Figure 7A . Cells derived from tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( # 4953 ) and Alk - F1178S ; Th - MYCN ( # 4938 ) mice are sensitive to both lorlatinib and brigatinib . The effect of increasing concentrations of each ALK TKI ( as indicated ) on cell confluence was analyzed by IncuCyte Live Cell Analysis . Data is presented as mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - tailed paired student t - test . B . Tumour volume changes over time for Rosa26 _ Alkal2 ; Th - MYCN mice treated with lorlatinib ( 10 mg / kg ; twice daily ) or vehicle control . Tumour volume was measured by ultrasound on Day 0 and 7 , and by direct measurement at Day 14 . Day 0 ( lorlatinib n = 7 , Ctrl n = 6 ) , Day 7 ( lorlatinib n = 2 , Ctrl n = 5 ) and Day 14 ( lorlatinib n = 7 , Ctrl n = 6 ) . Data shown represents mean ± SD . * * p < 0 . 005 ; two - tailed unpaired student t - test . C . Rosa26 _ Alkal2 ; Th - MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls . Data shown represents mean ± SD . D . Representative ultrasound images of tumours observed in Rosa26 _ Alkal2 ; Th - MYCN mice with annotated measurements at Day 0 and Day 7 . Tumours arise in the retroperitoneal space ventral to the aorta , Ao . E . Representative MRI imaging of Rosa26 _ Alkal2 ; Th - MYCN tumours in response to lorlatinib at 4d and 14d . F . Rosa26 _ Alkal2 ; Th - MYCN tumours from lorlatinib or vehicle controls were analysed for pHis . A representative field of view for each tumour at 40x ( 175740μm2 ) were manually counted . Data shown represents mean ± 95 % CI . p = 0 . 0286 ; Mann - Whitney test .

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Figure 7A. Cells derived from tumours arising in Rosa26_Alkal2;Th-MYCN (#4953) and Alk-F1178S;Th-MYCN (#4938) mice are sensitive to both lorlatinib and brigatinib. The effect of increasing concentrations of each ALK TKI (as indicated) on cell confluence was analyzed by IncuCyte Live Cell Analysis. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.B. Tumour volume changes over time for Rosa26_Alkal2;Th-MYCN mice treated with lorlatinib (10 mg/kg; twice daily) or vehicle control. Tumour volume was measured by ultrasound on Day 0 and 7, and by direct measurement at Day 14. Day 0 (lorlatinib n=7, Ctrl n=6), Day 7 (lorlatinib n=2, Ctrl n=5) and Day 14 (lorlatinib n=7, Ctrl n=6). Data shown represents mean ± SD. **p<0.005; two-tailed unpaired student t-test.C. Rosa26_Alkal2;Th-MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls. Data shown represents mean ± SD.D. Representative ultrasound images of tumours observed in Rosa26_Alkal2;Th-MYCN mice with annotated measurements at Day 0 and Day 7. Tumours arise in the retroperitoneal space ventral to the aorta, Ao.E. Representative MRI imaging of Rosa26_Alkal2;Th-MYCN tumours in response to lorlatinib at 4d and 14d.F. Rosa26_Alkal2;Th-MYCN tumours from lorlatinib or vehicle controls were analysed for pHis. A representative field of view for each tumour at 40x (175740μm2) were manually counted. Data shown represents mean ± 95% CI. p=0.0286; Mann-Whitney test.


Figure 1Schematic diagram of ATXN8OS ( top strand ) and ATXN8 ( bottom strand ) . RNA - seq read coverage visualized by Integrative Genomic Viewer across the ATXN8 / ATXN8OS locus is depicted . B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS . FPKM values for each transcript are normalized to FPKM value for ATXN8OS . ( * * * * ; p < 0 . 0001 ; mean ± SEM ; unpaired t test ) FPKM = fragments per kilo base of transcript per million mapped reads . D Immunohistochemistry of 10 - 12 month old end - stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein ( detected by unique antibody to the polySer protein C - terminus , α - SerCT ) in cerebellar white matter , brainstem , hippocampus and layer II of the frontal cortex . Representative polySer aggregates are indicated by black arrows . No aggregates are found in age matched non - transgenic ( NT ) littermates ( n = 6 for each cohort ) . F Immunohistochemistry of human SCA8 autopsy tissue ( n = 4 - 7 ) shows accumulation of novel polySer RAN protein in cerebellar white matter , brainstem and frontal cortex .

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 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 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

Figure 1Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand). RNA-seq read coverage visualized by Integrative Genomic Viewer across the ATXN8/ATXN8OS locus is depicted.B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS. FPKM values for each transcript are normalized to FPKM value for ATXN8OS. (****; p<0.0001; mean ± SEM; unpaired t test) FPKM= fragments per kilo base of transcript per million mapped reads.D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort).F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.


Figure 2A Immunohistochemistry ( IHC ) of end - stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells . Representative polySer aggregates are indicated by black arrows . Inset : higher magnification of molecular layer and white matter . B IF double staining shows no co - localization of polyGln and polySer in frontal cortex , pons or hippocampus of end - stage SCA8 BAC mice . C IF double staining of end - stage SCA8 BAC frontal cortex shows exclusive localization of polyGln ( mouse α - Gln , red , bottom panel ) in neurons ( rabbit α - NeuN , green , bottom panel ) . In contrast , polySer ( Rabbit α - SerCT , red , top panel ) shows widespread accumulation in the frontal cortex including within neurons ( mouse α - NeuN , green , top panel ) . D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells ( left panels ) while polyGln accumulates in Purkinje cells but not cerebellar white matter ( right panels ) .

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

Figure 2A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells. Representative polySer aggregates are indicated by black arrows. Inset: higher magnification of molecular layer and white matter.B IF double staining shows no co-localization of polyGln and polySer in frontal cortex, pons or hippocampus of end-stage SCA8 BAC mice.C IF double staining of end-stage SCA8 BAC frontal cortex shows exclusive localization of polyGln (mouse α-Gln, red, bottom panel) in neurons (rabbit α-NeuN, green, bottom panel). In contrast, polySer (Rabbit α-SerCT, red, top panel) shows widespread accumulation in the frontal cortex including within neurons (mouse α-NeuN, green, top panel).D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).


Figure 3A Representative images of the vestibular nucleus ( upper panels ) , cuneate nucleus ( middle panels ) and motor cortex layers II / III ( lower panels ) of SCA8 BAC mice at 2 months ( left panels ) , 6 months ( middle panels ) and end - stage ( 10 months , right panels ) stained with α - SerCT . Representative aggregates are indicated by black arrows . Corresponding quantifications of polySer aggregates for each region on the right ( n = 3 ; mean ± SEM ; One - way ANOVA with Tukey ' s post - hoc test ; * p < 0 . 025 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 ) B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance ( cm ) and ambulatory time ( s ) and a significant increase in resting time ( s ) ( NT n = 19 , SCA8 BAC n = 23 , * * p < 0 . 01 , * * * p < 0 . 001 ; mean ± SEM ; unpaired t test ) .

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 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

Figure 3A Representative images of the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT. Representative aggregates are indicated by black arrows. Corresponding quantifications of polySer aggregates for each region on the right (n=3; mean ± SEM; One-way ANOVA with Tukey's post-hoc test; * p<0.025, ** p<0.005, *** p<0.0005, **** p<0.0001)B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance (cm) and ambulatory time (s) and a significant increase in resting time (s) (NT n=19, SCA8 BAC n=23, ** p<0.01, *** p<0.001; mean ± SEM; unpaired t test).


Figure 4A Disease - specific sensitivity to vacuolization after prolonged storage in ethanol shown by H & E in cerebellar white matter and brainstem of 10 - 12 month old end - stage SCA8 BAC mice but not in age matched NT ( n = 3 ) ( panels 1 & 2 ) . Demyelination shown by luxol fast blue staining ( LFB ) ( panel 3 ) and axonal degeneration shown by α - SMI - 32 ( panel 4 ; degenerated axons are indicated by black arrows ) observed in sites of polySer accumulation shown by α - SerCT ( panel 5 ; representative aggregates are indicated by black arrows ) in deep cerebellar white matter and brainstem in SCA8 BAC mice ( lower panels ) but not in NT mice ( upper panels ) ( n = 3 ) . Demyelination shown by luxol fast blue staining ( LFB ) ( panel 1 ) and axonal degeneration shown by α - SMI - 32 ( panel 2 ) observed sites of polySer accumulation shown by a - SerCT2 ( panel 3 ) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains ( n = 3 ) . C Immunohistochemistry using CC1 ( α - APC ) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice ( NT n = 5 , SCA8 BAC n = 5 ; * * * * p < 0 . 0001 ; mean ± SEM ; unpaired t test ) . D Immunofluorescence using α - GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice ( NT n = 3 , SCA8 BAC n = 3 , * * p < 0 . 01 ; mean ± SEM ; unpaired t test ) . E Cell death measured by lactase dehydrogenase ( LDH ) assay in T98 cells transfected with codon replacement constructs expressing polyGln ( light blue ) or polySer ( dark blue ) . NT = nontransfected , EV = empty vector ( n = 5 , * * p < 0 . 003 ; * * * p < 0 . 0003 ; mean ± SEM ; unpaired t test ) .

