C ) Nuclear and cytoplasmic full - length RNA - Seq data from AC16 cells kept at 37 ° C or exposed to 18 ° C for 24h for REV - ERBα and CRY2 . Data for each track have been normalized to show counts per million aligned counts ( CPM ) for each nucleotide . Number of 3 ' end RNA - seq sample replicates : 37 ° C ( C : 6 , N : 7 ) , 18 ° C 5h ( 4 ) , 10h ( 4 ) , 24h ( 6 ) , 18 ° C 24h then 37 ° C 2h ( 4 ) , 5h ( 2 ) , 10h ( 2 ) , 24h ( 2 ) . Number of cytoplasmic ( C ) and nuclear ( N ) samples are the same for each condition except 37 °

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°C


Western blot of Flag - tagged REV - ERBα levels ( short and long exposure ) in whole cell extracts from AC16 cells transferred from 37 ° C to 18 ° C for the time periods indicated and also returned after each of these time periods to 37 ° C for 2h . TP53 was used as a loading control as its transcript levels show minimal changes in response to 18 ° C exposure Ponceau stain is shown as an additional loading control .

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

Western blot of Flag-tagged REV-ERBα levels (short and long exposure) in whole cell extracts from AC16 cells transferred from 37°C to 18°C for the time periods indicated and also returned after each of these time periods to 37°C for 2h. TP53 was used as a loading control as its transcript levels show minimal changes in response to 18°C exposure Ponceau stain is shown as an additional loading control.


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV -

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Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for CRY2 ( B )

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

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for CRY2 (B)


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for TP53 ( C )

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for TP53 (C)


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) U2OS cells ( D ) , exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV -

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Representative maximum intensity z-stack-projected images of DAPI-stained (blue) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα


Mean REV - ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( REV - ERBα ) : 37 ° C ( 65 ) , 18 ° C 5h ( 30 ) , 18 ° C 5h then 37 ° C 2h ( 21 ) , 18 ° C 24h ( 63 ) , 18 ° C 24h then 37 ° C 2h ( 64 )

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

Mean REV-ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (REV-ERBα): 37°C (65), 18°C 5h (30), 18°C 5h then 37°C 2h (21), 18°C 24h (63), 18°C 24h then 37°C 2h (64)


Mean CRY2 ( F ) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . ; AC16 cells ( CRY2 ) : 37 ° C ( 43 ) , 18 ° C 24h ( 42 ) , 18 ° C 24h then 37 ° C 2h ( 42 )

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

Mean CRY2 (F) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. ; AC16 cells (CRY2): 37°C (43), 18°C 24h (42), 18°C 24h then 37°C 2h (42)


Mean TP53 ( G ) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( TP53 ) : 37 ° C ( 47 ) , 18 ° C 24h ( 45 ) , 18 ° C 24h then 37 ° C 2h ( 48 )

Mean TP53 (G) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (TP53): 37°C (47), 18°C 24h (45), 18°C 24h then 37°C 2h (48)


Mean REV - ERBα per U2OS ( H ) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . U2OS cells ( REV - ERBα ) : 37 ° C ( 61 ) , 18 ° C 5h ( 61 ) , 18 ° C 5h then 37 ° C 2h ( 69 ) , 18 ° C 24h ( 53 ) , 18 ° C 24h then 37 ° C 2h ( 65 )

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

Mean REV-ERBα per U2OS (H) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. U2OS cells (REV-ERBα): 37°C (61), 18°C 5h (61), 18°C 5h then 37°C 2h (69), 18°C 24h (53), 18°C 24h then 37°C 2h (65)


A , B ) Mean baseline - detrended bioluminescence profiles from plate wells containing either PER2 : : LUC U2OS cells with ( WT , left panel ) or without REV - ERBα ( KO , right panel ) recorded at 37 ° C before and after transfer to 18 ° C for 24h ( A ) or 5h ( B ) ( grey region ) from biological repeat 1 . 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18 ° C exposure ( time zero ) .

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

A,B) Mean baseline-detrended bioluminescence profiles from plate wells containing either PER2::LUC U2OS cells with (WT, left panel) or without REV-ERBα (KO, right panel) recorded at 37°C before and after transfer to 18°C for 24h (A) or 5h (B) (grey region) from biological repeat 1. 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18°C exposure (time zero).


D ) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37 ° C or transferred to 18 ° C for 24h ( top panel ) or 5h ( bottom panel ) from biological repeats 1 and 2 . Points are colored according to their distinct phase prior to 18 ° C exposure . Colored lines ( bottom panel ) show the change in the mean for the points from each phase . Boxplots display median ( central line ) , interquartile range ( IQR , box edges ) and range ( whiskers ) . Number of wells : top panel WT cells : 37 ° C ( 120 ) , 18 ° C 24h ( 119 ) ; top panel KO cells : 37 ° C ( 120 ) , 18 ° C 24h ( 120 ) ; bottom panel WT cells : 37 ° C ( 58 ) , 18 ° C 5h ( 120 ) ; top panel KO cells : 37 ° C ( 60 ) , 18 ° C 5h ( 120 ) .

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D) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37°C or transferred to 18°C for 24h (top panel) or 5h (bottom panel) from biological repeats 1 and 2. Points are colored according to their distinct phase prior to 18°C exposure. Colored lines (bottom panel) show the change in the mean for the points from each phase. Boxplots display median (central line), interquartile range (IQR, box edges) and range (whiskers). Number of wells: top panel WT cells: 37°C (120), 18°C 24h (119); top panel KO cells: 37°C (120), 18°C 24h (120); bottom panel WT cells: 37°C (58), 18°C 5h (120); top panel KO cells: 37°C (60), 18°C 5h (120).


E ) Bar graphs showing the mean amplitude change ( left axis ) following 18 ° C exposure for 5h for each group of plate well profiles ( grouped according to their distinct phase ( a - f ) prior to 18 ° C exposure , 10 wells per group ) for both WT and KO cells from biological repeats 1 and 2 . Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2 : LUC ( dashed line ) and predicted REV - ERBα expression level ( right axis ) ( grey shaded region ( light grey indicates REV - ERBα deletion ) ) .

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E) Bar graphs showing the mean amplitude change (left axis) following 18°C exposure for 5h for each group of plate well profiles (grouped according to their distinct phase (a-f) prior to 18°C exposure, 10 wells per group) for both WT and KO cells from biological repeats 1 and 2. Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2:LUC (dashed line) and predicted REV-ERBα expression level (right axis) (grey shaded region (light grey indicates REV-ERBα deletion)).


B Spermatocyte chromosomes that underwent condensation ( right ) within a MII oocyte ( MII chromosome mass , left ) . In this study , spermatocytes picked up for injection had tetrad chromosomes ready for condensation . C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes . In the spermatocyte - injected groups , normality was improved by reducing the ooplasm mass ( * P < 0 . 005 by Fisher ' s exact probability test ) . Arrows in the right figure indicate prematurely separated chromatids . The numbers of oocytes observed are indicated on the top of the bars .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Spermatocyte chromosomes that underwent condensation (right) within a MII oocyte (MII chromosome mass, left). In this study, spermatocytes picked up for injection had tetrad chromosomes ready for condensation. C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes. In the spermatocyte-injected groups, normality was improved by reducing the ooplasm mass (*P < 0.005 by Fisher's exact probability test). Arrows in the right figure indicate prematurely separated chromatids. The numbers of oocytes observed are indicated on the top of the bars.


B Chromosome tracking in 3D . The reconstructed images are viewed from the side of the metaphase plate . Signals are interpolated in the Z axis for visualization . White and red arrowheads , as well as red surfaces , indicate univalent - like chromosomes that underwent unbalanced predivision ( premature segregation of sister chromatids ) . Scale bar = 4 μm . C Halving the recipient ooplasmic mass rescued chromosome alignment . The numbers of misaligned chromosomes and their parental origin were determined in 3D ( n = 39 and 17 oocytes ) . Error bars show the standard deviation . Student ' s t - test was used to compare means . * P < 0 . 05 . N . S . , not significant .

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B Chromosome tracking in 3D. The reconstructed images are viewed from the side of the metaphase plate. Signals are interpolated in the Z axis for visualization. White and red arrowheads, as well as red surfaces, indicate univalent-like chromosomes that underwent unbalanced predivision (premature segregation of sister chromatids). Scale bar = 4 μm. C Halving the recipient ooplasmic mass rescued chromosome alignment. The numbers of misaligned chromosomes and their parental origin were determined in 3D (n = 39 and 17 oocytes). Error bars show the standard deviation. Student's t-test was used to compare means. *P < 0.05. N.S., not significant.


C Univalent - like chromosomes . Images were 3D - reconstructed and viewed from the top of the metaphase plate . Red surfaces with white arrowheads indicate univalent - like chromosomes . Scale bar = 3 μm .

