D - F , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( D ) Adipose tissue per total section ( average of three sections / biological replicate ) ( E ) adipocyte ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) ( Δ1 + / + n = 3 , Δ2 n = 8 ) . scaf1Δ1 scaf1Δ2 are represented with circles squares , respectively .

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D-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively.


Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( F ) Representative of ventral fat deposits ( dotted lines ) .

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Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (F) Representative images of ventral fat deposits (dotted lines).


G , Number of eggs per clutch ( standard diet Δ1 + / + n = 8 , Δ1 - / - n = 8 , Δ2 + / + n = 7 , Δ2 - / - n = 12 , double diet Δ1 + / + n = 7 , Δ1 - / - n = 8 , Δ2 + / + n = 8 , Δ2 - / - n = 9 ) .

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G, Number of eggs per clutch (standard diet Δ1 +/+ n=8, Δ1 -/- n=8, Δ2 +/+ n=7, Δ2 -/- n=12, double diet Δ1 +/+ n=7, Δ1 -/- n=8, Δ2 +/+ n=8, Δ2 -/- n=9).


H , Representative of H & E - stained ovaries . Black dotted lines outline ovaries , blue dotted indicates a mature follicle . I , of mature ovary follicles per ovary section ( average of three sections / biological sample ) ( Δ1 n = 2 - 3 , Δ2 n = 6 ) .

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H, Representative images of H&E-stained ovaries. Black dotted lines outline the ovaries, blue dotted line indicates a mature follicle. I, Quantification of mature ovary follicles per ovary section (average of three sections/biological sample) (Δ1 n=2-3, Δ2 n=6).


A , B , Representative of scaf1 - / - scaf1 + / + ( A ) ( B ) fed with indicated diets .

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A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.


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C, D, Length of females (C) and males (D) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).


E, F, Weight of females (E) and males (F) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).


G , number of eggs per clutch ( standard diet : Δ1 + / + n = 4 , Δ1 - / - n = 3 , Δ2 + / + n = 3 , Δ2 - / - n = 2 , double diet : Δ1 + / + n = 5 , Δ1 - / - , n = 5 Δ2 + / + n = 1 , Δ2 - / - n = 2 ) . H , of mature ovary follicles per ovary section in haematoxylin - eosin ( H & E ) histological sections ( average of three sections / biological sample ) ( Δ1 n = 3 , Δ2 n = 3 ) .

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G, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2). H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3).


adipose tissue in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in : adipose tissue ( I , J )

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adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males : adipose tissue area (I, J)


adipose tissue in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in adipocyte ( K , L ) .

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adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males adipocyte size (K, L).


A , Immunoblot of indicated of BNGE from scaf1 + / + scaf1 - / - whole zebrafish mitochondria for indicated diet ( representative of n = 2 ) .

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A, Immunoblot of the indicated proteins of BNGE from female scaf1+/+ and scaf1-/- whole zebrafish mitochondria for the indicated diet (representative of n=2).


B , Maximum uncoupled ( FCCP ) oxygen consumption rate in whole zebrafish mitochondria ( Δ1 n = 4 Δ2 n = 4 , same number for their respective controls ) with glutamate ( Glu ) , malate ( Mal ) succinate ( Succ ) .

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B, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ).


RNAseq data from scaf1 + / + scaf1 - / - skeletal muscle for indicated diet ( standard diet scaf1 + / + scaf1 - / - n = 4 , double diet scaf1 + / + n = 3 , scaf1 - / - n = 4 ) . ( C - F ) Volcano plots of differentially expressed genes ( DEGs ) . ( C ) Comparison between scaf1 - / - scaf1 + / + zebrafish in standard diet . ( D ) Comparison between scaf1 - / - scaf1 + / + zebrafish in double diet . ( E ) Comparison of scaf1 + / + zebrafish in double diet standard diet . ( F ) Comparison of scaf1 - / - zebrafish in double diet standard diet . In blue , significant DEGs ( p - adj < 0 . 05 , log2FC > | 1 | ) , in grey not significant DEGs , red circles represent non - significant differentially regulated OXPHOS genes , green circles represent significant differentially regulated OXPHOS genes , purple scaf1 ( cox7a2l ) .

