indicates which TFs significantly affects rosette area , growth rate , shoot biomass , flowering time and seed yield and whether the effects of TFs are dependent on salt treatment . TFs are hierarchically clustered using Euclidean distance . ( C ) Rosette area of mutant allele of BPC4 from day 7 to day 21 post - germination . Linear model for two - way ANOVA considers AGI , salt treatment , day post - germination and their interactions . The AGI and AGI : Salt Condition terms are statistically signification ( P < 0 . 001 , P < 0 . 01 , respectively ) . Heat map under line plot indicates which term in linear model is statistically significant ( P < 0 . 05 ) using two - way ANOVA for each day from 9 biological replicates per condition . Solid lines , Col - 0 ; dashed line , bpc4 ; circle , control condition ; triangle , 50 mM NaCl .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Heat map indicates which TFs significantly affects rosette area, growth rate, shoot biomass, flowering time and seed yield and whether the effects of TFs are dependent on salt treatment. TFs are hierarchically clustered using Euclidean distance.(C) Rosette area of mutant allele of BPC4 from day 7 to day 21 post-germination. Linear model for two-way ANOVA considers AGI, salt treatment, day post-germination and their interactions. The AGI and AGI:Salt Condition terms are statistically signification (P < 0.001, P < 0.01, respectively). Heat map under line plot indicates which term in linear model is statistically significant (P < 0.05) using two-way ANOVA for each day from 9 biological replicates per condition. Solid lines, Col-0; dashed line, bpc4; circle, control condition; triangle, 50 mM NaCl.


Figure 1C ) Western Blot showing the levels of the PgP and control ( α - TUBB ) in RPE - 1 parental and Taxol resistant clones . E ) Representative smRNA - FISH images of RPE - 1 Parental and clones for the ABCB1 gene and DAPI . The images are projections of 0 , 5μm sections and a total 5 μm in thickness . Scale bar , 15μm .

O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C) Western Blot showing the levels of the PgP and control (α-TUBB) in RPE-1 parental and Taxol resistant clones.E) Representative smRNA-FISH images of RPE-1 Parental and clones for the ABCB1 gene and DAPI. The images are projections of 0,5μm sections and a total 5 μm in thickness. Scale bar, 15μm.


Figure 2A ) TLA analysis for ABCB1 contacts in RPE - 1 parental and Taxol resistant clone sg6C9 covering the whole genome . Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6 . B ) TLA analysis for RPE - 1 Parental and sg6C9 . Zoom in in the region of chr6 with de novo interaction for sg6C9 . Image modified from IGV viewer . D ) PCR products using the primers in C ( 1 ) over the ABCB1 and EEF1A1 regions in RPE - 1 Parental and the different Taxol resistant clones . E ) PCR products using the primers in C ( 2 ) over the specific break site in the ABCB1 promoter in RPE - 1 Parental and the different Taxol resistant clones . G ) Percentage of RPE - 1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration . Percentages were calculated from data in Fig 1A . Each dot represents a CFA replicate experiment . Horizontal bars represent the mean of lentiviral integratation per sgRNA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6. B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer.D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones. E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones.G) Percentage of RPE-1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration. Percentages were calculated from data in Fig 1A. Each dot represents a CFA replicate experiment. Horizontal bars represent the mean of lentiviral integratation per sgRNA.


Figure 3A ) H3K9me3 ChIP - sequencing tracks from the q21 . 12 arm of chromosome 7 in RPE - 1 cells ( Parental and Taxol - resistant clones ) . The ABCB1 gene region is shown in pink . Each colored track shows the H3k9me3 profile of a different Taxol - resistant clone . B ) H3K9me3 ChIP - qPCR in the ABCB1 promoter region . Bar graph show primer pairs amplifying the sgRNA # 6 break and spanning this region ( + / - base pairs ) for each Taxol - resistant clone compared to the parental . Bar plots show the mean of H3K9me3 relative enrichment . Each dot represents a technical replicate ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3).