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 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 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 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 O O O O O O O O O O O O O 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 Disease-specific sensitivity to vacuolization after prolonged storage in ethanol shown by H&E in cerebellar white matter and brainstem of 10-12 month old end-stage SCA8 BAC mice but not in age matched NT (n=3) (panels 1&2). Demyelination shown by luxol fast blue staining (LFB) (panel 3) and axonal degeneration shown by α-SMI-32 (panel 4; degenerated axons are indicated by black arrows) observed in sites of polySer accumulation shown by α-SerCT (panel 5; representative aggregates are indicated by black arrows) in deep cerebellar white matter and brainstem in SCA8 BAC mice (lower panels) but not in NT mice (upper panels) (n=3).Demyelination shown by luxol fast blue staining (LFB) (panel 1) and axonal degeneration shown by α-SMI-32 (panel 2) observed sites of polySer accumulation shown by a-SerCT2 (panel 3) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains (n=3).C Immunohistochemistry using CC1 (α- APC) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice (NT n=5, SCA8 BAC n=5; **** p<0.0001; mean ± SEM; unpaired t test).D Immunofluorescence using α-GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice (NT n=3, SCA8 BAC n=3, ** p<0.01; mean ± SEM; unpaired t test).E Cell death measured by lactase dehydrogenase (LDH) assay in T98 cells transfected with codon replacement constructs expressing polyGln (light blue) or polySer (dark blue). NT=nontransfected, EV=empty vector (n=5, ** p<0.003; *** p< 0.0003; mean ± SEM; unpaired t test).


Figure 5qRT - PCR showing Eif3f expression levels are increased 2 - fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter ( n = 3 , * * p < 0 . 01 ; mean ± SEM ; unpaired t test ) . C Dot blot detection of polySer expressionin using α - FLAG antibody showing decrease in RAN polySer but not ATG - polySer when HEK293 cells are co - transfected with eIF3F siRNA . D Quantification of polySer detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± SEM ; unpaired t test ) . E Detection of polyAla expression using α - HA antibody showing decrease in RAN polyAla but not ATG - polyAla when HEK293 cells are co - transfected with eIF3F siRNA . F Detection of polyGP expression using α - FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co - transfected with eIF3F siRNA . . G Quantification of polyAla and polyGP detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± SEM ; unpaired t test ) .

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Figure 5qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.01; mean ± SEM; unpaired t test).C Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA. D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test). E Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA. F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA.. G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).


Figure 1 ( B ) Volcano plot of 8 , 701 mRNA isoforms from 2 , 931 genes with multiple isoforms . X - axis indicates a difference in percentage of each mRNA isoform within the gene ( Δ % smg - 2 - N2 ) . Y - axis indicates - log10 of p - value in Fisher ' s exact tests . Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg - 2 mutant ( p < 0 . 05 , Δ % smg - 2 - N2 < - 10 ) . Red symbols indicate 420 isoforms significantly enriched in the smg - 2 mutant ( p < 0 . 05 , Δ % smg - 2 - N2 > 10 ) . ( C , D ) Length distribution of 3 ' UTRs ( C ) and proportions of putative PTC - containing isoforms ( D ) in 10 , 136 mRNA isoforms detected in N2 and / or smg - 2 ( All ) , the 219 isoforms depleted from and the 420 isoforms enriched in the smg - 2 mutant .

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 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

Figure 1(B) Volcano plot of 8,701 mRNA isoforms from 2,931 genes with multiple isoforms. X-axis indicates a difference in percentage of each mRNA isoform within the gene (Δ%smg-2-N2). Y-axis indicates -log10 of p-value in Fisher's exact tests. Blue symbols indicate 219 mRNA isoforms significantly depleted from the smg-2 mutant (p<0.05, Δ%smg-2-N2<-10). Red symbols indicate 420 isoforms significantly enriched in the smg-2 mutant (p<0.05, Δ%smg-2-N2>10).(C, D) Length distribution of 3'UTRs (C) and proportions of putative PTC-containing isoforms (D) in 10,136 mRNA isoforms detected in N2 and/or smg-2 (All), the 219 isoforms depleted from and the 420 isoforms enriched in the smg-2 mutant.


Figure 2 ( B ) Alternative splicing of sams - 3 , sams - 4 and sams - 5 in unfed and fed worms without or with a protein synthesis inhibitor emetine . Synchronized L1 larvae of an NMD - deficient strain KH1668 : smg - 2 ( yb979 ) ( lanes 1 - 3 ) and a wild - type strain N2 ( lanes 4 - 6 ) were incubated in S - complete medium alone ( lanes 1 , 4 ) , with a standard E . coli strain OP50 ( OD600 = 10 . 0 ) ( lanes 2 , 5 ) or OP50 supplemented with 10 mg / ml emetine ( lanes 3 , 6 ) for 3 hours at 20 ° C . Total RNAs were extracted from whole animals and subjected to semi - quantitative RT - PCR , whose products were analyzed by capillary electrophoresis . Representative gel - like presentation is indicated ( n = 3 ) . Schematic structure of each PCR product is indicated on the right . Open reading frames ( ORFs ) for full - length and truncated proteins are in orange and cyan , respectively . rpl - 12 was used as an unaffected control .

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

Figure 2(B) Alternative splicing of sams-3, sams-4 and sams-5 in unfed and fed worms without or with a protein synthesis inhibitor emetine. Synchronized L1 larvae of an NMD-deficient strain KH1668: smg-2 (yb979) (lanes 1-3) and a wild-type strain N2 (lanes 4-6) were incubated in S-complete medium alone (lanes 1, 4), with a standard E. coli strain OP50 (OD600=10.0) (lanes 2, 5) or OP50 supplemented with 10 mg/ml emetine (lanes 3, 6) for 3 hours at 20°C. Total RNAs were extracted from whole animals and subjected to semi-quantitative RT-PCR, whose products were analyzed by capillary electrophoresis. Representative gel-like presentation is indicated (n=3). Schematic structure of each PCR product is indicated on the right. Open reading frames (ORFs) for full-length and truncated proteins are in orange and cyan, respectively. rpl-12 was used as an unaffected control.


Figure 3 ( A ) Alternative splicing of sams - 3 , sams - 4 and sams - 5 in smg - 2 ( yb979 ) and smg - 2 ( yb979 ) ; sams - 5 ( gk147 ) ; sams - 1 ( ok2946 ) mutants . Synchronized L1 larvae of each strain were incubated in S - complete medium alone ( lanes 1 and 5 ) , with OP50 ( OD600 = 10 . 0 ) ( lanes 2 and 6 ) and with OP50 supplemented with 25 mM L - Met ( lanes 3 and 7 ) or 25 mM cycloleucine ( cLeu ) ( lanes 4 and 8 ) for 3 hours at 20 ° C . The splicing patterns were analyzed and presented as in Figure 2B ( n = 3 ) . ( B , C ) Western blot analysis of SAMS - 1 , SAMS - 3 and SAMS - 4 during larval development in the smg - 2 ( yb979 ) ( B ) and wild - type ( C ) backgrounds . Genotypes of the worms are smg - 2 ( w ) and smg - 2 ; sams - 5 ; sams - 1 ( s ) in ( B ) and wild - type ( w ) and sams - 1 ( s ) in ( C ) . Synchronized L1 larvae of each strain were incubated with OP50 at 20 ° C and subjected to Western blot analysis at indicated time points . Anti - β - tubulin ( B ) or coomassie brilliant blue ( CBB ) staining ( C ) was used as a loading control . Specificity of the antibodies is confirmed in Appendix Figure S12 . Note that upregulation of SAMS - 1 protein in the wild type during larval development in ( C ) is consistent with feeding - induced upregulation of sams - 1 mRNA in Figure EV3D .

