( G ) Brightfield ( left middle ) MHC ( right ) of indicated cultures treated or not with bezafibrate . Arrowheads point to bezafibrate - rescued Rb − / − myotubes .

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(G) Brightfield images (left and middle) and MHC staining (right) of the indicated cultures treated or not with bezafibrate. Arrowheads point to bezafibrate-rescued Rb−/− myotubes.


( A ) Immunostaining for MHC ( green ) in indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine . Note shortened myotubes in presence of lonidamine .

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(A) Immunostaining for MHC (green) in the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine. Note the shortened myotubes in the presence of lonidamine.


( B ) Representative immunoblot ( left ) mean expression of myogenin MCK normalized for MHC expression ( right ; n = 3 ) .

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(B) Representative immunoblot (left) and mean expression of myogenin and MCK normalized for MHC expression (right; n = 3).


( C ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre immunoblotted for pRb . Tubulin was used as a loading control .

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(C) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunoblotted for pRb. Tubulin was used as a loading control.


( D ) Rbf / f myoblasts transduced with Ad . GFP or Ad . Cre immunostained for pRb ( red ) . ( insets ) DAPI for nuclei ( blue ) .

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(D) Rbf/f myoblasts transduced with Ad.GFP or Ad.Cre and immunostained for pRb (red). (insets) DAPI staining for nuclei (blue).


( E ) Immunostaining for MHC in Rbf / f myoblasts transduced with Ad . Cre induced to differentiate in normoxia ( top middle ) or hypoxia ( bottom ) .

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(E) Immunostaining for MHC in Rbf/f myoblasts transduced with Ad.Cre and induced to differentiate in normoxia (top and middle) or hypoxia (bottom).


( F , top ) Brightfield of Rbf / f myoblasts transduced with Ad . EV or Ad . Cre induced to differentiate under hypoxia . ( bottom ) Larger view of boxed field . Arrowheads point to rescued Rb − / − myotubes . This field is shown in Video 6 .

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(F, top) Brightfield images of Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. (bottom) Larger view of the boxed field. Arrowheads point to rescued Rb−/− myotubes. This field is shown in Video 6.


( G ) of myotube formation in Rbf / f myoblasts transduced with Ad . EV or Ad . Cre induced to differentiate under hypoxia . are mean of six representative fields ( n = 3 ) . * , P = 0 . 004 .

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(G) Quantification of myotube formation in Rbf/f myoblasts transduced with Ad.EV or Ad.Cre and induced to differentiate under hypoxia. Counts are mean of six representative fields (n = 3). *, P = 0.004.


( H ) of myotube formation in indicated myoblasts induced to differentiate under indicated conditions . Myotube are mean of six fields ( n = 4 ) .

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(H) Quantification of myotube formation in the indicated myoblasts induced to differentiate under the indicated conditions. Myotube counts are the mean of six fields (n = 4).


( I ) MHC ( red ) of indicated myoblasts transduced with Ad . NF - κB / HIF - 1α induced to differentiate in normoxia .

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(I) MHC staining (red) of the indicated myoblasts transduced with Ad.NF-κB/HIF-1α and induced to differentiate in normoxia.


( J ) of myotube formation in indicated myoblasts transduced with Ad . EV or Ad . NF - κB / HIF - 1α induced to differentiate in normoxia . Myotube are mean of six fields ( n = 3 ) . Error bars represent SD .

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(J) Quantification of myotube formation in the indicated myoblasts transduced with Ad.EV or Ad.NF-κB/HIF-1α and induced to differentiate in normoxia. Myotube counts are the mean of six fields (n = 3). Error bars represent SD.


B . Volcano plot showing DE 1hr after NB1 ( top ) IMR32 ( bottom ) treatment with ALKAL2 . Dashed lines show DE thresholds . Up - / downregulated genes indicated in blue . Six genes that are DE in both lines sensitive to ALK inhibitor lorlatinib are indicated labelled in red .

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B. Volcano plot showing DE 1hr after NB1 (top) and IMR32 (bottom) cell treatment with ALKAL2. Dashed lines show DE thresholds. Up-/downregulated genes indicated in blue. Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red.


D . Temporal dynamics of ALKAL2 - induced transcription of ARC , EGR1 - 3 , FOS FOSB in NB1 IMR32 in presence absence of lorlatinib , as indicated .

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D. Temporal dynamics of ALKAL2-induced transcription of ARC, EGR1-3, FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib, as indicated.


E . Immunoblot validation of ALKAL2 induction of EGR1 FOS at level in NB1 . were treated for 0 , 1 6h in presence absence of lorlatinib as indicated .

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E. Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells. Cells were treated for 0, 1 and 6h in the presence and absence of lorlatinib as indicated.


G . Immunoblot of ALKAL2 induction of pSRF in NB1 . were treated for 0 1h in presence absence of lorlatinib as indicated . - Seq was performed using 3 biological repeats . Immunoblots are representative of at least 3 independent experiments .

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G. Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells. Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated. RNA-Seq analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.


B . Volcano plots showing differential expression 1hr 24hr after ALKAL2 + / - lorlatinib stimulation as indicated . Dashed lines indicate differential expression thresholds . Differentially expressed indicated in blue . Most pronounced responding indicated in black labelled .

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B. Volcano plots showing differential protein expression 1hr and 24hr after ALKAL2 +/- lorlatinib stimulation as indicated. Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled.


C , D . Immunoblot of VGF in NB . ( C ) NB1 after 24h stimulation with ALKAL2 in presence or absence of lorlatinib . ( D ) CLB - BAR CLB - GE after 24h inhibition with lorlatinib .

