Figure 2A . UCSC genome browser visualization of the mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 71 , 000 , 000 - 74 , 100 , 000 bp , 3 . 1 Mb ; galGal5 ) . RT weighted average ( WA ) values for the wt and the βA - globin + β - actin cell lines are shown . Early - replicated regions ( E ) are represented in orange and late replicating regions ( L ) in blue . Annotated genes ( Ref Seq genes ) are represented below . The mid - late insertion site is indicated with a red arrow and dotted line . B . UCSC genome browser visualization of the mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 72 , 450 , 000 - 72 , 650 , 000 bp ; 200 kb ; galGal5 ) . Tracks of nascent strands ( NS ) enrichments in the four S phase fractions were represented separately ( S1 to S4 ) for the wt and the 2x ( βA - globin + β - actin ) cell lines . NS enriched and depleted regions for each fraction are represented in purple and blue respectively . Single reads form SNS aligned and track of replication origins ( Ori peaks ) determined in ( Massip et al , 2019 ) are reported in between . The three mid - late insertion sites 1 , 2 and 3 are indicated with red arrows . Annotated genes and CpG Islands are shown below . Initiation zones ( IZ ) and termination zones ( TZ ) are reported .

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

Figure 2A. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:71,000,000-74,100,000 bp, 3.1 Mb; galGal5). RT weighted average (WA) values for the wt and the βA-globin+β-actin cell lines are shown. Early-replicated regions (E) are represented in orange and late replicating regions (L) in blue. Annotated genes (Ref Seq genes) are represented below. The mid-late insertion site is indicated with a red arrow and dotted line.B. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:72,450,000-72,650,000 bp; 200 kb; galGal5). Tracks of nascent strands (NS) enrichments in the four S phase fractions were represented separately (S1 to S4) for the wt and the 2x(βA-globin+β-actin) cell lines. NS enriched and depleted regions for each fraction are represented in purple and blue respectively. Single reads form SNS aligned and track of replication origins (Ori peaks) determined in (Massip et al, 2019) are reported in between. The three mid-late insertion sites 1, 2 and 3 are indicated with red arrows. Annotated genes and CpG Islands are shown below. Initiation zones (IZ) and termination zones (TZ) are reported.


Figure 3A . Distribution of - ΔL + ΔE values for clonal cell lines containing one advanced replicon ( βA - globin + β - actin ) inserted at site 1 ( i , N = 8 ) or 3 ( iii , N = 3 ) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA - globin promoter / origin linked to a 1 . 6 kb fragment of human chromosome 7 ( h . K7 ) inserted at site 2 , either on the same chromosome as 1 and 3 ( 1 + 2 + 3 ) , or on the other chromosome ( 1 + 3 ) ( ii , N = 5 ) . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) . C . Interaction profiles of allele 1 + 3 / 2 with chr1 for combined Hi - C libraries : distance - corrected interactions , i . e . observed / expected ( OE ) , binned at 500 kb with corresponding compartment ( EV1 ) tracks . Red arrows represent the loci that show preferred interactions with allele 1 + 3 and that coincide with A compartment . D . OE interactions of allele 1 + 3 ( orange rectangles ) and allele 2 ( blue rectangles ) averaged over A and B compartments for p and q - arms at 100kb bin size . A random compartment status permutation test was made and then we calculated the one - sided p - value for the observed value of ' difference allele 1 + 3 / 2 A - B ' . Values corresponding to OE interactions of allele 1 + 3 and allele 2 averaged over shuffled A ( red circles , N = 1000 ) and B compartments ( blue circles , N = 1000 ) for p and q - arms . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 . E . Scatterplot of density of interactions between allele 1 + 3 and allele 2 with chromosomes larger than 5 , 000 , 000 bp .