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C Univalent-like chromosomes. Images were 3D-reconstructed and viewed from the top of the metaphase plate. Red surfaces with white arrowheads indicate univalent-like chromosomes. Scale bar = 3 μm.


D Halving the ooplasm volume suppressed the premature separation of paternal chromosomes . Oocytes were categorized based on whether the chromosomes exhibited premature separation into univalent - like structures prior to segregation errors . Data from 39 and 17 oocytes , respectively .

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

D Halving the ooplasm volume suppressed the premature separation of paternal chromosomes. Oocytes were categorized based on whether the chromosomes exhibited premature separation into univalent-like structures prior to segregation errors. Data from 39 and 17 oocytes, respectively.


D Effect of forced SAC activation . Oocytes expressing MAD1 - mNeonGreen - CENP - C ( green ) , together with major satellite - mClover ( green ) and H2B - mCherry ( red ) , were imaged . Time after the start of imaging ( h : mm ) is shown . Oocytes were categorized based on anaphase figures ( n = 24 , 23 oocytes ) .

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

D Effect of forced SAC activation. Oocytes expressing MAD1-mNeonGreen-CENP-C (green), together with major satellite-mClover (green) and H2B-mCherry (red), were imaged. Time after the start of imaging (h:mm) is shown. Oocytes were categorized based on anaphase figures (n = 24, 23 oocytes).


( A ) Western blot analysis for pRb in DM - 2 cultures .

(A) Western blot analysis for pRb in DM-2 cultures.


( B ) Immunostaining for MHC in DM - 3 and DM - 6 differentiating myotubes . Nuclei were stained with DAPI ( blue ) . ( C ) Mean number of myotubes on the indicated days after differentiation . Each time point is mean ± SD of six fields from six independent experiments .

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(B) Immunostaining for MHC in DM-3 and DM-6 differentiating myotubes. Nuclei were stained with DAPI (blue). (C) Mean number of myotubes on the indicated days after differentiation. Each time point is mean ± SD of six fields from six independent experiments.


( D ) TUNEL staining . Arrowheads indicate TUNEL - positive nuclei ( green ) .

(D) TUNEL staining. Arrowheads indicate TUNEL-positive nuclei (green).


( E ) LC3 - RFP expression in control ( ctrl ) and Rb − / − myotubes . Insets show nuclear DAPI stain ( blue ) .

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(E) LC3-RFP expression in control (ctrl) and Rb−/− myotubes. Insets show nuclear DAPI stain (blue).


( F ) Representative Western blot ( n = 3 ) for LC3 . Chloroquine was added 12 h before cell harvest .

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(F) Representative Western blot (n = 3) for LC3. Chloroquine was added 12 h before cell harvest.


( G ) Representative Western blot for LC3 in E16 . 5 control and mgRb : Rb − / − muscles . ( H ) Quantification of LC3 - II / tubulin ratio in E16 . 5 mgRb : Rb − / − skeletal muscles relative to control as depicted in G ( mean ± SD ; n = 3 ) . * , P = 0 . 016 by Student ' s t test .

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(G) Representative Western blot for LC3 in E16.5 control and mgRb:Rb−/− muscles. (H) Quantification of LC3-II/tubulin ratio in E16.5 mgRb:Rb−/− skeletal muscles relative to control as depicted in G (mean ± SD; n = 3). *, P = 0.016 by Student's t test.


( I ) Representative Western blot for LC3 - I and LC3 - II expression in whole cell lysates from the indicated myotubes transduced with Ad . GFP or Ad . RbΔK11 . ( J ) Quantification of LC3 - II / tubulin ratio by Western blots as shown in I . Data represent mean ± SD ( n = 7 ) . * , P = 0 . 047 ; * * , P = 0 . 005 by analysis of variance .

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

(I) Representative Western blot for LC3-I and LC3-II expression in whole cell lysates from the indicated myotubes transduced with Ad.GFP or Ad.RbΔK11. (J) Quantification of LC3-II/tubulin ratio by Western blots as shown in I. Data represent mean ± SD (n = 7). *, P = 0.047; **, P = 0.005 by analysis of variance.


( A , left ) MitoTracker staining . ( right ) MitoTracker staining after bezafibrate treatment . ( B ) Quantification of myotubes with perinuclear MitoTracker - positive aggregates in untreated ( vehicle ) or bezafibrate - treated myotubes at DM - 2 . Data represent mean ± SD ( n = 3 ) . * , P < 0 . 001 by Student ' s t test .