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C, D. Immunoblot analysis of VGF protein in NB cells. (C) NB1 cells after 24h stimulation with ALKAL2 in the presence or absence of lorlatinib. (D) CLB-BAR and CLB-GE cells after 24h inhibition with lorlatinib.


Dashed lines indicate differential expression thresholds . Differentially expressed indicated in blue . Most pronounced responding indicated in black labelled . E . Volcano plots showing differential phosphorylation . Labelling colors as in ( B ) .

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Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled. E. Volcano plots showing differential phosphorylation. Labelling colors as in (B).


G . Graphical representation of FOXO3 dynamics , indicating S253 phosphorylation total FOXO3 in response to ALKAL2 stimulation , in presence or absence of lorlatinib .

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G. Graphical representation of FOXO3 dynamics, indicating S253 phosphorylation and total FOXO3 protein levels in response to ALKAL2 stimulation, in the presence or absence of lorlatinib.


H - I . Immunoblot validation of FOXO3a STAT3 in response to ALKAL2 stimulation in presence or absence of lorlatinib as indicated . slower migrating FOXO3a band in SDS - PAGE in ( H ) likely reflects FOXO3a phosphorylation that is not seen in presence of lorlatinib . Proteomic was performed using 3 biological repeats . Immunoblots are representative of at least 3 independent experiments .

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H-I. Immunoblot validation of FOXO3a and STAT3 in response to ALKAL2 stimulation in the presence or absence of lorlatinib as indicated. The slower migrating FOXO3a band in SDS-PAGE in (H) likely reflects FOXO3a phosphorylation that is not seen in the presence of lorlatinib. Proteomic analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.


A . Hematoxylin eosine of longitudinal sections of P9 pups at central part of left celiac ganglion . B . of of celiac ganglia cross - section . Largest sections from central part of left celiac ganglions of different individuals were chosen for . * * p < 0 . 01 , one - way ANOVA followed by Tukey multiple comparison . Data shown represents mean ± SD . C . Hyperplasia in central sections of P9 left celiac ganglions shown as a of hyperplastic regions areas per ganglion cross - section . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Tukey multiple comparison . Data shown represents mean ± SD .

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A. Hematoxylin and eosine staining of longitudinal sections of P9 pups at the central part of the left celiac ganglion. B. Quantification of the area of celiac ganglia cross-section. Largest sections from the central part of left celiac ganglions of different individuals were chosen for the analysis. **p<0.01, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD. C. Hyperplasia quantification in central sections of P9 left celiac ganglions shown as a percent of hyperplastic regions areas per ganglion cross-section. **p<0.01, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD.


D . Kaplan - Meier survival curve of mice resulting from intercrosses of Th - MYCN hemizygotes Alk - F1178S heterozygote mice ( p < 0 . 0001 ; log - rank ) . Wild type were excluded .

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D. Kaplan-Meier survival curve of mice resulting from intercrosses of Th-MYCN hemizygotes and Alk-F1178S heterozygote mice (p<0.0001; log-rank test). Wild type littermates were excluded.


E . Gross appearance , hematoxylin eosin as well as Ki67 stained sections of representative Th - MYCN Alk - F1178S ; Th - MYCN tumours . Scale bars indicate 250μm .

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E. Gross appearance, hematoxylin and eosin as well as Ki67 stained sections of representative Th-MYCN and Alk-F1178S;Th-MYCN tumours. Scale bars indicate 250μm.


F . Ki67 immunohistochemically of P9 celiac ganglia in mice of indicated genotype ( quantified in G ) . G . of hyperplastic areas ( shown as of hyperplastic regions per ganglion cross - section ) Ki67 expression ( shown as positive for Ki67 areas per total of section through ganglion central part at P9 ) . ( * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA followed by Tukey multiple comparison . Data shown represents mean ± SD ) .

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F. Ki67 immunohistochemically staining of P9 celiac ganglia in mice of the indicated genotype (quantified in G). G. Quantification of hyperplastic areas (shown as percent of hyperplastic regions per ganglion cross-section) and Ki67 expression (shown as positive for Ki67 staining areas per total area of the section through the ganglion central part at P9). (*p<0.05, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD).


A . oncogenic activity of MYCN is potentiated by overexpression of ALKAL2 . Kaplan - Meier survival curves for Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN Th - MYCN mice . Also shown are Rosa26 _ Alkal2N , Alk - F1178S control ( Ctrl ) mice . Comparison of survival of Th - MYCN alone Rosa26 _ Alkal2 ; Th - MYCN curves showed a significant difference ( p = 0 . 003 ; log - rank ) .

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A. The oncogenic activity of MYCN is potentiated by overexpression of ALKAL2. Kaplan-Meier survival curves for Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice. Also shown are Rosa26_Alkal2N, Alk-F1178S and control (Ctrl) mice. Comparison of survival of Th-MYCN alone and Rosa26_Alkal2;Th-MYCN curves showed a significant difference (p=0.003; log-rank test).


Tumours harvested from Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN Th - MYCN mice express NB markers . Tumours from three genotypes were large , in most cases filling abdominal cavity ( B ) .

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Tumours harvested from Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice express NB markers. Tumours from all three genotypes were large, in most cases filling the abdominal cavity (B).


Dissection post - mortem revealed that majority of Rosa26 _ Alkal2 ; Th - MYCN ( 18 / 20 ) Alk - F1178S ; Th - MYCN ( 7 / 10 ) tumours did not involve adrenal ( C ) .

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Dissection post-mortem revealed that the majority of Rosa26_Alkal2;Th-MYCN (18/20) and Alk-F1178S;Th-MYCN (7/10) tumours did not involve the adrenal glands (C).