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

Figure 3A. Distribution of -ΔL+ΔE values for clonal cell lines containing one advanced replicon (βA-globin+β-actin) inserted at site 1 (i, N=8) or 3 (iii, N=3) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter/origin linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2, either on the same chromosome as 1 and 3 (1+2+3), or on the other chromosome (1+3) (ii, N=5). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).C. Interaction profiles of allele 1+3/2 with chr1 for combined Hi-C libraries: distance-corrected interactions, i.e. observed/expected (OE), binned at 500 kb with corresponding compartment ( EV1) tracks. Red arrows represent the loci that show preferred interactions with allele 1+3 and that coincide with A compartment.D. OE interactions of allele 1+3 (orange rectangles) and allele 2 (blue rectangles) averaged over A and B compartments for p and q-arms at 100kb bin size. A random compartment status permutation test was made and then we calculated the one-sided p-value for the observed value of 'difference allele 1+3 / 2 A - B'. Values corresponding to OE interactions of allele 1+3 and allele 2 averaged over shuffled A (red circles, N=1000) and B compartments (blue circles, N=1000) for p and q-arms. ns, not significant; **p<0.01;***p<0.001.E. Scatterplot of density of interactions between allele 1+3 and allele 2 with chromosomes larger than 5,000,000 bp.


Figure 4A . Distribution of - ΔL + ΔE values for clonal cell lines containing one β - actin construct at site 1 ( i , N = 7 ) or at site 1 and 3 ( ii , N = 5 ) , on the same chromosome . Data information : Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) . B . Distribution of - ΔL + ΔE values for clonal cell lines containing one βA - globin construct at site 1 ( i , N = 8 ) or at site 1 and 3 ( ii , N = 6 ) , on the same chromosome . Data information : Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

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

Figure 4A. Distribution of -ΔL+ΔE values for clonal cell lines containing one β-actin construct at site 1 (i, N=7) or at site 1 and 3 (ii, N=5), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).B. Distribution of -ΔL+ΔE values for clonal cell lines containing one βA-globin construct at site 1 (i, N=8) or at site 1 and 3 (ii, N=6), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


Figure 5C . After 1 , 8 , 24 and 48 h of 4 - hydroxytamoxifen treatment , genomic DNA was extracted and quantified by semi - quantitative PCR ( see Appendix Figure S6 ) . Error bars correspond to the standard deviation for PCR duplicates . The percentages of cells with the large 1 + 3 and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) or β - actin ( 1 + 3 ) excision at each time point are shown for different cell lines .

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

Figure 5C. After 1, 8, 24 and 48 h of 4-hydroxytamoxifen treatment, genomic DNA was extracted and quantified by semi-quantitative PCR (see Appendix Figure S6). Error bars correspond to the standard deviation for PCR duplicates. The percentages of cells with the large 1+3 and loxP_LE (1) + βA-globin+β-actin (3) or β-actin (1+3) excision at each time point are shown for different cell lines.


Figure 6A , B . Clones 1 + 2 + 3 , 1 + 3 ( A ) and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) ( B ) were used to assess chromatin accessibility within the transgenes . The positions of the amplicons used for quantification in each shifting construct individually ( thick black lines ; 5 ' 2xFIV - 1 or - 3 , β - actin pro - 1 or - 3 , BsR gene , PuroR gene , LoxP site ) , in both shifting constructs ( βA pro 1 and 2 , IL2R gene , 3 ' 2xFIV1 - 3 ) or in the GFP reporter construct ( βA - GFP , GFP gene , h . K7 ) are shown . The insertion sites on the wt chromosome ( insertion sites 1 , 2 and 3 ) and a genomic region located 5 kb downstream from the insertion site ( insertion site + 5kb ) were used as controls for the targeted genomic region . The endogenous active MED14 promoter was analyzed as a control . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Quantifications by real - time qPCR of total chromatin ( input ) extracted from two 1 + 2 + 3 , 1 + 3 and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) clonal cell lines , after digestion with micrococcal nuclease ( MNAse , 2 . 5 U , 10 U , 40 U , 160 U / mL ) and size selection were shown . Error bars indicate the standard deviation for qPCR triplicates , for two independent clones . Data are presented as total chromatin input relative to the condensed genomic regions ( cond1 and cond2 ) used for the normalization .