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Figure 3(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).(B, C) Western blot analysis of SAMS-1, SAMS-3 and SAMS-4 during larval development in the smg-2 (yb979) (B) and wild-type (C) backgrounds. Genotypes of the worms are smg-2 (w) and smg-2; sams-5; sams-1 (s) in (B) and wild-type (w) and sams-1 (s) in (C). Synchronized L1 larvae of each strain were incubated with OP50 at 20°C and subjected to Western blot analysis at indicated time points. Anti-β-tubulin (B) or coomassie brilliant blue (CBB) staining (C) was used as a loading control. Specificity of the antibodies is confirmed in Appendix Figure S12. Note that upregulation of SAMS-1 protein in the wild type during larval development in (C) is consistent with feeding-induced upregulation of sams-1 mRNA in Figure EV3D.


Figure 4 ( C ) Alternative splicing of sams - 3 , sams - 4 and sams - 5 in smg - 2 ( yb979 ) and smg - 2 ( yb979 ) ; mett - 10 ( ok2204 ) mutants . Synchronized L1 larvae of each strain were incubated in S - complete medium alone ( lanes 1 and 3 ) or with OP50 ( lanes 2 and 4 ) for 3 hours at 20 ° C and the splicing patterns were analyzed and presented as in Figure 2B .

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

Figure 4(C) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); mett-10 (ok2204) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 3) or with OP50 (lanes 2 and 4) for 3 hours at 20°C and the splicing patterns were analyzed and presented as in Figure 2B.


Figure 5 ( A ) Mass chromatograms of RNA fragments to detect m6A ( top ) or unmodified A ( bottom ) after in vitro incubation of 127 - nt sams - 3 / sams - 4 pre - mRNA ( sequence available in Fig EV2 ) with recombinant full - length METT - 10 protein in the presence ( left ) or absence ( right ) of 1 mM SAM . The sequence , m / z value , and charge state for each fragment are indicated on the right . Asterisks indicate non - specific signals . ( B ) The negatively - charged ions of RNase T1 fragment is decomposed in the instrument by collision - induced dissociation ( CID ) using helium gas . The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel . Nomenclature of the product ions are described in the literature ( McLuckey et al , 1992 ) . Product ions of c and y series derive from the 5 ' and 3 ' termini of the fragment , respectively .

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 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

Figure 5(A) Mass chromatograms of RNA fragments to detect m6A (top) or unmodified A (bottom) after in vitro incubation of 127-nt sams-3/sams-4 pre-mRNA (sequence available in Fig EV2) with recombinant full-length METT-10 protein in the presence (left) or absence (right) of 1 mM SAM. The sequence, m/z value, and charge state for each fragment are indicated on the right. Asterisks indicate non-specific signals.(B) The negatively-charged ions of RNase T1 fragment is decomposed in the instrument by collision-induced dissociation (CID) using helium gas. The product ions produced by CID are assigned on the sequence illustrated on the top right inset panel. Nomenclature of the product ions are described in the literature (McLuckey et al, 1992). Product ions of c and y series derive from the 5' and 3' termini of the fragment, respectively.


Figure 6A , B ) m6A - IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal / productive 3 ' SS . ( A ) Representative gel - like images ( n = 3 ) of semi - quantitative RT - PCR analysis of sams - 3 , sams - 4 and sams - 5 in input and immunoprecipitated ( IP ) RNAs from the smg - 2 ( yb979 ) mutant . The splicing patterns were analyzed and presented as in Figure 2B . ( B ) Relative enrichment of total and each of mRNA isoforms compared to eef - 1A . 1 mRNA by RT - qPCR . Error bars indicate standard error of mean ( n = 3 ) . ( C ) Examples of normalized Nanopore currents for unmodified and m6A - modified in - vitro transcribed sams - 3 / sams - 4 RNAs at nucleotide positions - 4 through + 4 relevant to the m6A site . One hundred reads are lotted for each . Color codes are indicated . ( D ) Distribution of mean ( top ) and standard deviation ( bottom ) of normalized Nanopore currents at nucleotide positions - 4 through + 4 relevant to the m6A site for 5 , 000 of the unmodified and 2 , 680 of the m6A - modified sams - 3 / sams - 4 RNAs and 85 of an endogenous sams - 3 mRNA isoform E2 / E3L . Color codes are indicated . Central bands represent the median , boxes represent the 25th and 75th percentiles , and whiskers represent the lowest and highest values . A red line at position 0 in the top panel indicates a cut - off line ( mean current = 1 . 7557 ) that discriminates between the unmodified and m6A - modified sams - 3 / sams - 4 RNAs with accuracies of 64 . 58 % for both . (

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 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

Figure 6A, B) m6A-IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal/productive 3'SS. (A) Representative gel-like images (n=3) of semi-quantitative RT-PCR analysis of sams-3, sams-4 and sams-5 in input and immunoprecipitated (IP) RNAs from the smg-2 (yb979) mutant. The splicing patterns were analyzed and presented as in Figure 2B. (B) Relative enrichment of total and each of mRNA isoforms compared to eef-1A.1 mRNA by RT-qPCR. Error bars indicate standard error of mean (n=3). ( C) Examples of normalized Nanopore currents for unmodified and m6A-modified in-vitro transcribed sams-3/sams-4 RNAs at nucleotide positions -4 through +4 relevant to the m6A site. One hundred reads are lotted for each. Color codes are indicated. (D) Distribution of mean (top) and standard deviation (bottom) of normalized Nanopore currents at nucleotide positions -4 through +4 relevant to the m6A site for 5,000 of the unmodified and 2,680 of the m6A-modified sams-3/sams-4 RNAs and 85 of an endogenous sams-3 mRNA isoform E2/E3L. Color codes are indicated. Central bands represent the median, boxes represent the 25th and 75th percentiles, and whiskers represent the lowest and highest values. A red line at position 0 in the top panel indicates a cut-off line (mean current = 1.7557) that discriminates between the unmodified and m6A-modified sams-3/sams-4 RNAs with accuracies of 64.58% for both. (


Figure 1 ( A RNA sequencing of CD31 and CD34 double - positive retinal endothelial cells from P5 wild - type mice . ( A ) RNA levels ( FPKM ; fragments per kilobase million ) of marker genes of endothelial cells ( green ; CD31 ( gene name PECAM1 ) , CD34 , von Willebrand Factor ( vWF ) , tyrosine - protein kinase receptor Tie2 ( TEK ) , VE - cadherin ( CDH5 ) , endoglin ( ENG ) , CD146 ( MCAM ) and VEGFR2 ( KDR ) ) , astrocytes ( blue , GFAP ) , immune cells ( red ; T - cells ( CD3E ) , B - cells ( CD19 ) , all leucocytes ( CD45 , PTPRC ) , and monocytes / macrophages ( F4 / 80 , EMR1 ) ) , Müller glia cells ( orange ; aquaporin 4 ( AQP4 ) ) , neurons ( yellow ; retinal ganglion cells ( RNA binding fox - 1 homolog 3 , RBFOX3 ) , amacrine cells ( parvalbumin , PVALB ) , bipolar cells ( PKC - α , PRKCA ) , horizontal cell ( calbindin , CALB1 ) , photoreceptors ( rods , CD73 ( NT5E ) ; cones , transducing ( GNAT1 ) ) , and retinal pigment epithelial cells ( magenta ; retinal pigment epithelium - specific 65 kDa protein ( RPE65 ) . B ) RNA sequencing of CD31 and CD34 double - positive retinal endothelial cells from P5 wild - type mice . ( B ) RNA levels of VASP ( red ) , EVL ( green ) and Mena ( blue ) in the CD31 and CD34 double - positive P5 retinal endothelial cells . Error bars represent SEM ; n = 18 animals ( 36 retinas ) from six independent litters ; four independent experiments . * * * p < 0 . 001 , one - way ANOVA with Bonferroni ' s multi comparison test . ( C - E ) Staining of Ena / VASP proteins in blood vessels of P5 mouse retinas . P5 wild - type mouse retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and VASP - ( C ) , EVL - ( D ) , or Mena - specific ( E ) antibodies ( red ) . Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells . No Mena protein expression was detected in P5 retinal endothelial cells ( E ) . Representative images from three independent experiments are shown . Scale bars , 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (A) RNA levels (FPKM; fragments per kilobase million) of marker genes of endothelial cells (green; CD31 (gene name PECAM1), CD34, von Willebrand Factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE-cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR)), astrocytes (blue, GFAP), immune cells (red; T-cells (CD3E), B-cells (CD19), all leucocytes (CD45, PTPRC), and monocytes/macrophages (F4/80, EMR1)), Müller glia cells (orange; aquaporin 4 (AQP4)), neurons (yellow; retinal ganglion cells (RNA binding fox-1 homolog 3, RBFOX3), amacrine cells (parvalbumin, PVALB), bipolar cells (PKC-α, PRKCA), horizontal cell (calbindin, CALB1), photoreceptors (rods, CD73 (NT5E); cones, transducing (GNAT1)), and retinal pigment epithelial cells (magenta; retinal pigment epithelium-specific 65 kDa protein (RPE65).  B) RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (B) RNA levels of VASP (red), EVL (green) and Mena (blue) in the CD31 and CD34 double-positive P5 retinal endothelial cells. Error bars represent SEM; n = 18 animals (36 retinas) from six independent litters; four independent experiments. ***p<0.001, one-way ANOVA with Bonferroni's multi comparison test.  (C-E) Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP - (C), EVL- (D), or Mena-specific (E) antibodies (red). Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells. No Mena protein expression was detected in P5 retinal endothelial cells (E). Representative images from three independent experiments are shown. Scale bars, 10 µm.