Histological examination of Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN Th - MYCN tumours revealed positive for NCAM1 , synaptophysin ( SYP ) Chromogranin A ( CGA ) . Scale bars indicate 100μm . ( D )

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Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours revealed positive staining for NCAM1, synaptophysin (SYP) and Chromogranin A (CGA). Scale bars indicate 100μm. (D)


Histological examination of Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN Th - MYCN tumours was confirmed for NCAM1 SYP along with MYCN , ALK ALKAL2 by immunoblotting ( E ) . Immunoblots are representative of 3 independent technical analyses .

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Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours was confirmed for NCAM1 and SYP along with MYCN, ALK and ALKAL2 by immunoblotting (E). Immunoblots are representative of 3 independent technical analyses.


F . Accumulated Kaplan - Meier survival curves are shown for monitored Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN Th - MYCN mice over time , estimating tumour penetrance ( p < 0 . 001 ; log - rank ) .

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F. Accumulated Kaplan-Meier survival curves are shown for all monitored Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice over time, estimating tumour penetrance (p<0.001; log-rank test).


- Seq differential gene expression of tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( Alkal2 ) [ n = 6 ] , Alk - F1178S ; Th - MYCN ( AlkF1174S ) [ n = 6 ] Th - MYCN ( MYCN ) mice [ n = 4 ] . See Table S4 for detailed results . A . Read coverage of codon - optimised Alkal2 transgene , confirming Alkal2 expression in Alkal2 tumours .

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RNA-Seq based differential gene expression analysis of tumours arising in Rosa26_Alkal2;Th-MYCN (Alkal2) [n=6], Alk-F1178S;Th-MYCN (AlkF1174S) [n=6] and Th-MYCN (MYCN) mice [n=4]. See Table S4 for detailed results. A. Read coverage of the codon-optimised Alkal2 transgene, confirming Alkal2 expression in Alkal2 tumours.


C - D . Volcano plot showing differential expression ( DE ) between Alkal2 MYCN tumours . Differentially expressed genes are shown in blue ( DE in Alkal2 tumours only ) or black ( DE in both Alkal2 AlkF1174S tumours , as shown in ( D ) ) . Top ranked genes labelled . Dashed lines represent DE cut - offs .

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C-D. Volcano plot showing differential expression (DE) between Alkal2 and MYCN tumours. Differentially expressed genes are shown in blue (DE in Alkal2 tumours only) or black (DE in both Alkal2 and AlkF1174S tumours, as shown in (D)). Top ranked genes labelled. Dashed lines represent DE cut-offs.


E . DE on unsupervised hierarchical clustering of 400 DE genes ( rows ) 16 samples ( columns , as indicated on top ) . Sample color legend as in ( A ) . Color key shown on top left .

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E. DE heatmap based on unsupervised hierarchical clustering of 400 DE genes (rows) and 16 samples (columns, as indicated on top). Sample color legend as in (A). Color key shown on top left.


F . Boxplot showing Vgf expression in 3 tumour types as indicated . Box plots indicate median values lower / upper quartiles with whiskers extending to 1 . 5 times interquartile range . P values calculated using Wald as reported by DESeq2 .

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F. Boxplot showing Vgf expression in the 3 tumour types as indicated. Box plots indicate median values and lower/upper quartiles with whiskers extending to 1.5 times the interquartile range. P values calculated using Wald test as reported by DESeq2.


G . Histological examination of Th - MYCN , Rosa26 _ Alkal2 ; Th - null Alk - F1178S ; Th - MYCN tumours revealing positive for VGF . Scale bars indicate 100μm .

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G. Histological examination of Th-MYCN, Rosa26_Alkal2;Th-null and Alk-F1178S;Th-MYCN tumours revealing positive staining for VGF. Scale bars indicate 100μm.


H . Kaplan - Meier relapse free ( RF ) event free ( EF ) survival probability curves from two different NB cohorts , Versteeg 88 cohort ( upper panel ) Kocak 649 cohort ( lower panel ) , as derived from R2 platform . with higher VGF expression are highlighted in blue , whereas with lower expression are highlighted in red . log - rank P values are indicated .

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H. Kaplan-Meier relapse free (RF) and event free (EF) survival probability curves from two different NB cohorts, the Versteeg 88 cohort (upper panel) and the Kocak 649 cohort (lower panel), as derived from the R2 platform. Patients with higher VGF expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P values are indicated.


Murine NB lines were generated from tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) Alk - F1178S ; Th - MYCN ( # 3456 ) mice . A . Alkal2 expression in derived from Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) Alk - F1178S ; Th - MYCN ( # 3456 ) NB . Immunoblotting for ALKAL2 tubulin in indicated mouse NB lines . Whole lysates ( 30μg ) were analysed in each lane .

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Murine NB cell lines were generated from tumours arising in Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) mice. A. Alkal2 expression in cells derived from Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) NB. Immunoblotting analysis for ALKAL2 and tubulin in the indicated mouse NB cell lines. Whole cell lysates (30μg) were analysed in each lane.


B . effect of increasing concentrations of brigatintib on confluence was analyzed by IncuCyte Live of both Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) Alk - F1178S ; Th - MYCN ( # 3456 ) lines . Data is presented as mean ± from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - paired student t - .

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B. The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) cell lines. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.


C - D . Brigatinib suppressed tumour spheroid formation spheroid viability . ( # 3456 or # 3540 ) were treated with brigatinib ( 0 , 150nM ) for 4d in ultra - low attachment plates . spheroid number was analyzed by IncuCyte ® Live Systems . Data are presented as means ± from three independent experiments . * p < 0 . 05 , two - unpaired student t - . Scale bar ( C ) is 200μm .