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

Figure 6A, B. Clones 1+2+3, 1+3 (A) and loxP_LE (1) + βA-globin+β-actin (3) (B) were used to assess chromatin accessibility within the transgenes. The positions of the amplicons used for quantification in each shifting construct individually (thick black lines; 5' 2xFIV-1 or -3, β-actin pro-1 or -3, BsR gene, PuroR gene, LoxP site), in both shifting constructs (βA pro 1 and 2, IL2R gene, 3' 2xFIV1-3) or in the GFP reporter construct (βA-GFP, GFP gene, h.K7) are shown. The insertion sites on the wt chromosome (insertion sites 1, 2 and 3) and a genomic region located 5 kb downstream from the insertion site (insertion site +5kb) were used as controls for the targeted genomic region. The endogenous active MED14 promoter was analyzed as a control. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Quantifications by real-time qPCR of total chromatin (input) extracted from two 1+2+3, 1+3 and loxP_LE (1)+ βA-globin+β-actin (3) clonal cell lines, after digestion with micrococcal nuclease (MNAse, 2.5 U, 10 U, 40 U, 160 U/mL) and size selection were shown. Error bars indicate the standard deviation for qPCR triplicates, for two independent clones. Data are presented as total chromatin input relative to the condensed genomic regions (cond1 and cond2) used for the normalization.


Figure 7A , B . UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites ( genomic positions : chr1 : 69 , 000 , 000 - 74 , 200 , 000 ; 5 . 2 Mb ( A ) and chr1 : 175 , 300 , 000 - 180 , 500 , 000 ; 5 . 2 Mb ( B ) ; galGal5 ) . RT weighted average ( WA ) values obtained after cells sorting into four S - phase fractions , BrdU pulse - labeled nascent strands ( NS ) immunoprecipitation and sequencing are shown . Early - replicated regions are represented in orange and late - replicating regions in blue . Below , track of eigenvector 1 values corresponding to A / B compartments at 100 kb resolution after Hi - C analysis in wt cells , track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) and annotated genes are reported . The three sites chosen for insertion are indicated with red arrows and dotted lines ( mid - late , late 1 ( A ) and late 2 ( B ) insertion ) . At the bottom , an enlargement of late insertion sites ( chr1 : 69 , 500 , 000 - 71 , 500 , 000 ( A ) , chr1 : 177 , 000 , 000 - 179 , 000 , 000 ( B ) ; galGal5 ) , displaying genes , mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) is shown . Tracks of RefSeq genes are represented in blue , tracks of Ensembl genes in red , tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black . Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions .

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

Figure 7A, B. UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites (genomic positions: chr1:69,000,000-74,200,000; 5.2 Mb (A) and chr1: 175,300,000-180,500,000; 5.2 Mb (B); galGal5). RT weighted average (WA) values obtained after cells sorting into four S-phase fractions, BrdU pulse-labeled nascent strands (NS) immunoprecipitation and sequencing are shown. Early-replicated regions are represented in orange and late-replicating regions in blue. Below, track of eigenvector 1 values corresponding to A/B compartments at 100 kb resolution after Hi-C analysis in wt cells, track of replication origins determined in (Massip et al, 2019) (Ori peaks) and annotated genes are reported. The three sites chosen for insertion are indicated with red arrows and dotted lines (mid-late, late 1 (A) and late 2 (B) insertion). At the bottom, an enlargement of late insertion sites (chr1: 69,500,000-71,500,000 (A), chr1: 177,000,000-179,000,000 (B); galGal5), displaying genes, mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in (Massip et al, 2019) (Ori peaks) is shown. Tracks of RefSeq genes are represented in blue, tracks of Ensembl genes in red, tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black. Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions.