Figure 2 ( A ) Wild - type and EVL - / - P5 retinas were digested , labelled with CD31 - , CD34 - and EVL - specific antibodies and analyzed by flow cytometry . Endothelial cells were defined as CD31 / CD34 - double positive cells ( gate indicated in the left panel ) and analyzed for EVL expression ( middle panel ) . Mean fluorescence intensity ( MFI ) of EVL in wild - type ( magenta ) and EVL - / - ( black ) cells is shown in the right panel . WT : n = 3 , EVL - / - : n = 2 ; two retinas per animal . Error bars represent SEM . ( B - C ) EVL protein expression in sparse / migrating wild - type ( WT ) and EVL - / - mouse brain endothelial cells ( MBEC ) and mouse lung endothelial cells ( MLEC ) . Actin was used as loading control . Western blots are representative of three independent experiments . ( D ) MLEC from wild - type ( upper panel ) and EVL - / - ( lower panel ) mice stimulated with 10 ng / ml VEGF were stained for actin ( cyan ) and EVL ( magenta ) . Asterisks indicate focal adhesions , white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia . Representative images from four independent experiments are shown . Scale bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type and EVL-/- P5 retinas were digested, labelled with CD31-, CD34- and EVL-specific antibodies and analyzed by flow cytometry. Endothelial cells were defined as CD31/CD34-double positive cells (gate indicated in the left panel) and analyzed for EVL expression (middle panel). Mean fluorescence intensity (MFI) of EVL in wild-type (magenta) and EVL-/- (black) cells is shown in the right panel. WT: n=3, EVL-/-: n=2; two retinas per animal. Error bars represent SEM.  (B-C) EVL protein expression in sparse/migrating wild-type (WT) and EVL-/- mouse brain endothelial cells (MBEC) and mouse lung endothelial cells (MLEC). Actin was used as loading control. Western blots are representative of three independent experiments.  (D) MLEC from wild-type (upper panel) and EVL-/- (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm.


Figure 3 ( A ) Isolectin B4 stained vasculature in whole mount retinas of wild - type ( WT ) and global EVL - / - mice on postnatal days 3 , 5 and 7 ( P3 , P5 , P7 ) assessed by confocal microscopy . Scale bars 200 µm . ( B ) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild - type littermates . ( C ) Impact of individual and combined VASP / EVL deletion on sprouting angiogenesis in the postnatal mouse retina at P5 ( EVL - deficient animals ( E - / - ) , green ; VASP - deficient animals ( V - / - ) , red ; VASP / EVL - double deficient animals ( EV - / - ) , orange ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 (A) Isolectin B4 stained vasculature in whole mount retinas of wild-type (WT) and global EVL-/- mice on postnatal days 3, 5 and 7 (P3, P5, P7) assessed by confocal microscopy. Scale bars 200 µm.  (B) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates. (C) Impact of individual and combined VASP/EVL deletion on sprouting angiogenesis in the postnatal mouse retina at P5 (EVL-deficient animals (E-/-), green; VASP-deficient animals (V-/-), red; VASP/EVL-double deficient animals (EV-/-), orange).


Figure 4 The retinal vasculature of endothelial cell specific EVL knockout mice ( EVLΔEC ) and littermate controls ( EVLfl / fl ) on P5 was analyzed by Isolectin B4 staining . Radial outgrowth of the retinal vasculature ; in global EVL - / - mice are shown for comparison ; 4 - 5 independent experiments . ( C ) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front . ( D ) Tipp cells numbers per field of view ( 387 , 5 x 387 , 5 µm ) . ( E ) Filopodia numbers normalized to the length of the angiogenic front . ( F ) Endothelial cell proliferation analyzed by BrdU incorporation ( red ) of ERG positive ( blue ) endothelial nuclei .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The retinal vasculature of endothelial cell specific EVL knockout mice (EVLΔEC) and littermate controls (EVLfl/fl) on P5 was analyzed by Isolectin B4 staining.  Radial outgrowth of the retinal vasculature; in global EVL-/- mice are shown for comparison; 4 - 5 independent experiments.  (C) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front. (D) Tipp cells numbers per field of view (387,5 x 387,5 µm).  (E) Filopodia numbers normalized to the length of the angiogenic front.  (F) Endothelial cell proliferation analyzed by BrdU incorporation (red) of ERG positive (blue) endothelial nuclei.


Figure 5 CD31 and CD34 double positive endothelial cells were isolated from wild - type or EVL - / - P5 retinas by FACS and analyzed by RNA - sequencing . Two independent experiments ; in total 18 retinas from WT mice and 18 retinas from EVL - / - mice from three different litters , each . ( A ) RNA levels ( FPKM ) of VASP , EVL and Mena in endothelial cells from wild - type or EVL - / - retinas . Error bars represent SEM . ( D ) Analysis of mRNA levels of Esm1 , Ptgs2 ( cyclooxygenase 2 ) , serpine 1 and paxillin in EVL - deficient MLEC relative to WT controls set to 100 . n = 3 - 4 independent experiments , error bars represent SEM , one sample t - test , * p < 0 . 05 ; * * p < 0 . 01 . ( E ) ESM1 protein expression in P5 retinas of wild - type ( WT ) and global EVL - / - mice . Retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and antibodies directed against the tip cell marker ESM1 ( red ) . Representative images from three independent experiments are shown . Scale bars , 100 µm . ( F ) Analysis of retinal ESM1 protein levels normalized to wild - type littermates . Error bars represent SEM ; * * * p < 0 . 001 , unpaired Student ' s t - test ; three different litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CD31 and CD34 double positive endothelial cells were isolated from wild-type or EVL-/- P5 retinas by FACS and analyzed by RNA-sequencing. Two independent experiments; in total 18 retinas from WT mice and 18 retinas from EVL-/- mice from three different litters, each. (A) RNA levels (FPKM) of VASP, EVL and Mena in endothelial cells from wild-type or EVL-/- retinas. Error bars represent SEM.  (D) Analysis of mRNA levels of Esm1, Ptgs2 (cyclooxygenase 2), serpine 1 and paxillin in EVL-deficient MLEC relative to WT controls set to 100. n = 3-4 independent experiments, error bars represent SEM, one sample t-test, *p<0.05; **p<0.01.  (E) ESM1 protein expression in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against the tip cell marker ESM1 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.  (F) Analysis of retinal ESM1 protein levels normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; three different litters.