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C-D. Brigatinib suppressed tumour spheroid formation and spheroid viability. Cells (#3456 or #3540) were treated with brigatinib (0, 150nM) for 4d in ultra-low attachment plates. The spheroid number was analyzed by IncuCyte® Live Cell Analysis Systems. Data are presented as means ± SEM from three independent experiments. *p<0.05, two-tailed unpaired student t-test. Scale bar (C) is 200μm.


E . Tumour spheroids formed from either Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) or Alk - F1178S ; Th - MYCN ( # 3456 ) were formed at indicated number in ultra - low attachment plates for 3d followed by brigatinib ( 0 , 150nM ) for 10d . Inhibitor was re - fed every other day . viability was determined by CellTiter - Glo 3D viability kit data is presented as mean ± from five independent experiments . * p < 0 . 05 , * * * * p < 0 . 0001 , two - unpaired student t - .

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E. Tumour spheroids formed from either Rosa26_Alkal2;Th-MYCN (#3540) or Alk-F1178S;Th-MYCN (#3456) were formed at indicated cell number in ultra-low attachment plates for 3d and followed by brigatinib (0, 150nM) for 10d. Inhibitor was re-fed every other day. Cell viability was determined by CellTiter-Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments. *p<0.05, ****p<0.0001, two-tailed unpaired student t-test.


F . viability . Mouse tumour derived lines # 3540 ( Rosa26 _ Alkal2 ; Th - MYCN ) # 3456 ( Alk - F1178S ; Th - MYCN ) were treated with brigatinib ( 125nM 500nM ) viability was evaluated by using a resazurin - . Data is presented as mean ± from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - paired student t - .

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F. Cell viability. Mouse tumour derived cell lines #3540 (Rosa26_Alkal2;Th-MYCN) and #3456 (Alk-F1178S;Th-MYCN) were treated with brigatinib (125nM and 500nM) and viability was evaluated by using a resazurin-based assay. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.


G . Brigatinib treatment ( 0 or 150 nM ) for 6h resulted in inhibition of ALK phosphorylation , of activation of downstream signaling ( ERK1 / 2 ) , as well as MYCN expression . lysates ( Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) Alk - F1178S ; Th - MYCN ( # 3456 ) ) were immunoblotted with indicated antibodies .

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G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.


A . derived from tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( # 4953 ) Alk - F1178S ; Th - MYCN ( # 4938 ) mice are sensitive to both lorlatinib brigatinib . effect of increasing concentrations of each ALK TKI ( as indicated ) on confluence was analyzed by IncuCyte Live . Data is presented as mean ± from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - paired student t - .

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A. 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 7 , by direct measurement at Day 14 . Day 0 ( lorlatinib n = 7 , Ctrl n = 6 ) , Day 7 ( lorlatinib n = 2 , Ctrl n = 5 ) Day 14 ( lorlatinib n = 7 , Ctrl n = 6 ) . Data shown represents mean ± SD . * * p < 0 . 005 ; two - unpaired student t - .

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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 treated with lorlatinib did not display any significant loss of compared with vehicle controls . Data shown represents mean ± SD .

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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 of tumours observed in Rosa26 _ Alkal2 ; Th - MYCN mice with annotated measurements at Day 0 Day 7 . Tumours arise in retroperitoneal space ventral to aorta , Ao .

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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 14d .

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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 .

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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.


Schematic diagram of ATXN8OS ( top strand ) ATXN8 ( bottom strand ) . - seq read coverage visualized by Integrative Genomic Viewer across ATXN8 / ATXN8OS locus is depicted .

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Schematic 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 of ATXN8 ATXN8OS . FPKM values for each transcript are normalized to FPKM value for ATXN8OS . ( * * * * ; p < 0 . 0001 ; mean ± ; unpaired t ) FPKM = fragments per kilo base of transcript per million mapped reads .

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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 ( detected by unique antibody to polySer C - terminus , α - SerCT ) in cerebellar matter , brainstem , hippocampus layer II of frontal cortex . Representative polySer are indicated by black arrows . No are found in matched non - transgenic ( NT ) ( n = 6 for each cohort ) .

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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 in cerebellar matter , brainstem frontal cortex .

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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.


A Immunohistochemistry ( ) of end - stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in molecular layer cerebellar matter that polyGln but not polySer accumulates in Purkinje . Representative polySer are indicated by black arrows . Inset : higher magnification of molecular layer matter .

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A 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 shows no co - localization of polyGln polySer in frontal cortex , pons or hippocampus of end - stage SCA8 BAC mice .

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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 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 frontal cortex including within neurons ( mouse α - NeuN , green , top panel ) .

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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 of SCA8 human cerebellum shows that PolySer accumulates in matter but not in Purkinje ( left panels ) while polyGln accumulates in Purkinje but not cerebellar matter ( right panels ) .

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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).


A Representative of vestibular nucleus ( upper panels ) , cuneate nucleus ( middle panels ) motor cortex layers II / III ( lower panels ) of SCA8 BAC mice at 2 months ( left panels ) , 6 months ( middle panels ) end - stage ( 10 months , right panels ) stained with α - SerCT . Representative are indicated by black arrows . Corresponding quantifications of polySer for each region on right ( n = 3 ; mean ± ; One - way ANOVA with Tukey ' s post - hoc ; * p < 0 . 025 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 )

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A 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 of 2 month old show a significant decrease in ambulatory ( cm ) ambulatory time ( s ) a significant increase in resting time ( s ) ( NT n = 19 , SCA8 BAC n = 23 , * * p < 0 . 01 , * * * p < 0 . 001 ; mean ± ; unpaired t ) .

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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).