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RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (C-F) Volcano plots of differentially expressed genes (DEGs). (C) Comparison between scaf1-/- and scaf1+/+ zebrafish in standard diet. (D) Comparison between scaf1-/- and scaf1+/+ zebrafish in double diet. (E) Comparison of scaf1+/+ zebrafish in double diet and standard diet. (F) Comparison of scaf1-/- zebrafish in double diet and standard diet. In blue, significant DEGs (p-adj < 0.05, log2FC > |1|), in grey not significant DEGs, red circles represent non-significant differentially regulated OXPHOS genes, green circles represent significant differentially regulated OXPHOS genes, purple scaf1 (cox7a2l).


HMRs express RNAs . Heat maps show expression in 16 . 5 dpc prospermatogonia . Only uniquely mapped - seq reads were used for analyses .

HMRs express RNAs. Heat maps show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses.


metaplots ( B ) show expression in 16 . 5 dpc prospermatogonia . Only uniquely mapped - seq reads were used for analyses . Positions ( 1 ) - ( 4 ) in ( A B ) ( green arrows ) indicate same positions ( ± 2 kbp from centers ) . lower chart in ( B ) represents mappability defined as of unique sequences in possible 50 - nt sequences ( see Appendix Figure S2B ) . TSS isoforms are included in 55798 TSSs . Low mappability at centers of HMR _ - L1s is due to high occurrence of repeat ( retrotransposon ) sequences . RPM , reads per million mapped reads .

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metaplots (B) show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. Positions (1)-(4) in (A and B) (green arrows) indicate the same positions (±2 kbp from the centers). The lower chart in (B) represents mappability defined as the percentage of unique sequences in all possible 50-nt sequences (see Appendix Figure S2B). TSS isoforms are included in 55798 TSSs. Low mappability at the centers of HMR_-L1s is due to high occurrence of repeat (retrotransposon) sequences. RPM, reads per million mapped reads.


marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons ( HMR _ - L1s ) . Metaplots show average ChIP - seq signals across HMR _ - L1s , transcription start sites ( TSSs ) , randomly selected sites . signals were normalized by either H3 or input signal . results shown in heat maps were not normalized by H3 or input .

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Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_-L1s). Metaplots show the average ChIP-seq signals across HMR_-L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heat maps were not normalized by H3 or input.


initiation form of Pol2 with an unphosphorylated carboxy terminal domain ( Pol2 null - P CTD ) is highly enriched in HMR _ - L1s .

The initiation form of Pol2 with an unphosphorylated carboxy terminal domain (Pol2 null-P CTD) is highly enriched in HMR_-L1s.


- immunoprecipitation ( RIP ) analyses of EGFP - MIWI2 EGFP . EGFP - MIWI2 EGFP were immunoprecipitated from 16 . 5 dpc prospermatogonia , long RNAs bound to these input RNAs were sequenced . RNAs expressed from HMR _ - L1s piRNA clusters , but not Ensembl prospermatogonium - expressed mRNAs , are overall enriched by immunoprecipitation ( IP ) of EGFP - MIWI2 ( left ) . This enrichment is not observed when EGFP is immunoprecipitated ( right ) . numbers of mRNAs , piRNA clusters , HMR _ - L1s analyzed were shown in parentheses . Prospermatogonium - expressed mRNAs are those that are highly specifically expressed in prospermatogonia . bottom top of boxes represent 25 % percentile 75 % percentile , respectively . in box represents median . Circles represent outliers , bottom ( top ) of whiskers represents minimum ( maximum ) values except for outliers .

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RNA-immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_-L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_-L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers.


RIP analyses of EGFP - MIWI2 in newborn testes from WT Mili KO mice . bound to EGFP - MIWI2 input were sequenced . RNAs derived from each class was counted for this .

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RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA were sequenced. All RNAs derived from each class was counted for this analysis.


HMR _ - L1s preferentially generate piRNAs . X - Y - axes represent expression of long RNAs ( 16 . 5 dpc prospermatogonia ) as determined by - seq MIWI2 - bound piRNAs ( 18 . 5 dpc testes ) as determined by small - seq , respectively . Circles in charts represent mRNAs , piRNA clusters HMR _ - L1s . More than half of HMR _ - L1s ( right ) are located in of y > x ( upper part of diagonal ) , whereas most mRNAs ( left ) are located in of y < x ( lower part of diagonal ) . RPKM , reads per kilobase per million mapped reads .

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HMR_-L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters and HMR_-L1s. More than half of the HMR_-L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads.


Frequent 10 - nt overlap between sense antisense piRNAs derived from HMR _ - L1s . Top 100 HMR _ - L1s that generate largest number of unique piRNAs were analyzed . Frequencies of 1 − 18 - nt overlaps were analyzed for each HMR _ - L1 .