Figure 6 ( A ) Proliferation of WT and EVL - / - MLEC was assessed by BrdU incorporation . The number of BrdU positive cells ( red ) was normalized to the total number of cells ( white ) . Eight fields of view per condition and genotype for each of 5 independent WT and EVL - / - cell batches ; error bars represent SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , # p = 0 . 171 non - significant vs . Basal EVL - / - , one - way ANOVA with Bonferroni ' s multi comparison test ; scale bar = 50 µm . ( C ) Sprouting of WT and EVL - / - primary MLEC spheres in the absence ( Basal ) or presence of VEGF ( 50 ng / ml ) ; asterisks indicate sprouts ; scale bars , 100 µm . ( D ) Solid bars represent proportion of WT and EVL - / - MLEC spheres forming sprouts in the absence ( − ) or presence ( + ) of VEGF . n - numbers are indicated ; n . s . non - significant ( p = 0 . 557 ) ; * * * p < 0 . 001 , Fisher ' s exact test . ( E ) Quantification of MLEC sphere sprout length without or with VEGF . Dots represent individual sprouts . Horizontal lines represent mean , error bars represent SEM . n = 20 - 30 beads ( from three independent experiments ) . * * * p < 0 . 001 , n . s . non - significant ( p = 0 . 543 ) , one - way ANOVA with Bonferroni ' s multi comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation of WT and EVL-/- MLEC was assessed by BrdU incorporation. The number of BrdU positive cells (red) was normalized to the total number of cells (white). Eight fields of view per condition and genotype for each of 5 independent WT and EVL-/- cell batches; error bars represent SEM; **p<0.01, ***p<0.001, #p=0.171 non-significant vs. Basal EVL-/-, one-way ANOVA with Bonferroni's multi comparison test; scale bar = 50 µm.  (C) Sprouting of WT and EVL-/- primary MLEC spheres in the absence (Basal) or presence of VEGF (50 ng/ml); asterisks indicate sprouts; scale bars, 100 µm. (D) Solid bars represent proportion of WT and EVL-/- MLEC spheres forming sprouts in the absence (−) or presence (+) of VEGF. n-numbers are indicated; n.s. non-significant (p=0.557); ***p<0.001, Fisher's exact test. (E) Quantification of MLEC sphere sprout length without or with VEGF. Dots represent individual sprouts. Horizontal lines represent mean, error bars represent SEM. n = 20-30 beads (from three independent experiments). ***p<0.001, n.s. non-significant (p=0.543), one-way ANOVA with Bonferroni's multi comparison test.


Figure 7 ( A ) VEGFR2 protein levels in wild - type ( WT ) and EVL - deficient ( EVL - / - ) endothelial cells . Actin was used as loading control . The lower panel shows quantification of VEGFR2 to actin ratios from five independent cell batches ; error bars represent SEM ; n . s . non - significant ( p = 0 . 388 ) , unpaired Student ' s t - test . ( B ) Antibody feeding assay to monitor VEGFR2 internalization after VEGF stimulation . Left panel : schematic diagram showing the two step staining procedure of surface and internalized VEGFR2 antibodies , which are displayed in yellow and green in the merged confocal images of WT and EVL - / - endothelial cells ( middle panel ) , respectively ; scale bar , 10 µm . The bar graphs show quantification of the number of VEGFR2 clusters and the ratio of internalized to total VEGFR2 ratios normalized to wild - type ; n = 4 - 6 independent cell batches , error bars represent SEM ; * * p < 0 . 01 , * * * p < 0 . 001 , unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 (A) VEGFR2 protein levels in wild-type (WT) and EVL-deficient (EVL-/-) endothelial cells. Actin was used as loading control. The lower panel shows quantification of VEGFR2 to actin ratios from five independent cell batches; error bars represent SEM; n.s. non-significant (p=0.388), unpaired Student's t-test.  (B) Antibody feeding assay to monitor VEGFR2 internalization after VEGF stimulation. Left panel: schematic diagram showing the two step staining procedure of surface and internalized VEGFR2 antibodies, which are displayed in yellow and green in the merged confocal images of WT and EVL-/- endothelial cells (middle panel), respectively; scale bar, 10 µm. The bar graphs show quantification of the number of VEGFR2 clusters and the ratio of internalized to total VEGFR2 ratios normalized to wild-type; n = 4-6 independent cell batches, error bars represent SEM; **p<0.01, ***p<0.001, unpaired Student's t-test.


Figure 8 ( A ) Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL - / - endothelial cells under basal and VEGF - stimulated conditions ( 80 ng / ml ) ; n = 5 independent cell batches , error bars represent SEM ; * p < 0 . 05 , one - way ANOVA with Bonferroni ' s multi comparison test . ( B ) Western blots and quantification of ERK1 / 2 phosphorylation levels relative to total ERK levels in WT and EVL - / - endothelial cells under basal and VEGF - stimulated conditions ( 80 ng / ml ) ; n = 6 independent cell batches , error bars represent SEM ; * p < 0 . 05 , one - way ANOVA with Bonferroni ' s multi comparison test . ( C ) ERK1 / 2 phosphorylation in P5 retinas of wild - type ( WT ) and global EVL - / - mice . Retinas were fixed and stained with isolectin B4 ( IB4 , green ) to visualize endothelial cells and antibodies directed against phospho - ERK1 / 2 ( red ) . Representative images from three independent experiments are shown . Scale bars , 100 µm . ( D ) Analysis of endothelial ERK1 / 2 - phosphorylation normalized to wild - type littermates . Error bars represent SEM ; * * * p < 0 . 001 , unpaired Student ' s t - test ; two different litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8 (A) Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 5 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test.  (B) Western blots and quantification of ERK1/2 phosphorylation levels relative to total ERK levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 6 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test.  (C) ERK1/2 phosphorylation in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against phospho-ERK1/2 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm. (D) Analysis of endothelial ERK1/2-phosphorylation normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; two different litters.


Figure 1 ( B ) Representative high content microscopy images of RIPosomes in THP - 1 cells infected with S . flexneri ( MOI 1 : 25 , 8 h ) . About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well in 3 technical replicates . Right panel , the graph depicts an average number of RIPosomes / cell , which is calculated from 3 biological replicates , Mean ± SD . * * * p < 0 . 005 , Student ' s unpaired t - test . ( C ) Western blot analysis of soluble and insoluble fractions of S . flexneri infected THP - 1 cells ( MOI 1 : 25 , 8 h ) . Arrow indicates oligomers of RIPK2 . ( D ) Left panel , representative high content microscopy image of control and RIPK2 knockdown THP - 1 cells infected with S . flexneri ( MOI 1 : 25 , 8 h ) . Right panel , the graph depicts an average number of RIPosomes / cell , which is calculated from 3 biological replicates , Mean ± SD . * * * p < 0 . 0005 , Student ' s unpaired t - test . ( E - F ) Western blot analysis of soluble and insoluble fractions of S . flexneri infected ( E ) control siRNA and RIPK2 siRNA transfected THP - 1 cells ( F ) Control CRISPR ( RIPK2 + / + ) cells and RIPK2 CRISPR knockout ( RIPK2 - / - ) HT - 29 cells . ( G ) Representative confocal images of RIPosomes in mouse BMDMs infected with S . flexneri ( MOI 1 : 25 , 8 h ) . Line profile : co - localization analysis using line intensity profile . Scale bar , 5 µm . Zoom panels are digital magnifications . DNA is stained with DAPI ( pseudo - colored red for better contrast ) . ( I ) Representative confocal images of RIPosomes in THP - 1 cells infected with RFP expressing Salmonella typhimurium ( MOI 1 : 5 , 4 h ) . Line profile : co - localization analysis using line intensity profile . Scale bar , 8 µm . Zoom panels are digital magnifications . DNA is stained with DAPI . ( J ) Left panel , representative high - content microscopy images ( yellow masks represent software algorithms - defined cell boundaries ) of RIPosomes in THP - 1 cells infected with S . typhimurium ( MOI 1 : 5 , 4 h ) . Right panel , the graph depicts an average number of RIPosomes / cell . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test . DNA is stained with DAPI . Representative confocal images of RIPosomes in THP - 1 cells infected with ( K ) E . coli LF82 strain ( MOI 1 : 10 , 8h ) ( LPS antibody is used to stain LF82 ) Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . DNA is stained with DAPI . Representative confocal images of ( M ) GFP expressing M . tuberculosis ( MOI 1 : 10 , 8 h ) . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . DNA is stained with DAPI . ( N ) Western blot analysis of soluble and insoluble fractions from THP - 1 cells , uninfected or infected with E . coli DH5α ( MOI 1 : 10 , 8h ) or E . coli LF82 strains ( MOI 1 : 10 , 8h ) .