Figure 8A , B . RT profiles of chromosomal alleles following the targeted integration of a βA - globin + β - actin construct into two late - replicating loci , 1 ( A ) and 2 ( B ) in two or three clonal cell lines respectively . Nascent strands ( NS ) were quantified by real - time qPCR in four S phase fractions . Specific primer pairs determine the RT profile for the modified allele ( With ) , the wt allele ( Without ) and both alleles ( Both ) . The endogenous β - globin locus was analyzed as an early - replicated control . The modified ( Mod ) and wild - type ( wt ) alleles are shown . Difference - ΔL + ΔE values calculated at the target site following transgene integration are indicated ( see Appendix Figure S1 ) . Error bars correspond to the standard deviation for qPCR duplicates . The black vertical bars represent the precise insertion position .

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

Figure 8A, B. RT profiles of chromosomal alleles following the targeted integration of a βA-globin+β-actin construct into two late-replicating loci, 1 (A) and 2 (B) in two or three clonal cell lines respectively. Nascent strands (NS) were quantified by real-time qPCR in four S phase fractions. Specific primer pairs determine the RT profile for the modified allele (With), the wt allele (Without) and both alleles (Both). The endogenous β-globin locus was analyzed as an early-replicated control. The modified (Mod) and wild-type (wt) alleles are shown. Difference -ΔL+ΔE values calculated at the target site following transgene integration are indicated (see Appendix Figure S1). Error bars correspond to the standard deviation for qPCR duplicates. The black vertical bars represent the precise insertion position.


Figure 1B , C Both mRNA and protein levels of RBM47 were elevated in SeV - infected ( B ) or IFN - α - stimulated ( C ) 293T , HFF , HUVEC , and THP - 1 cells . Cells were infected with SeV ( MOI = 1 ) or stimulated with IFN - α for 6 or 12 h , and the expression of RBM47 mRNA ( at 6 and 12 h post treatment ) and protein ( at 6 and 12 h post treatment ) were tested . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 1B, C Both mRNA and protein levels of RBM47 were elevated in SeV-infected (B) or IFN-α-stimulated (C) 293T, HFF, HUVEC, and THP-1 cells. Cells were infected with SeV (MOI = 1) or stimulated with IFN-α for 6 or 12 h, and the expression of RBM47 mRNA (at 6 and 12 h post treatment) and protein (at 6 and 12 h post treatment) were tested. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 2C , D Levels of viral genome , protein , and progeny of VSV ( C ) or HSV - 1 ( D ) were quantified in virus - infected 293T cells with or without RBM47 ectopic expression at the indicated time points . The viral protein levels were tested 12 h post VSV infection and 24 h post HSV - 1 infection . The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 2C, D Levels of viral genome, protein, and progeny of VSV (C) or HSV-1 (D) were quantified in virus-infected 293T cells with or without RBM47 ectopic expression at the indicated time points. The viral protein levels were tested 12 h post VSV infection and 24 h post HSV-1 infection.The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments.**P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 3D , E mRNA expression of IFIT1 , ISG15 , and Cig5 in SeV - infected 293T cells overexpressing RBM47 ( D ) or RBM47 knocked down cells ( E ) . Cells were infected with SeV for 24 h , and gene expression was determined using qRT - PCR . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . F The expression of IFIT1 , ISG15 , and Cig5 in RBM47 - overexpressing or control 293T cells stimulated by IFN - α for 12 h . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . G The expression of IFIT1 , ISG15 , and Cig5 in WT and RBM47 + / - peritoneal macrophages stimulated by IFN - α for 12 h . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 3D, E mRNA expression of IFIT1, ISG15, and Cig5 in SeV-infected 293T cells overexpressing RBM47 (D) or RBM47 knocked down cells (E). Cells were infected with SeV for 24 h, and gene expression was determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).F The expression of IFIT1, ISG15, and Cig5 in RBM47-overexpressing or control 293T cells stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).G The expression of IFIT1, ISG15, and Cig5 in WT and RBM47+/- peritoneal macrophages stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 4B IFNAR1 mRNA stability after DRB treatment . 293T cells were transfected with RBM47 - Flag or empty vectors and treated with DRB ( 50 μg / mL ) . Total RNA was harvested at 0 , 15 , 30 , 45 , and 60 min , and IFNAR1 mRNA levels were measured using qRT - PCR and normalized to β - actin . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : Data are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 4B IFNAR1 mRNA stability after DRB treatment. 293T cells were transfected with RBM47-Flag or empty vectors and treated with DRB (50 μg/mL). Total RNA was harvested at 0, 15, 30, 45, and 60 min, and IFNAR1 mRNA levels were measured using qRT-PCR and normalized to β-actin. The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: Data are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 5A mRNA of IFNAR1 , but not IFNAR2 or STAT1 , was stabilized by RBM47 ectopic expression in 293T cells with or without IFN - α stimulation . Cells were treated with IFN - α or PBS for 6 h , and the relative gene expression levels were determined using qRT - PCR . The PCR results are represented as the means ± SD of n = 3 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . The data shown are representative of three independent experiments . F , G Protein levels of IFNAR1 and phosphorylation levels of TYK2 , STAT1 , and STAT2 in RBM47 - overexpressing ( F ) or knockdown ( G ) 293T cells . Cells were treated with PBS or IFN - α for 6 h , and the relative band densities were analyzed using ImageJ software and normalized to GAPDH . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) . The data shown are representative of three independent experiments .