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Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_-L1s. Top 100 HMR_-L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1−18-nt overlaps were analyzed for each HMR_-L1.


piRNAs derived from HMR _ - L1s are enriched in adenine at their 10th nucleotide . HMR _ - L1s generating more than 40 unique piRNAs were analyzed ( 678 HMR _ - L1s ) . proportions of piRNAs with adenine ( left ) uridine ( right ) at indicated positions were analyzed for each HMR _ - L1 . See A for explanation of box plots .

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piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.


methylation status of a portion of Rasgrf1 differentially methylated region ( DMR ) in 0 − 2 - dpp Mili + / − Mili − / − prospermatogonia . Regions corresponding to B , C , D , E , F are demethylated in Mili − / − prospermatogonia . Vertical bars represent methylation at CpG sites determined by whole genome bisulfite sequencing ( see below ) . top gray bar represents a portion of Rasgrf1 gametic DMR ( Tomizawa et al , 2011 ) . CpG cluster is shown . deleted RMER4B is indicated .

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DNA methylation status of a portion of the Rasgrf1 differentially methylated region (DMR) in 0−2-dpp Mili+/− and Mili−/− prospermatogonia. Regions corresponding to B, C, D, E, and F are demethylated in Mili−/− prospermatogonia. Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing (see below). The top dark gray bar represents a portion of the Rasgrf1 gametic DMR (Tomizawa et al, 2011). The CpG cluster is shown. The deleted RMER4B sequence is indicated.


methylation status of Rasgrf1 DMR in Rasgrf1ΔRMER4B / ΔRMER4B control spermatogonia are shown . regions examined are shown in ( A ) . Black ( ) circles represent methylated ( unmethylated ) CpGs .

DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B/ΔRMER4B and control spermatogonia are shown. The regions examined are shown in (A). Black (white) circles represent methylated (unmethylated) CpGs.


Rasgrf1 RMER4B functions in cis . Wild - type mutant alleles of Rasgrf1 DMR in Rasgrf1 + / ΔRMER4B spermatogonia were analyzed separately .

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Rasgrf1 RMER4B functions in cis. Wild-type and mutant alleles of the Rasgrf1 DMR in Rasgrf1+/ΔRMER4B spermatogonia were analyzed separately.


Structure of chr7 piRNA cluster . Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP - seq , - seq , small - seq . Only uniquely mapped reads were used for displaying sequencing result . For - seq small - seq , only results of minus strand are shown , because plus strand does not express appreciable level of . A promoter region deleted is shown above putative piRNA precursor . RPM , Read Per Million .

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Structure of the chr7 piRNA cluster. Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP-seq, RNA-seq, and small RNA-seq. Only uniquely mapped reads were used for displaying the sequencing result. For RNA-seq and small RNA-seq, only the results of minus strand are shown, because plus strand does not express appreciable level of RNA. A promoter region deleted is shown above the putative piRNA precursor. RPM, Read Per Million.


Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter / Δpromoter testes . Unchanged expression of piRNAs from chr10 cluster ( bottom ) shows chr7 cluster is specifically affected . Only uniquely mapped piRNAs are shown .

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Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter/Δpromoter testes. Unchanged expression of piRNAs from chr10 cluster (bottom) shows chr7 cluster is specifically affected. Only uniquely mapped piRNAs are shown.


methylation status of Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔpromoter / Δpromoter . See Figures 4A B for information about region examined .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔpromoter/Δpromoter. See Figures 4A and B for information about the region examined.


Depletion of piRNAs from chr7 piRNA cluster RMER4B in chr7 piRNA clusterΔRMER4B / ΔRMER4B mice . Top panel shows overall view of chr7 piRNA cluster , lower panel shows magnified view of RMER4B region in chr7 piRNA cluster . Only uniquely mapped piRNAs are shown .

Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. Top panel shows overall view of chr7 piRNA cluster, and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster. Only uniquely mapped piRNAs are shown.


methylation status of Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice .

DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.


methylation status of Rasgrf1 differentially methylated region ( DMR ) in spermatocytes , spermatids , sperms ( D ) from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice .

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DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatocytes, spermatids, and sperms (D) from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.


methylation pattern in progeny of Rasgrf1ΔRMER4B / ΔRMER4B mice . methylation status of Rasgrf1 DMR in E12 . 5 whole is shown . E12 . 5 whole were obtained by crosses shown in panel . Two independent bisulfite - treated sample were examined for Homo13 .

DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.


A Domain architecture of S . pombe bromodomain AAA - ATPases . D1 D2 ATPase domains ( AAA ) are shaded blue orange , respectively , bromodomain ( BD ) is grey . similarity to each other indicated was determined by FASTA comparison [ 74 ] using scoring matrix BLOSUM50 .

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A Domain architecture of S. pombe bromodomain AAA-ATPases. The D1 and D2 ATPase domains (AAA) are shaded blue and orange, respectively, and the bromodomain (BD) is grey. The percentage similarity to each other and the indicated proteins was determined by FASTA sequence comparison [74] using the scoring matrix BLOSUM50.


B Venn diagrams showing overlap between genes up - regulated ( 1 . 5 fold ) in abo1Δ mutants with genes up - regulated under indicated condition , along with significance of overlaps ( on hypergeometric distribution ) .

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B Venn diagrams showing overlap between genes up-regulated (1.5 fold) in abo1Δ mutants with genes up-regulated under the indicated condition, along with the significance of the overlaps (based on hypergeometric distribution).


A Normalised cumulative nucleosome ( 150 ± 30 bp class ) position frequency profiles for 4013 S . pombe genes aligned at transcription start site ( TSS ) plotted from low MNase ( biorep1 ) high MNase ( biorep 2 ) datasets . P - values ( calculated using a two - unpaired t - ) for difference in means between random n = 100 subsets of frequency values at points indicated by dotted lines ( " - 4 " nucleosome " + 4 " nucleosome ) are shown .

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A Normalised cumulative nucleosome (150 ± 30 bp size class) position frequency profiles for 4013 S. pombe genes aligned at the transcription start site (TSS) plotted from low MNase (biorep1) and high MNase (biorep 2) datasets. P-values (calculated using a two-tailed unpaired t-test) for the difference in means between random n = 100 subsets of the frequency values at the points indicated by the dotted lines ("-4" nucleosome and "+4" nucleosome) are shown.


B Nucleosome position frequency values for coding regions of 4013 S . pombe genes were k - means clustered ( k = 9 ) using abo1Δ data from biorep 1 ( low MNase ) displayed with positive values coloured yellow other values coloured blue ( left - panel ) . cluster order was then used to display equivalent wild type frequency values in right - panel .

B Nucleosome position frequency values for the coding regions of 4013 S. pombe genes were k-means clustered (k = 9) using the abo1Δ data from biorep 1 (low MNase) and displayed with positive values coloured yellow and other values coloured blue (left-hand panel). The cluster order was then used to display the equivalent wild type frequency values in the right-hand panel.


C Level of histone gene mRNAs was determined by qRT - PCR . Data are mean of four independent biological repeats error bars are ± . Two - unpaired t - showed no significant differences ( P > 0 . 05 ) between wild type abo1Δ .

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C Level of histone gene mRNAs was determined by qRT-PCR. Data are the mean of four independent biological repeats and error bars are ±SEM. Two-tailed unpaired t-tests showed no significant differences (P > 0.05) between wild type and abo1Δ cells.


D Whole extracts were subjected to western blotting with histone H3 ( Abcam ) , histone H2A ( Abcam ) tubulin antibodies . Examples of data are shown ( left ) along with a of histone H2A H3 normalized to tubulin ( right ) . Data are mean of at least three independent repeats error bars represent ± . P - values were calculated using a two - unpaired t - .

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D Whole cell extracts were subjected to western blotting with histone H3 (Abcam), histone H2A (Abcam) and tubulin antibodies. Examples of the primary data are shown (left) along with a quantification of histone H2A and H3 levels normalized to tubulin (right). Data are the mean of at least three independent repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test.


A Whole extracts ( WCE ) were prepared from indicated strains , immunoprecipitated ( IP ) with anti - GFP antibody subjected to western blotting with anti - V5 - Pk ( Serotec ) anti - GFP ( Life Technologies ) antibodies . Data are representative of three independent biological repeats . B As for A .

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A Whole cell extracts (WCE) were prepared from the indicated strains, immunoprecipitated (IP) with anti-GFP antibody and subjected to western blotting with anti-V5-Pk (Serotec) and anti-GFP (Life Technologies) antibodies. Data are representative of three independent biological repeats.B As for A.