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 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 O O O O O O 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 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 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 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 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 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

Figure 1(B) Representative high content microscopy images of RIPosomes in THP-1 cells infected with S. flexneri (MOI 1:25, 8 h). About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well in 3 technical replicates. Right panel, the graph depicts an average number of RIPosomes/cell, which is calculated from 3 biological replicates, Mean ± SD. ***p < 0.005, Student's unpaired t-test.(C) Western blot analysis of soluble and insoluble fractions of S. flexneri infected THP-1 cells (MOI 1:25, 8 h). Arrow indicates oligomers of RIPK2.(D) Left panel, representative high content microscopy image of control and RIPK2 knockdown THP-1 cells infected with S. flexneri (MOI 1:25, 8 h). Right panel, the graph depicts an average number of RIPosomes/cell, which is calculated from 3 biological replicates, Mean ± SD. ***p < 0.0005, Student's unpaired t-test.(E-F) Western blot analysis of soluble and insoluble fractions of S. flexneri infected (E) control siRNA and RIPK2 siRNA transfected THP-1 cells (F) Control CRISPR (RIPK2+/+) cells and RIPK2 CRISPR knockout (RIPK2-/-) HT-29 cells.(G) Representative confocal images of RIPosomes in mouse BMDMs infected with S. flexneri ( MOI 1:25, 8 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI (pseudo-colored red for better contrast).(I) Representative confocal images of RIPosomes in THP-1 cells infected with RFP expressing Salmonella typhimurium (MOI 1:5, 4 h). Line profile: co-localization analysis using line intensity profile. Scale bar, 8 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.(J) Left panel, representative high-content microscopy images (yellow masks represent software algorithms-defined cell boundaries) of RIPosomes in THP-1 cells infected with S. typhimurium (MOI 1:5, 4 h). Right panel, the graph depicts an average number of RIPosomes/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test. DNA is stained with DAPI.Representative confocal images of RIPosomes in THP-1 cells infected with (K) E. coli LF82 strain (MOI 1: 10, 8h) (LPS antibody is used to stain LF82) Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.Representative confocal images of (M) GFP expressing M. tuberculosis (MOI 1:10, 8 h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. DNA is stained with DAPI.(N) Western blot analysis of soluble and insoluble fractions from THP-1 cells, uninfected or infected with E. coli DH5α (MOI 1:10, 8h) or E. coli LF82 strains (MOI 1:10, 8h).


Figure 2 ( B ) Left panel , representative high - content microscopy images of HEK293T cells transfected with GFP - NOD1 , GFP - NOD2 , and GFP - RIPK2 plasmids for 9 h . Right panel , the graph depicts the average number of RIPosomes per GFP - positive cell . About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) . ( C ) Western blot analysis of soluble and insoluble fractions of HEK293T cells transfected with the CARD domain - containing region of NOD1 , NOD2 , and RIPK2 . ( E ) The soluble and insoluble fractions of S . flexneri infected THP - 1 ( MOI 1 : 25 , 8 h ) cell lysates were subjected to Western blot analysis with indicated antibodies . ( F ) Representative confocal images of immunofluorescence assays conducted with THP - 1 cells infected with RFP expressing S . flexneri ( pseudo - colored , blue ) ( MOI 1 : 25 , 8h ) . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . ( G ) Representative confocal images of immunofluorescence assays performed with HEK293T cells transfected with GFP - RIPK2 and Flag - NOD2 ( upper panel ) or Flag - NOD1 ( lower panel ) . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 or 10 µm as indicated in figures . Inset zoom panels are digital magnifications . ( H - I ) HEK293T cells were transfected with ( H ) full length and ( I ) CARD domains of NOD1 , NOD2 , and RIPK2 followed by Western blot analysis with soluble and insoluble fractions using indicated antibodies . ( J - K ) Western blot analysis of soluble and insoluble fractions of RIPK2 + / + and RIPK2 - / - HT - 29 cells infected with S . flexneri ( MOI 1 : 25 , 8 h ) with indicated antibodies . ( L - M ) Representative immunofluorescence images of doxycycline - inducible GFP - RIPK2 expressing HeLa cells . ( L ) Upper panel , uninfected . Lower panel , S . flexneri - infected MOI 1 : 25 , 4h ) . Immunostaining was performed with the p65 antibody ( red ) and DNA stained with DAPI ( Blue ) . ( M ) The graph indicates % of cells that are RIPosomes positive or negative with nuclear / cytoplasmic p65 ( 5 fields ( each group ) , Mean ± SD , n = 3 ) . * * * * p < 0 . 00005 , Student ' s unpaired t - test .

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 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 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 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 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 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 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

Figure 2(B) Left panel, representative high-content microscopy images of HEK293T cells transfected with GFP-NOD1, GFP-NOD2, and GFP -RIPK2 plasmids for 9 h. Right panel, the graph depicts the average number of RIPosomes per GFP-positive cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).(C) Western blot analysis of soluble and insoluble fractions of HEK293T cells transfected with the CARD domain-containing region of NOD1, NOD2, and RIPK2.(E) The soluble and insoluble fractions of S. flexneri infected THP-1 (MOI 1:25, 8 h) cell lysates were subjected to Western blot analysis with indicated antibodies.(F) Representative confocal images of immunofluorescence assays conducted with THP-1 cells infected with RFP expressing S. flexneri (pseudo-colored, blue) (MOI 1:25, 8h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications.(G) Representative confocal images of immunofluorescence assays performed with HEK293T cells transfected with GFP-RIPK2 and Flag-NOD2 (upper panel) or Flag-NOD1 (lower panel). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 or 10 µm as indicated in figures. Inset zoom panels are digital magnifications.(H-I) HEK293T cells were transfected with (H) full length and (I) CARD domains of NOD1, NOD2, and RIPK2 followed by Western blot analysis with soluble and insoluble fractions using indicated antibodies.(J-K) Western blot analysis of soluble and insoluble fractions of RIPK2 +/+ and RIPK2-/- HT-29 cells infected with S. flexneri (MOI 1:25, 8 h) with indicated antibodies.(L-M) Representative immunofluorescence images of doxycycline-inducible GFP-RIPK2 expressing HeLa cells. (L) Upper panel, uninfected. Lower panel, S. flexneri -infected MOI 1:25, 4h). Immunostaining was performed with the p65 antibody (red) and DNA stained with DAPI (Blue). (M) The graph indicates % of cells that are RIPosomes positive or negative with nuclear/cytoplasmic p65 (5 fields (each group), Mean ± SD, n=3). ****p < 0.00005, Student's unpaired t-test.


Figure 3 ( A - B ) Western blot analysis with cell lysate of uninfected and S . flexneri infected ( MOI 1 : 25 , 8 h ) ( A ) control and ATG5 siRNA knockdown THP - 1 cells , ( B ) wild type ( WT ) and ATG5 knockout ( ATG5 - / - ) MEF cells with indicated antibodies . Densitometric analysis was performed using Image J software . FC , fold change . ( C ) Left panel , representative high - content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin ( 500 nM , 4h ) or cells treated with rapamycin ( 500 nM , 4h ) and bafilomycin A1 ( BafA1 , 300 nM ) . Right panel , the graph depicts the average number of RIPosomes / cell . About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * p < 0 . 005 and , * * * p < 0 . 0005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) . ( D ) Left panel , representative high - content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP - 1 cells infected with S . flexneri ( 8 h ) . Right panel , the graph depicts average number of RIPosomes / cell . About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * * p < 0 . 0005 , Student ' s unpaired t - test . ( E ) The soluble and insoluble fractions of S . flexneri infected ( MOI 1 : 25 , 8 h ) control and ATG5 siRNA knockdown THP - 1 cells were subjected to Western blot analysis with indicated antibodies . ( F ) Representative confocal images of HEK293T cells transfected with GFP - RIPK2 and immunostained with anti - FK2 antibodies . Line profile : co - localization analysis using line intensity profiles . Scale bar , 3 µm . ( G ) Autophagy receptor screen using co - immunoprecipitation ( Co - IP ) assay to analyze the interaction between GFP - RIPK2 and Flag - p62 or Flag - NBRI or Flag - NDP52 or Flag - TAX1BP1 or Flag - OPTINEURIN in HEK293T cell lysate . ( H ) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S . flexneri infected THP - 1 cells ( MOI 1 : 25 , 8 h ) . ( I ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 ( 6 h ) immunostained with p62 and LC3B antibodies . Zoom panels are digital magnifications . ( J ) GST pull - down assay using purified GST or GST - RIPK2 proteins with in - vitro translated 35S radiolabeled myc - ULK1 or myc - ATG16L or myc - BECLIN1 . Representative confocal images of HEK293T cells transfected with GFP - RIPK2 ( 9 h ) and immunofluorescence assay performed with antibodies specific to ( K ) ULK1 Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications . Representative confocal images of HEK293T cells transfected with GFP - RIPK2 or mcherry - RIPK2 ( 9 h ) and immunofluorescence assay performed with antibodies specific to ( L ) ATG16L ( M ) BECLIN1 . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications . ( N ) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF - κB luciferase reporter vector pGL4 . 32 NFκB - RE , GFP - RIPK2 , and Flag - NOD1 plasmids . Mean ± SD , n = 3 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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