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

Figure 5A mRNA of IFNAR1, but not IFNAR2 or STAT1, was stabilized by RBM47 ectopic expression in 293T cells with or without IFN-α stimulation. Cells were treated with IFN-α or PBS for 6 h, and the relative gene expression levels were determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.F, G Protein levels of IFNAR1 and phosphorylation levels of TYK2, STAT1, and STAT2 in RBM47-overexpressing (F) or knockdown (G) 293T cells. Cells were treated with PBS or IFN-α for 6 h, and the relative band densities were analyzed using ImageJ software and normalized to GAPDH. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.


Figure 6G RBM47 + / + and RBM47 + / - mice were infected with HSV - 1 ( 1 × 107 pfu ) via caudal vein injection . Viral genome DNA and progeny titers were tested using blood samples from infected mice . Data are represented as the means ± SD of n = 6 biological replicates . H qRT - PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV - 1 - infected mice . The PCR results are represented as the means ± SD of n = 6 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 6G RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates. H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 7D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient ( IFNAR1 - / - ) mice ( n = 6 per group ) infected with VSV . Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h . Each dot represents one mouse . The PCR results are represented as the means ± SD of n = 6 biological replicates . Data information : The data shown are representative of n = 3 independent experiments . NS , non - significant ; * * P ≤ 0 . 01 , and * * * P ≤ 0 . 001 ( Student ' s t - test ) .

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

Figure 7D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient (IFNAR1-/-) mice (n = 6 per group) infected with VSV. Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h. Each dot represents one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates. Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).