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(A, left) MitoTracker staining. (right) MitoTracker staining after bezafibrate treatment. (B) Quantification of myotubes with perinuclear MitoTracker-positive aggregates in untreated (vehicle) or bezafibrate-treated myotubes at DM-2. Data represent mean ± SD (n = 3). *, P < 0.001 by Student's t test.


( C ) MitoTracker ( red ) and LC3immunostaining ( green ) of myotubes at DM - 2 . Chloroquine and 3 - MA ( where indicated ) were added 12 h before staining . Note the extensive overlap of MitoTracker and LC3 in Rb − / − myotubes ( yellow ) , which is inhibited by 3 - MA . ( D ) Mean percentage of MitoTracker ( red ) to LC3 ( green ) overlap in 25 myotubes from two independent cultures in control versus Rb − / − myotubes ( total ) or Rb − / − myotubes with perinuclear mitochondrial aggregation ( high ) . * , P = 7 × 10 − 5 by Student ' s t test .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 O O O O O O O O O O O O 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) MitoTracker (red) and LC3immunostaining (green) of myotubes at DM-2. Chloroquine and 3-MA (where indicated) were added 12 h before staining. Note the extensive overlap of MitoTracker and LC3 in Rb−/−myotubes (yellow), which is inhibited by 3-MA. (D) Mean percentage of MitoTracker (red) to LC3 (green) overlap in 25 myotubes from two independent cultures in control versus Rb−/−myotubes (total) or Rb−/−myotubes with perinuclear mitochondrial aggregation (high). *, P = 7 × 10−5 by Student's t test.


( E ) Ultrastructural analysis of myotubes treated or not with 3 - MA at DM - 2 . Arrowheads point to mitochondria . Asterisks indicate electron - dense autolysosomes . N , nucleus . ( F ) Ratio of mitochondrialDNA / nuclearDNA in the indicated cultures ( n = 3 ) . ( G ) Ratio of mitochondrialDNA / nuclearDNA in E16 . 5 skeletal muscle ( n = 3 ; * , P = 0 . 009 ) . ( H ) Quantification of ATP in E16 . 5 skeletal muscle ( n = 3 ; * , P = 0 . 011 ) . ( F - H ) Error bars represent SD . * , P < 0 . 001 .

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(E) Ultrastructural analysis of myotubes treated or not with 3-MA at DM-2. Arrowheads point to mitochondria. Asterisks indicate electron-dense autolysosomes. N, nucleus. (F) Ratio of mitochondrialDNA/nuclearDNA in the indicated cultures (n = 3). (G) Ratio of mitochondrialDNA/nuclearDNA in E16.5 skeletal muscle (n = 3; *, P = 0.009). (H) Quantification of ATP in E16.5 skeletal muscle (n = 3; *, P = 0.011). (F-H) Error bars represent SD. *, P < 0.001.


( A ) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad . GFP or Ad . Bcl - 2 . Each point represents the mean ± SD of six fields from six independent experiments . Bcl - 2 - rescued , twitching Rb − / − myotubes are shown in Video 2 . ( inset ) Western blot for Bcl - 2 in Ad . Bcl2 - transduced versus uninfected primary myoblasts .

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(A) Mean number of myotubes per field in indicated myoblast cultures induced to differentiate after transduction with Ad.GFP or Ad.Bcl-2. Each point represents the mean ± SD of six fields from six independent experiments. Bcl-2-rescued, twitching Rb−/− myotubes are shown in Video 2. (inset) Western blot for Bcl-2 in Ad.Bcl2-transduced versus uninfected primary myoblasts.


( B ) Immunostaining for MCK ( green ) in Ad . Bcl - 2 - transduced control ( ctrl ) and Rb − / − myotubes at DM - 6 .

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(B) Immunostaining for MCK (green) in Ad.Bcl-2-transduced control (ctrl) and Rb−/− myotubes at DM-6.


( C ) Western blot for MCK , myogenin , MHC , and troponin T in indicated cultures at DM - 6 after transduction with Ad . Bcl - 2 . Tubulin was used as a loading control . Black lines indicate that intervening lanes have been spliced out .

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(C) Western blot for MCK, myogenin, MHC, and troponin T in indicated cultures at DM-6 after transduction with Ad.Bcl-2. Tubulin was used as a loading control. Black lines indicate that intervening lanes have been spliced out.