Figure 3(A-B) Western blot analysis with cell lysate of uninfected and S. flexneri infected (MOI 1:25, 8 h) (A) control and ATG5 siRNA knockdown THP-1 cells, (B) wild type (WT) and ATG5 knockout (ATG5-/-) MEF cells with indicated antibodies. Densitometric analysis was performed using Image J software. FC, fold change.(C) Left panel, representative high-content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin (500 nM, 4h) or cells treated with rapamycin (500 nM, 4h) and bafilomycin A1 (BafA1, 300 nM). Right panel, the graph depicts the average number of RIPosomes/cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), **p < 0.005 and, ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test).(D) Left panel, representative high-content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP-1 cells infected with S. flexneri (8 h). Right panel, the graph depicts average number of RIPosomes/cell. About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.(E) The soluble and insoluble fractions of S. flexneri infected (MOI 1:25, 8 h) control and ATG5 siRNA knockdown THP-1 cells were subjected to Western blot analysis with indicated antibodies.(F) Representative confocal images of HEK293T cells transfected with GFP-RIPK2 and immunostained with anti-FK2 antibodies. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm.(G) Autophagy receptor screen using co-immunoprecipitation (Co-IP) assay to analyze the interaction between GFP-RIPK2 and Flag-p62 or Flag-NBRI or Flag-NDP52 or Flag-TAX1BP1 or Flag-OPTINEURIN in HEK293T cell lysate.(H) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S. flexneri infected THP-1 cells (MOI 1:25, 8 h).(I) Representative confocal images of HEK293T cells expressing GFP-RIPK2 (6 h) immunostained with p62 and LC3B antibodies. Zoom panels are digital magnifications.(J) GST pull-down assay using purified GST or GST-RIPK2 proteins with in-vitro translated 35S radiolabeled myc-ULK1 or myc-ATG16L or myc-BECLIN1.Representative confocal images of HEK293T cells transfected with GFP-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (K) ULK1 Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.Representative confocal images of HEK293T cells transfected with GFP-RIPK2 or mcherry-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (L) ATG16L (M) BECLIN1. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.(N) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32 NFκB-RE, GFP-RIPK2, and Flag-NOD1 plasmids. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


Figure 4 ( A - B ) The THP - 1 cell lysates were subjected to immunoprecipitation analysis ( A ) untreated and treated with iE - DAP ( 40 μg / ml , 6 h ) , ( B ) untreated and treated with MDP ( 40 μg / ml , 6 h ) . IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies . IgG ( H ) ; IgG heavy chain . ( C - D ) Co - IP analysis of the interactions between ( C ) GFP - RIPK2 and Flag - IRGM or ( D ) Flag - NOD1 and GFP - IRGM in HEK293T cell lysates . ( E ) GST pull - down assay using purified GST and GST - IRGM and in - vitro translated 35S radiolabeled myc - NOD2 , myc - NOD1 , and myc - RIPK2 . ( F ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 and mCherry or mCherry - IRGM . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Representative confocal images of THP - 1 cells infected with RFP expressing S . flexneri , ( MOI 1 : 25 , 8 h ) and immunostained with , ( H ) IRGM and RIPK2 antibody . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . In image H , for better contrast , RFP expressing Shigella is pseudocolored to blue . ( J ) Representative confocal images of HEK293T cells expressing GFP - RIPK2 , Flag - NOD1 , and mCherry - IRGM . Line profile : co - localization analysis using line intensity profiles . Scale bar , 3 µm . Zoom panels are digital magnifications . ( M ) A co - IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain / s of RIPK2 interacting with IRGM . Asterisk indicate the main band of overexpressed protein . ( O ) A co - IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain / s of NOD1 interacting with IRGM . Asterisk indicate the main band of overexpressed

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

Figure 4(A-B) The THP-1 cell lysates were subjected to immunoprecipitation analysis (A) untreated and treated with iE-DAP (40 μg/ml, 6 h), (B) untreated and treated with MDP (40 μg/ml, 6 h). IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies. IgG (H); IgG heavy chain.(C-D) Co-IP analysis of the interactions between (C) GFP-RIPK2 and Flag-IRGM or (D) Flag-NOD1 and GFP-IRGM in HEK293T cell lysates.(E) GST pull-down assay using purified GST and GST-IRGM and in-vitro translated 35S radiolabeled myc-NOD2, myc-NOD1, and myc-RIPK2.(F) Representative confocal images of HEK293T cells expressing GFP-RIPK2 and mCherry or mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.Representative confocal images of THP-1 cells infected with RFP expressing S. flexneri, (MOI 1:25, 8 h) and immunostained with, (H) IRGM and RIPK2 antibody. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. In image H, for better contrast, RFP expressing Shigella is pseudocolored to blue.(J) Representative confocal images of HEK293T cells expressing GFP-RIPK2, Flag-NOD1, and mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm. Zoom panels are digital magnifications.(M) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain/s of RIPK2 interacting with IRGM. Asterisk indicate the main band of overexpressed protein.(O) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain/s of NOD1 interacting with IRGM. Asterisk indicate the main band of overexpressed protein


Figure 5 ( A ) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR - Cas9 mediated IRGM partial knockout ( IRGM + / - ) HT - 29 cells ( 2 clones were tested ) . Densitometric analysis was performed using Image J software . FC , fold change . ( B ) Left panel , soluble and insoluble fractions of S . flexneri ( MOI 1 : 25 , 8 h ) infected mouse BMDMs from Irgm1 + / + and Irgm1 - / - mice were subjected to immunoblot analysis with antibodies as indicated . Right panel , Western blot analysis with the colon lysates from Irgm1 + / + and Irgm1 - / - mice with indicated antibodies . The graph indicate ratio of band intensity ( measured using ImageJ ) and actin ( n = 5 , Mean ± SD , * p < 0 . 05 , * * p < 0 . 005 , and * * * * p < 0 . 00005 , Student ' s unpaired t - test ) ( C ) Doxycycline - inducible stable GFP - RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S . flexneri ( MOI 1 : 25 , 4h ) . The cells were fixed and subjected to high - content microscopy to quantitate the number of RIPosomes formed . The graph depicts an average number of RIPosome / cell . About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test . Western blot analysis with the cell lysate of ( D ) HT - 29 clones stably expressing Flag - vector control or Flag - IRGM Densitometric analysis was performed using Image J software . FC , fold change . Western blot analysis with the cell lysate of ( E ) THP - 1 cells transiently transfected with GFP or GFP - IRGM for 6 h . Densitometric analysis was performed using Image J software . FC , fold change . ( F ) Left panel , representative high - content microscopy images of NODo - RIPosomes in HEK293T cells transfected with GFP - RIPK2 and myc - NOD2 ( upper panel ) or GFP - RIPK2 , myc - NOD2 , and Flag - IRGM ( lower panel ) . Right panel , the graph depicts average number of NODo - RIPosome / cell . About 15000 cells plated per well and RIPosomes were screened in 35 fields per well . Mean ± SD , n = 5 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - test . ( G ) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies . ( H ) Representative confocal images of HEK293T cells transfected with mCherry and GFP - RIPK2CARD or mCherry - IRGM and GFP - RIPK2CARD for 9 h . Line profile : co - localization analysis using line intensity profiles . Scale bar , 5 µm . ( I - K ) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin , Flag , Myc , and , GFP antibodies as indicated . Densitometric analysis was performed using Image J software . FC , fold change . ( L ) Left panels , representative high - content microscopy images of HEK293T cells transfected with GFP - RIPK2 or GFP - RIPK2 and Flag - IRGM or GFP - RIPK2 and Flag - IRGM ( S47N ) . The graph depicts the average number of RIPosome / cell . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons test ) .