Figure 1A ) Upper panels : Representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of AC16 cells kept at 37 ° C or exposed to different cold temperatures for 24h . Lower panels : DAPI signal segmented into chromatin and interchromatin regions according to relative intensity . Scale bar : 5 μm . Arrows mark chromatin concentrated at the nuclear periphery ( P ) and around nucleoli ( N ) . B ) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions . Median ( central line ) , interquartile range ( IQR , box edges ) , most extreme data point no more than 1 . 5x IQR from box edges ( whiskers ) . Number of nuclei : 37 ° C ( 31 ) , 28 ° C 24h ( 26 ) , 18 ° C 5h ( 19 ) , 18 ° C 24h ( 22 ) , 18 ° C 24h then 37 ° C 2h ( 25 ) , 8 ° C 24h ( 23 ) . C ) Left and central panels : representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of cells kept at 37 ° C or exposed to 18 ° C for 24h showing the spatial distribution of the immunofluorescence ( IF ) signal of hnRNPC ( green ) and nuclear pore complexes ( NPCs , red ) . Right panel : DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z - plane shown . Scale bar whole nuclei : 5 μm . Scale bar enlarged section : 1 μm . D ) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition . Number of distances : 37 ° C ( 112511 ) , 28 ° C 24h ( 94521 ) , 18 ° C 5h ( 72340 ) , 18 ° C 24h ( 74998 ) , 18 ° C 24h then 37 ° C 2h ( 92896 ) , 8 ° C 24h ( 80570 ) .

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

Figure 1A) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N). B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23). C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm. D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).


Figure 2A ) Number of genes showing significant ( adjusted p < 0 . 05 , Wald test ) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h . B ) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h . Points are color - coded from low to high density ( black < blue < red < yellow ) . C ) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37 ° C to 28 ° C or 18 ° C ( top ) , 28 ° C or 8 ° C ( middle ) and 18 ° C or 8 ° C ( bottom ) for 24h in both the nucleus and cytoplasm . Points are shaded from low to high density ( grey < black ) . Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles , respectively .

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

Figure 2A) Number of genes showing significant (adjusted p < 0.05, Wald test) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37°C to different cold temperatures for 24h.B) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow).C) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37°C to 28°C or 18°C (top), 28°C or 8°C (middle) and 18°C or 8°C (bottom) for 24h in both the nucleus and cytoplasm. Points are shaded from low to high density (grey < black). Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles, respectively.


Figure 3A ) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18 ° C for 24h time point . Range ( whiskers ) , interquartile range ( IQR , box edges ) , median + / - IQR / [ square root ( number of genes ) ] ( notch ) and median ( central line ) . B ) Log2 fold change in cytoplasmic ( black line , left axis ) and nuclear ( red line , right axis ) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h relative to cells kept at 37 ° C . Error bars show the standard error . 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 ° CWestern 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18°C for 24h time point. Range (whiskers), interquartile range (IQR, box edges), median +/-IQR/[square root(number of genes)] (notch) and median (central line).B) Log2 fold change in cytoplasmic (black line, left axis) and nuclear (red line, right axis) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h relative to cells kept at 37°C. Error bars show the standard error.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°CWestern 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.


Figure 4Representative 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 - ERBαRepresentative 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 ) U2OS cells ( D ) , exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV - ERBαMean 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 ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative 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-ERBαRepresentative 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) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBαMean 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)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)


Figure 5A , 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 ) . 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 ) ) .

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

Figure 5A,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).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)).


Figure 1B 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B 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.


Figure 2B 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 .

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

Figure 2B 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.


Figure 3C 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C 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.


Figure 4D 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D 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).


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

Figure 1.(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.(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).(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.(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.


Figure 2 . ( 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 . ( 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 .

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

Figure 2.(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.(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.


Figure 3 . ( 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 . ( 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 ) . ( 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 . ( 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 .

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

Figure 3.(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.(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).(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.(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.


Figure 4 . ( 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 ) . ( 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 ) . ( 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 . ( 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 ) . ( 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 .

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

Figure 4.(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).(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).(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.(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).(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.


Figure 1B . 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 . 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 .

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

Figure 1B. 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.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.


Figure 2B . 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 . 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 . 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 .

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

Figure 2B. 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.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.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.


Figure 3A . 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 . 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 ) .

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

Figure 3A. 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.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).


Figure 4A . 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 ) . 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 ) 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 ) .

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

Figure 4A. 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).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)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).


Figure 5RNA - 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 . 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 . 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 .

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

Figure 5RNA-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.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.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.


Figure 6Murine 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 . 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 . 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 .

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

Figure 6Murine 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.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.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.