( D ) Mean number of Rb − / − myotubes after 3 - MA treatment , bezafibrate , or DMSO / vehicle as indicated . Counts are mean ± SD of six fields ( n = 4 ) .

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(D) Mean number of Rb−/− myotubes after 3-MA treatment, bezafibrate, or DMSO/vehicle as indicated. Counts are mean ± SD of six fields (n = 4).


( E , left and middle ) Brightfield images of the indicated myotubes treated or not with 3 - MA at DM - 8 . ( right ) Immunostaining for MHC ( red ) at DM - 14 in the presence of 3 - MA . Arrowheads label 3 - MA - rescued Rb − / − myotubes at DM - 8 . 3 - MA - rescued , twitching Rb − / − myotubes are shown in Video 4 .

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(E, left and middle) Brightfield images of the indicated myotubes treated or not with 3-MA at DM-8. (right) Immunostaining for MHC (red) at DM-14 in the presence of 3-MA. Arrowheads label 3-MA-rescued Rb−/− myotubes at DM-8. 3-MA-rescued, twitching Rb−/− myotubes are shown in Video 4.


( F ) Immunostaining for BrdU ( green ) and MHC ( red ) of 3 - MA - treated cultures at DM - 2 or DM - 10 . Rb − / − myotubes incorporated BrdU at DM - 2 ( arrowheads ) but not DM - 10 .

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(F) Immunostaining for BrdU (green) and MHC (red) of 3-MA-treated cultures at DM-2 or DM-10. Rb−/− myotubes incorporated BrdU at DM-2 (arrowheads) but not DM-10.


( 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 .

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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 the indicated myoblasts induced to differentiate under normoxia or hypoxia treated or not with lonidamine . Note the shortened myotubes in the 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 ) and mean expression of myogenin and 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 and 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 and immunostained for pRb ( red ) . ( insets ) DAPI staining 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 and induced to differentiate in normoxia ( top and 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 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 .

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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 ) 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 .

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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 ) 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 ) .

(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 staining ( red ) of the indicated myoblasts transduced with Ad . NF - κB / HIF - 1α and 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 ) 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 .

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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 ) 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 .

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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 and FOSB in NB1 and IMR32 cells in the presence and absence of the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 proteins indicated in blue . Most pronounced responding proteins indicated in black and 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 and total FOXO3 protein levels in response to ALKAL2 stimulation , in the 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 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 .

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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 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 .

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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 and Alk - F1178S heterozygote mice ( p < 0 . 0001 ; log - rank test ) . Wild type littermates 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 and eosin as well as Ki67 stained sections of representative Th - MYCN and 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 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 ) .

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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 . 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 ) .

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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 and Th - MYCN mice express NB markers . Tumours from all three genotypes were large , in most cases filling the 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 the majority of Rosa26 _ Alkal2 ; Th - MYCN ( 18 / 20 ) and Alk - F1178S ; Th - MYCN ( 7 / 10 ) tumours did not involve the adrenal glands ( 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 and Th - MYCN tumours revealed positive staining for NCAM1 , synaptophysin ( SYP ) and 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 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 .

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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 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 ) .

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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).


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 .

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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 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 .

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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 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 .

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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 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 .

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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 and Alk - F1178S ; Th - MYCN tumours revealing positive staining 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 ) 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 , 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 .

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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 . 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 .

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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 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 .

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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 animals treated with lorlatinib did not display any significant loss of body weight 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 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 .

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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 and 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 test .

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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 ) and ATXN8 ( bottom strand ) . RNA - seq read coverage visualized by Integrative Genomic Viewer across the 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 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 .

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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 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 ) .

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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 protein in cerebellar white matter , brainstem and 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 ( 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 .

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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 staining shows no co - localization of polyGln and 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 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 ) .

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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 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 ) .

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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 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 )

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 ± SEM ; unpaired t test ) .

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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 ± SEM ; unpaired t test ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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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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 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 ) .

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(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 .

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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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O B-MEASURED_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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) . (

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

A, 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 O O O O O O O O O B-MEASURED_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 B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_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 O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

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

(A 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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

(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 ) 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

O O O O O O O O O O O O O O O O O O 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-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 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 .

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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 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 .

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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 ) 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 .

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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 ) and global EVL - / - mice on postnatal days 3 , 5 and 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 ) 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 ) .

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

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


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

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

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 .

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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 ) .

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

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(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 the length of the angiogenic front .

(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 .

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


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 .

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

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 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 .

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

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 ) 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 .

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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.