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

Figure 5(A) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR-Cas9 mediated IRGM partial knockout (IRGM+/-) HT-29 cells (2 clones were tested). Densitometric analysis was performed using Image J software. FC, fold change.(B) Left panel, soluble and insoluble fractions of S. flexneri (MOI 1:25, 8 h) infected mouse BMDMs from Irgm1+/+ and Irgm1-/- mice were subjected to immunoblot analysis with antibodies as indicated. Right panel, Western blot analysis with the colon lysates from Irgm1+/+ and Irgm1-/- mice with indicated antibodies. The graph indicate ratio of band intensity (measured using ImageJ) and actin (n=5, Mean ± SD, *p < 0.05, **p < 0.005, and ****p < 0.00005, Student's unpaired t-test)(C) Doxycycline-inducible stable GFP-RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S. flexneri (MOI 1:25, 4h). The cells were fixed and subjected to high-content microscopy to quantitate the number of RIPosomes formed. The graph depicts an average number of RIPosome/cell. About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test.Western blot analysis with the cell lysate of (D) HT-29 clones stably expressing Flag-vector control or Flag-IRGM Densitometric analysis was performed using Image J software. FC, fold change.Western blot analysis with the cell lysate of (E) THP-1 cells transiently transfected with GFP or GFP-IRGM for 6 h. Densitometric analysis was performed using Image J software. FC, fold change.(F) Left panel, representative high-content microscopy images of NODo-RIPosomes in HEK293T cells transfected with GFP-RIPK2 and myc-NOD2 (upper panel) or GFP-RIPK2, myc-NOD2, and Flag-IRGM (lower panel). Right panel, the graph depicts average number of NODo-RIPosome/cell. About 15000 cells plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=5 (biological replicates), ****p < 0.00005, Student's unpaired t-test.(G) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies.(H) Representative confocal images of HEK293T cells transfected with mCherry and GFP-RIPK2CARD or mCherry-IRGM and GFP-RIPK2CARD for 9 h. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.(I-K) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin, Flag, Myc, and, GFP antibodies as indicated. Densitometric analysis was performed using Image J software. FC, fold change.(L) Left panels, representative high-content microscopy images of HEK293T cells transfected with GFP-RIPK2 or GFP-RIPK2 and Flag-IRGM or GFP-RIPK2 and Flag-IRGM (S47N). The graph depicts the average number of RIPosome/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


Figure 7 ( C ) Left panel , representative pictures of colons of Irgm1 + / + and Irgm - / - mice untreated or treated with GSK583 infected with S . flexneri . Right panel , the graph depicts the average colon lengths of the mice groups . Mean ± SD , n = 6 , * * p < 0 . 005 , * * * p < 0 . 0005 , Student ' s unpaired t - test . ( D ) Representative microscopic images of H & E staining of colon tissues of Irgm1 + / + and Irgm - / - mice untreated or treated with GSK583 administrated with DSS . The graph depicts the combined histological scores . Mean ± SD , n = 3 ( DSS ) , * * p < 0 . 005 , Student ' s unpaired t - test . Scale bar , 200 µm ( E ) The soluble and insoluble fractionations of lysates from colon tissues of Shigella - infected or DSS - treated Irgm1 + / + and Irgm - / - mice treated with GSK583 as indicated , were subjected to immunoblot analysis with indicated antibodies . ( H - I ) Western blot analysis with the colon tissue lysates of DSS - treated or S . flexneri infected mice groups as indicated .

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 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 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 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

Figure 7(C) Left panel, representative pictures of colons of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 infected with S. flexneri. Right panel, the graph depicts the average colon lengths of the mice groups. Mean ± SD, n=6, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.(D) Representative microscopic images of H&E staining of colon tissues of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 administrated with DSS. The graph depicts the combined histological scores. Mean ± SD, n=3 (DSS), **p < 0.005, Student's unpaired t-test. Scale bar, 200 µm(E) The soluble and insoluble fractionations of lysates from colon tissues of Shigella-infected or DSS-treated Irgm1+/+ and Irgm-/- mice treated with GSK583 as indicated, were subjected to immunoblot analysis with indicated antibodies.(H-I) Western blot analysis with the colon tissue lysates of DSS-treated or S. flexneri infected mice groups as indicated.


Figure 1A . Arginine methylase inhibitor decreases SIRT7 methylation . HEK293T cells expressing Flag - tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs . After immunoprecipitated with Flag - beads , SIRT7 was analyzed by western blot and detected by an antibody against mono - methylarginine ( α - me1 ) . Relative methylation ratio ( Ratio ) was calculated by normalizing arginine methylation level against the Flag - SIRT7 protein . WCL , whole cell lysateC . PRMT inhibitor reduces arginine methylation of wildtype SIRT7 , but not its RK / RF mutants . HEK293T cells expressing wildtype SIRT7 or its RK / RF mutants were treated with or without AdOx for 24 hrs . Arginine methylation of immunopurified SIRT7 was determined by western blotD . SIRT7 is mono - and asymmetrically di - methylated at R388 . Flag - tagged SIRT7 and its RK / RF mutants were expressed in HEK293T cells . Immunopurified SIRT7 was blotted with antibodies against mono - methylarginine ( α - me1 ) , asymmetric di - methylarginine ( α - me2a ) , and symmetric di - methylarginine ( α - me2s ) , respectivelyE . R388 site - specific methylation antibody recognizes R388 - methylated peptide , but not the unmodified peptide . The unmodified peptide corresponding to R388 , R388 - monomethylated ( R388me1 ) peptide , R388 - asymmetrically di - methylated ( R388me2a ) peptide were blotted with α - meSIRT7 ( R388 ) antibodyF . R388 - methylated peptide , but not the unmodified peptide , blocks α - meSIRT7 ( R388 ) antibody . After incubation with unmodified peptide or R388 - methylated peptide ( R388me2a ) , α - meSIRT7 ( R388 ) antibody was used to detect immunopurified SIRT7G . RK and RF mutations disrupt SIRT7 methylation . Flag - tagged wildtype SIRT7 and its RK / RF mutants were immunoprecipitated and blotted with α - meSIRT7 ( R388 ) antibodyH . SIRT7 is methylated at R388 . HEK293T cells ectopically expressing Flag - tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated . SIRT7 protein was pulled down by Flag - beads . Arginine methylation of SIRT7 was detected with a site - specific antibody against R388 methylation [ α - meSIRT7 ( R388 ) ] I - J . AdOx treatment decreases R388 methylation of SIRT7 . MEF and L02 cells were treated with AdOx for 24 hrs . Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot ( I ) . The band intensity of western blots was measured to quantify relative R388 methylation ratio ( J ) ( mean ± SD , n = 3 experimental replicates , * * * p < 0 . 001 , unpaired two - tailed t - test ) K - L . Quantification of R388 - methylated endogenous SIRT7 in HEK293T and L02 cells . R388 - methylated endogenous SIRT7 was captured from HEK293T and L02 cells by using R388 site - specific methylation antibody ( K ) . Input , output , and immunoprecipitate were analyzed by western blot to quantify the percentage of R388 - methylated protein ( L ) ( mean ± SD , n = 3 experimental replicates )

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 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 O O O O O O O O O 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 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 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 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 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

Figure 1A. Arginine methylase inhibitor decreases SIRT7 methylation. HEK293T cells expressing Flag-tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs. After immunoprecipitated with Flag-beads, SIRT7 was analyzed by western blot and detected by an antibody against mono-methylarginine (α-me1). Relative methylation ratio (Ratio) was calculated by normalizing arginine methylation level against the Flag-SIRT7 protein. WCL, whole cell lysateC. PRMT inhibitor reduces arginine methylation of wildtype SIRT7, but not its RK/RF mutants. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. Arginine methylation of immunopurified SIRT7 was determined by western blotD. SIRT7 is mono- and asymmetrically di-methylated at R388. Flag-tagged SIRT7 and its RK/RF mutants were expressed in HEK293T cells. Immunopurified SIRT7 was blotted with antibodies against mono-methylarginine (α-me1), asymmetric di-methylarginine (α-me2a), and symmetric di-methylarginine (α-me2s), respectivelyE. R388 site-specific methylation antibody recognizes R388-methylated peptide, but not the unmodified peptide. The unmodified peptide corresponding to R388, R388-monomethylated (R388me1) peptide, R388-asymmetrically di-methylated (R388me2a) peptide were blotted with α-meSIRT7(R388) antibodyF. R388-methylated peptide, but not the unmodified peptide, blocks α-meSIRT7(R388) antibody. After incubation with unmodified peptide or R388-methylated peptide (R388me2a), α-meSIRT7(R388) antibody was used to detect immunopurified SIRT7G. RK and RF mutations disrupt SIRT7 methylation. Flag-tagged wildtype SIRT7 and its RK/RF mutants were immunoprecipitated and blotted with α-meSIRT7(R388) antibodyH. SIRT7 is methylated at R388. HEK293T cells ectopically expressing Flag-tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated. SIRT7 protein was pulled down by Flag-beads. Arginine methylation of SIRT7 was detected with a site-specific antibody against R388 methylation [α-meSIRT7(R388)]I-J. AdOx treatment decreases R388 methylation of SIRT7. MEF and L02 cells were treated with AdOx for 24 hrs. Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot (I). The band intensity of western blots was measured to quantify relative R388 methylation ratio (J) (mean ± SD, n=3 experimental replicates, ***p < 0.001, unpaired two-tailed t-test)K-L. Quantification of R388-methylated endogenous SIRT7 in HEK293T and L02 cells. R388-methylated endogenous SIRT7 was captured from HEK293T and L02 cells by using R388 site-specific methylation antibody (K). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (L) (mean±SD, n=3 experimental replicates)