A Disease - specific sensitivity to vacuolization after prolonged storage in ethanol shown by H & E in cerebellar matter brainstem of 10 - 12 month old end - stage SCA8 BAC mice but not in matched NT ( n = 3 ) ( panels 1 & 2 ) . Demyelination shown by luxol fast blue ( LFB ) ( panel 3 ) 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 are indicated by black arrows ) in deep cerebellar matter brainstem in SCA8 BAC mice ( lower panels ) but not in NT mice ( upper panels ) ( n = 3 ) .

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A 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 ( LFB ) ( panel 1 ) axonal degeneration shown by α - SMI - 32 ( panel 2 ) observed sites of polySer accumulation shown by a - SerCT2 ( panel 3 ) in deep cerebellar matter was found in SCA8 autopsy tissue but not in control brains ( n = 3 ) .

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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 ± ; unpaired t ) .

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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 ± ; unpaired t ) .

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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 measured by lactase dehydrogenase ( LDH ) in T98 transfected with codon replacement constructs expressing polyGln ( blue ) or polySer ( blue ) . NT = nontransfected , EV = empty vector ( n = 5 , * * p < 0 . 003 ; * * * p < 0 . 0003 ; mean ± ; unpaired t ) .

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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).


qRT - PCR showing Eif3f expression are increased 2 - fold in SCA8 cerebellar matter compared to SCA8 cerebellar grey matter ( n = 3 , * * p < 0 . 01 ; mean ± ; unpaired t ) .

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qRT-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 are co - transfected with eIF3F siRNA . D of polySer detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± ; unpaired t ) .

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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 are co - transfected with eIF3F siRNA . F Detection of polyGP expression using α - FLAG antibody showing a decrease in RAN polyGP when HEK293 are co - transfected with eIF3F siRNA . . G of polyAla polyGP detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± ; unpaired t ) .

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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).


( B ) Volcano plot of 8 , 701 mRNA isoforms from 2 , 931 genes with multiple isoforms . X - axis indicates a difference in of each mRNA isoform within gene ( Δ % smg - 2 - N2 ) . Y - axis indicates - log10 of p - value in Fisher ' s exact . Blue symbols indicate 219 mRNA isoforms significantly depleted from smg - 2 mutant ( p < 0 . 05 , Δ % smg - 2 - N2 < - 10 ) . Red symbols indicate 420 isoforms significantly enriched in smg - 2 mutant ( p < 0 . 05 , Δ % smg - 2 - N2 > 10 ) .

O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O

(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 ) distribution of 3 ' UTRs ( C ) proportions of putative PTC - containing isoforms ( D ) in 10 , 136 mRNA isoforms detected in N2 / or smg - 2 ( ) , 219 isoforms depleted from 420 isoforms enriched in smg - 2 mutant .

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(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.


( B ) Alternative splicing of sams - 3 , sams - 4 sams - 5 in unfed fed worms without or with a synthesis inhibitor emetine . Synchronized L1 larvae of an NMD - deficient KH1668 : smg - 2 ( yb979 ) ( lanes 1 - 3 ) a wild - type N2 ( lanes 4 - 6 ) were incubated in S - complete medium alone ( lanes 1 , 4 ) , with a standard E . coli 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 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 right . Open reading frames ( ORFs ) for full - truncated are in orange cyan , respectively . rpl - 12 was used as an unaffected control .

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(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.


( A ) Alternative splicing of sams - 3 , sams - 4 sams - 5 in smg - 2 ( yb979 ) smg - 2 ( yb979 ) ; sams - 5 ( gk147 ) ; sams - 1 ( ok2946 ) mutants . Synchronized L1 larvae of each were incubated in S - complete medium alone ( lanes 1 5 ) , with OP50 ( OD600 = 10 . 0 ) ( lanes 2 6 ) with OP50 supplemented with 25 mM L - Met ( lanes 3 7 ) or 25 mM cycloleucine ( cLeu ) ( lanes 4 8 ) for 3 hours at 20 ° C . splicing patterns were analyzed presented as in Figure 2B ( n = 3 ) .

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(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 of SAMS - 1 , SAMS - 3 SAMS - 4 during larval development in smg - 2 ( yb979 ) ( B ) wild - type ( C ) backgrounds . Genotypes of worms are smg - 2 ( w ) smg - 2 ; sams - 5 ; sams - 1 ( s ) in ( B ) wild - type ( w ) sams - 1 ( s ) in ( C ) . Synchronized L1 larvae of each were incubated with OP50 at 20 ° C subjected to Western blot at indicated time points . Anti - β - tubulin ( B ) or coomassie brilliant blue ( CBB ) ( C ) was used as a loading control . Specificity of antibodies is confirmed in Appendix Figure S12 . Note that upregulation of SAMS - 1 in wild type during larval development in ( C ) is consistent with feeding - induced upregulation of sams - 1 mRNA in Figure EV3D .

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(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.


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

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(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.


( A ) Mass chromatograms of fragments to detect m6A ( top ) or unmodified A ( bottom ) after in vitro incubation of 127 - nt sams - 3 / sams - 4 pre - mRNA ( available in Fig EV2 ) with recombinant full - METT - 10 in presence ( left ) or absence ( right ) of 1 mM SAM . , m / z value , charge state for each fragment are indicated on right . Asterisks indicate non - specific signals .

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(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 ) negatively - charged ions of RNase T1 fragment is decomposed in instrument by collision - induced dissociation ( CID ) using helium . product ions produced by CID are assigned on illustrated on top right inset panel . Nomenclature of product ions are described in literature ( McLuckey et al , 1992 ) . Product ions of c y series derive from 5 ' 3 ' termini of fragment , respectively .

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(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.


A , B ) m6A - IP specifically enriched sams mRNA isoforms that retain AG dinucleotide at distal / productive 3 ' SS . ( A ) Representative gel - like ( n = 3 ) of semi - quantitative RT - PCR of sams - 3 , sams - 4 sams - 5 in input immunoprecipitated ( IP ) RNAs from smg - 2 ( yb979 ) mutant . splicing patterns were analyzed presented as in Figure 2B . ( B ) Relative enrichment of total each of mRNA isoforms compared to eef - 1A . 1 mRNA by RT - qPCR . Error bars indicate standard error of mean ( n = 3 ) . (

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A, 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 m6A - modified in - vitro transcribed sams - 3 / sams - 4 RNAs at nucleotide positions - 4 through + 4 relevant to m6A site . One hundred reads are lotted for each . Color codes are indicated . ( D ) Distribution of mean ( top ) standard deviation ( bottom ) of normalized Nanopore currents at nucleotide positions - 4 through + 4 relevant to m6A site for 5 , 000 of unmodified 2 , 680 of m6A - modified sams - 3 / sams - 4 RNAs 85 of an endogenous sams - 3 mRNA isoform E2 / E3L . Color codes are indicated . Central bands represent median , boxes represent 25th 75th percentiles , whiskers represent lowest highest values . A red at position 0 in top panel indicates a cut - off ( mean current = 1 . 7557 ) that discriminates between unmodified m6A - modified sams - 3 / sams - 4 RNAs with accuracies of 64 . 58 % for both . (

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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. (


( A sequencing of CD31 CD34 double - positive retinal endothelial from P5 wild - type mice . ( A ) ( FPKM ; fragments per kilobase million ) of marker genes of endothelial ( green ; CD31 ( gene name PECAM1 ) , CD34 , von Willebrand Factor ( vWF ) , tyrosine - kinase receptor Tie2 ( TEK ) , VE - cadherin ( CDH5 ) , endoglin ( ENG ) , CD146 ( MCAM ) VEGFR2 ( KDR ) ) , astrocytes ( blue , GFAP ) , immune ( red ; T - ( CD3E ) , B - ( CD19 ) , leucocytes ( CD45 , PTPRC ) , monocytes / macrophages ( F4 / 80 , EMR1 ) ) , Müller glia ( orange ; aquaporin 4 ( AQP4 ) ) , neurons ( yellow ; retinal ganglion ( binding fox - 1 homolog 3 , RBFOX3 ) , amacrine ( parvalbumin , PVALB ) , bipolar ( PKC - α , PRKCA ) , horizontal ( calbindin , CALB1 ) , photoreceptors ( rods , CD73 ( NT5E ) ; cones , transducing ( GNAT1 ) ) , retinal pigment epithelial ( magenta ; retinal pigment epithelium - specific 65 kDa ( RPE65 ) .

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(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 ) sequencing of CD31 CD34 double - positive retinal endothelial from P5 wild - type mice . ( B ) of VASP ( red ) , EVL ( green ) Mena ( blue ) in CD31 CD34 double - positive P5 retinal endothelial . Error bars represent ; n = 18 ( 36 retinas ) from six independent litters ; four independent experiments . * * * p < 0 . 001 , one - way ANOVA with Bonferroni ' s multi comparison .

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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 ) of Ena / VASP in blood vessels of P5 mouse retinas . P5 wild - type mouse retinas were fixed stained with isolectin B4 ( IB4 , green ) to visualize endothelial VASP - ( C ) , EVL - ( D ) , or Mena - specific ( E ) antibodies ( red ) . Yellow color in merged indicates expression of VASP EVL in endothelial . No Mena expression was detected in P5 retinal endothelial ( E ) . Representative from three independent experiments are shown . Scale bars , 10 µm .

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(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.


( C - E ) of Ena / VASP in blood vessels of P5 mouse retinas . P5 wild - type mouse retinas were fixed stained with isolectin B4 ( IB4 , green ) to visualize endothelial

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(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


( A ) Wild - type EVL - / - P5 retinas were digested , labelled with CD31 - , CD34 - EVL - specific antibodies analyzed by flow cytometry . Endothelial were defined as CD31 / CD34 - double positive ( gate indicated in left panel ) analyzed for EVL expression ( middle panel ) . Mean ( MFI ) of EVL in wild - type ( magenta ) EVL - / - ( black ) is shown in right panel . WT : n = 3 , EVL - / - : n = 2 ; two retinas per . Error bars represent .

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(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 expression in sparse / migrating wild - type ( WT ) EVL - / - mouse brain endothelial ( MBEC ) mouse lung endothelial ( MLEC ) . Actin was used as loading control . Western blots are representative of three independent experiments .

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(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 ) EVL - / - ( lower panel ) mice stimulated with 10 ng / ml VEGF were stained for actin ( cyan ) EVL ( magenta ) . Asterisks indicate focal adhesions , arrows indicate filopodia arrowheads indicate leading edge of lamellipodia . Representative from four independent experiments are shown . Scale bar , 20 µm .

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(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.


( A ) Isolectin B4 stained vasculature in whole mount retinas of wild - type ( WT ) global EVL - / - mice on postnatal days 3 , 5 7 ( P3 , P5 , P7 ) assessed by confocal microscopy . Scale bars 200 µm .

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(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 ) of radial vascular outgrowth relative to retinal radius normalized to wild - type . ( C ) Impact of combined VASP / EVL deletion on sprouting angiogenesis in postnatal mouse retina at P5 ( EVL - deficient ( E - / - ) , green ; VASP - deficient ( V - / - ) , red ; VASP / EVL - double deficient ( EV - / - ) , orange ) .

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(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).


( B ) of radial vascular outgrowth relative to retinal radius normalized to wild - type .

(B) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates.


retinal vasculature of endothelial specific EVL knockout mice ( EVLΔEC ) littermate controls ( EVLfl / fl ) on P5 was analyzed by Isolectin B4 .

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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 retinal vasculature ; in global EVL - / - mice are shown for comparison ; 4 - 5 independent experiments .

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Radial outgrowth of the retinal vasculature; in global EVL-/- mice are shown for comparison; 4 - 5 independent experiments.


( C ) of branch points in a field of 170 μm x 300 μm directly behind vascular front . ( D ) Tipp numbers per field of view ( 387 , 5 x 387 , 5 µm ) .

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(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).


( C ) of branch points in a field of 170 μm x 300 μm directly behind vascular front .

(C) Quantification of the branch points in a field of 170 μm x 300 μm directly behind the vascular front.


( E ) Filopodia numbers normalized to of angiogenic front .

(E) Filopodia numbers normalized to the length of the angiogenic front.


( F ) Endothelial proliferation analyzed by BrdU incorporation ( red ) of ERG positive ( blue ) endothelial nuclei .

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(F) Endothelial cell proliferation analyzed by BrdU incorporation (red) of ERG positive (blue) endothelial nuclei.


CD31 CD34 double positive endothelial were isolated from wild - type or EVL - / - P5 retinas by FACS analyzed by - sequencing . Two independent experiments ; in total 18 retinas from WT mice 18 retinas from EVL - / - mice from three different litters , each . ( A ) ( FPKM ) of VASP , EVL Mena in endothelial from wild - type or EVL - / - retinas . Error bars represent .

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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.


CD31 CD34 double positive endothelial were isolated from wild - type or EVL - / - P5 retinas by FACS analyzed by - sequencing . Two independent experiments ; in total 18 retinas from WT mice 18 retinas from EVL - / - mice from three different litters , each .

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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.


( D ) of mRNA of Esm1 , Ptgs2 ( cyclooxygenase 2 ) , serpine 1 paxillin in EVL - deficient MLEC relative to WT controls set to 100 . n = 3 - 4 independent experiments , error bars represent , one sample t - , * p < 0 . 05 ; * * p < 0 . 01 .

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(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 expression in P5 retinas of wild - type ( WT ) global EVL - / - mice . Retinas were fixed stained with isolectin B4 ( IB4 , green ) to visualize endothelial antibodies directed against tip marker ESM1 ( red ) . Representative from three independent experiments are shown . Scale bars , 100 µm .

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(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 ) of retinal ESM1 normalized to wild - type . Error bars represent ; * * * p < 0 . 001 , unpaired Student ' s t - ; three different litters .

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(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.


( A ) Proliferation of WT EVL - / - MLEC was assessed by BrdU incorporation . number of BrdU positive ( red ) was normalized to total number of ( ) . Eight fields of view per condition genotype for each of 5 independent WT EVL - / - batches ; error bars represent ; * * p < 0 . 01 , * * * p < 0 . 001 , # p = 0 . 171 non - significant vs . Basal EVL - / - , one - way ANOVA with Bonferroni ' s multi comparison ; scale bar = 50 µm .

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(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 EVL - / - MLEC spheres in absence ( Basal ) or presence of VEGF ( 50 ng / ml ) ; asterisks indicate sprouts ; scale bars , 100 µm . ( D ) Solid bars represent proportion of WT EVL - / - MLEC spheres forming sprouts in absence ( − ) or presence ( + ) of VEGF . n - numbers are indicated ; n . s . non - significant ( p = 0 . 557 ) ; * * * p < 0 . 001 , Fisher ' s exact . ( E ) of MLEC sphere sprout without or with VEGF . Dots represent sprouts . Horizontal lines represent mean , error bars represent . 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 .

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(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.


( C ) Sprouting of WT EVL - / - MLEC spheres in absence ( Basal ) or presence of VEGF ( 50 ng / ml ) ; asterisks indicate sprouts ; scale bars , 100 µm .

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(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.


( A ) VEGFR2 in wild - type ( WT ) EVL - deficient ( EVL - / - ) endothelial . Actin was used as loading control . lower panel shows of VEGFR2 to actin ratios from five independent batches ; error bars represent ; n . s . non - significant ( p = 0 . 388 ) , unpaired Student ' s t - .

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(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 to monitor VEGFR2 internalization after VEGF stimulation . Left panel : schematic diagram showing two step procedure of surface internalized VEGFR2 antibodies , which are displayed in yellow green in merged confocal of WT EVL - / - endothelial ( middle panel ) , respectively ; scale bar , 10 µm . bar graphs show of number of VEGFR2 clusters of internalized to total VEGFR2 ratios normalized to wild - type ; n = 4 - 6 independent batches , error bars represent ; * * p < 0 . 01 , * * * p < 0 . 001 , unpaired Student ' s t - .

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(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.


( A ) Western blots of VEGFR2 phosphorylation relative to total VEGFR2 in WT EVL - / - endothelial under basal VEGF - stimulated conditions ( 80 ng / ml ) ; n = 5 independent batches , error bars represent ; * p < 0 . 05 , one - way ANOVA with Bonferroni ' s multi comparison .

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(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 of ERK1 / 2 phosphorylation relative to total ERK in WT EVL - / - endothelial under basal VEGF - stimulated conditions ( 80 ng / ml ) ; n = 6 independent batches , error bars represent ; * p < 0 . 05 , one - way ANOVA with Bonferroni ' s multi comparison .

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(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 ) global EVL - / - mice . Retinas were fixed stained with isolectin B4 ( IB4 , green ) to visualize endothelial antibodies directed against phospho - ERK1 / 2 ( red ) . Representative from three independent experiments are shown . Scale bars , 100 µm . ( D ) of endothelial ERK1 / 2 - phosphorylation normalized to wild - type . Error bars represent ; * * * p < 0 . 001 , unpaired Student ' s t - ; two different litters .

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(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.


( C ) ERK1 / 2 phosphorylation in P5 retinas of wild - type ( WT ) global EVL - / - mice . Retinas were fixed stained with isolectin B4 ( IB4 , green ) to visualize endothelial antibodies directed against phospho - ERK1 / 2 ( red ) . Representative from three independent experiments are shown . Scale bars , 100 µm .

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(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.


( B ) Representative high content microscopy of RIPosomes in THP - 1 infected with S . flexneri ( MOI 1 : 25 , 8 h ) . About 50000 were plated per well RIPosomes were screened in 35 fields per well in 3 technical replicates . Right panel , graph depicts an average number of RIPosomes / , which is calculated from 3 biological replicates , Mean ± SD . * * * p < 0 . 005 , Student ' s unpaired t - .

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(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 of soluble insoluble fractions of S . flexneri infected THP - 1 ( MOI 1 : 25 , 8 h ) . Arrow indicates oligomers of RIPK2 .

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(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 RIPK2 knockdown THP - 1 infected with S . flexneri ( MOI 1 : 25 , 8 h ) . Right panel , graph depicts an average number of RIPosomes / , which is calculated from 3 biological replicates , Mean ± SD . * * * p < 0 . 0005 , Student ' s unpaired t - .

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(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 of soluble insoluble fractions of S . flexneri infected ( E ) control siRNA RIPK2 siRNA transfected THP - 1 ( F ) Control CRISPR ( RIPK2 + / + ) RIPK2 CRISPR knockout ( RIPK2 - / - ) HT - 29 .

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(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 of RIPosomes in mouse BMDMs infected with S . flexneri ( MOI 1 : 25 , 8 h ) . profile : co - localization using profile . Scale bar , 5 µm . Zoom panels are digital magnifications . is stained with DAPI ( pseudo - colored red for better contrast ) .

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(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 of RIPosomes in THP - 1 infected with RFP expressing Salmonella typhimurium ( MOI 1 : 5 , 4 h ) . profile : co - localization using profile . Scale bar , 8 µm . Zoom panels are digital magnifications . is stained with DAPI .

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(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 ( yellow masks represent software algorithms - defined boundaries ) of RIPosomes in THP - 1 infected with S . typhimurium ( MOI 1 : 5 , 4 h ) . Right panel , graph depicts an average number of RIPosomes / . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - . is stained with DAPI .

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(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 of RIPosomes in THP - 1 infected with ( K ) E . coli LF82 ( MOI 1 : 10 , 8h ) ( LPS antibody is used to stain LF82 ) profile : co - localization using profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . 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 of ( M ) GFP expressing M . tuberculosis ( MOI 1 : 10 , 8 h ) . profile : co - localization using profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . is stained with DAPI .

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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 of soluble insoluble fractions from THP - 1 , uninfected or infected with E . coli DH5α ( MOI 1 : 10 , 8h ) or E . coli LF82 strains ( MOI 1 : 10 , 8h ) .

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(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).


( B ) Left panel , representative high - content microscopy of HEK293T transfected with GFP - NOD1 , GFP - NOD2 , GFP - RIPK2 plasmids for 9 h . Right panel , graph depicts average number of RIPosomes per GFP - positive . About 15000 were plated per well 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 ) .

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(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 of soluble insoluble fractions of HEK293T transfected with CARD domain - containing region of NOD1 , NOD2 , RIPK2 .

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(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 ) soluble insoluble fractions of S . flexneri infected THP - 1 ( MOI 1 : 25 , 8 h ) lysates were subjected to Western blot with indicated antibodies .

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(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 of immunofluorescence assays conducted with THP - 1 infected with RFP expressing S . flexneri ( pseudo - colored , blue ) ( MOI 1 : 25 , 8h ) . profile : co - localization using profiles . Scale bar , 5 µm . Zoom panels are digital magnifications .

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(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 of immunofluorescence assays performed with HEK293T transfected with GFP - RIPK2 Flag - NOD2 ( upper panel ) or Flag - NOD1 ( lower panel ) . profile : co - localization using profiles . Scale bar , 5 or 10 µm as indicated in figures . Inset 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 were transfected with ( H ) full ( I ) CARD domains of NOD1 , NOD2 , RIPK2 followed by Western blot with soluble insoluble fractions using indicated antibodies .

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(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 of soluble insoluble fractions of RIPK2 + / + RIPK2 - / - HT - 29 infected with S . flexneri ( MOI 1 : 25 , 8 h ) with indicated antibodies .

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(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 of doxycycline - inducible GFP - RIPK2 expressing HeLa . ( L ) Upper panel , uninfected . Lower panel , S . flexneri - infected MOI 1 : 25 , 4h ) . Immunostaining was performed with p65 antibody ( red ) stained with DAPI ( Blue ) . ( M ) graph indicates % of that are RIPosomes positive or negative with / p65 ( 5 fields ( each group ) , Mean ± SD , n = 3 ) . * * * * p < 0 . 00005 , Student ' s unpaired t - .

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(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.


( A - B ) Western blot with lysate of uninfected S . flexneri infected ( MOI 1 : 25 , 8 h ) ( A ) control ATG5 siRNA knockdown THP - 1 , ( B ) wild type ( WT ) ATG5 knockout ( ATG5 - / - ) MEF with indicated antibodies . Densitometric was performed using Image J software . FC , fold change .

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(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 of RIPosomes in HEK293T that are control or treated with rapamycin ( 500 nM , 4h ) or treated with rapamycin ( 500 nM , 4h ) bafilomycin A1 ( BafA1 , 300 nM ) . Right panel , graph depicts average number of RIPosomes / . About 15000 were plated per well RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * p < 0 . 005 , * * * p < 0 . 0005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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).