C purified from wild type , abo1Δ spt16 - 18 was analysed by northern blotting using a probe to 3 ′ end of spbc19c7 . 11 ( top panel ) . ( 5 µg ) used for northern blotting was analysed on an ethidium bromide stained 1 % TAE agarose gel ( bottom panel ) . Data are representative of two independent biological repeats .

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C RNA purified from wild type, abo1Δ and spt16-18 cells was analysed by northern blotting using a probe to the 3′ end of spbc19c7.11 (top panel). RNA (5 µg) used for northern blotting was analysed on an ethidium bromide stained 1% TAE agarose gel (bottom panel). Data are representative of two independent biological repeats.


D purified from wild type , abo1Δ was analysed by strand specific RT - PCR . from hip1Δ was analysed as a control . One primer , complementary to either forward or reverse transcripts , was included during reverse transcription step second primer was then added during PCR amplification . Control reactions omitting reverse transcription step ( - RT ) were included to demonstrate absence of contaminating genomic . Data are representative of two independent biological repeats .

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

D RNA purified from wild type, and abo1Δ cells was analysed by strand specific RT-PCR. RNA from hip1Δ cells was analysed as a control. One primer, complementary to either the forward or reverse transcripts, was included during the reverse transcription step the second primer was then added during PCR amplification. Control reactions omitting the reverse transcription step (-RT) were included to demonstrate the absence of contaminating genomic DNA. Data are representative of two independent biological repeats.


E indicated strains were subjected to ChIP with anti - GFP antibodies resulting was analysed by qPCR for indicated locus . Data are mean of three independent biological repeats error bars represent ± . P - values were calculated using a two - unpaired t - . Significant ( P < 0 . 05 ) values are indicated .

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

E The indicated strains were subjected to ChIP analysis with anti-GFP antibodies and the resulting DNA was analysed by qPCR for the indicated locus. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-values were calculated using a two-tailed unpaired t-test. Significant (P < 0.05) values are indicated.


F ChIP of histone H3 at act1 + was determined by qPCR . Data are mean of three independent biological repeats error bars represent ± . P - value was calculated using a two - unpaired t - .

F ChIP analysis of histone H3 levels at act1+ was determined by qPCR. Data are the mean of three independent biological repeats and error bars represent ±SEM. P-value was calculated using a two-tailed unpaired t-test.


G indicated strains were subjected to ChIP as described for E .

G The indicated strains were subjected to ChIP analysis as described for E.


H Log phase were subjected to five - fold serial dilution spotted onto rich ( YE5S ) agar or agar supplemented with MMS ( 0 . 006 % ) . Plates were incubated for 3 - 5 days at 30 ° C or 7 days at 20 ° C . are representative of three independent biological repeats .

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

H Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with MMS (0.006%). Plates were incubated for 3-5 days at 30°C or 7 days at 20°C. Images are representative of three independent biological repeats.


A position of ura4 + reporter alleles in centromere 1 is shown in top panel . Strains containing imr : : ura4 + allele were grown to log phase in YE5S medium , subjected to 5 - fold serial dilutions spotted onto YE5S agar or YE5S agar supplemented with 5 - FOA ( 1 mg / ml ) . Data are representative of three independent biological repeats .

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

A The position of ura4+ reporter alleles in centromere 1 is shown in the top panel. Strains containing the imr::ura4+ allele were grown to log phase in YE5S medium, subjected to 5-fold serial dilutions and spotted onto YE5S agar or YE5S agar supplemented with 5-FOA (1 mg/ml). Data are representative of three independent biological repeats.


B Strains carrying otr : : ura4 + were analysed as described for A .

B Strains carrying the otr::ura4+ were analysed as described for A.


C was purified from indicated strains level of centromeric transcripts determined by qRT - PCR . Data is mean of three independent repeats error bars indicate ± . P - value was calculated using a two - unpaired t - .

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

C RNA was purified from the indicated strains and the level of centromeric transcripts determined by qRT-PCR. Data is the mean of three independent repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired t-test.


D was purified from indicated strains subjected to qRT - PCR for subtelomeric gene , tlh1 + . Data is mean of three biological repeats error bars indicate ± . P - value was calculated using a two - unpaired Student ' s t - .

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

D RNA was purified from the indicated strains and subjected to qRT-PCR for the subtelomeric gene, tlh1+. Data is the mean of three biological repeats and error bars indicate ± SEM. P-value was calculated using a two-tailed unpaired Student's t-test.


E position of insertion of ade6 + reporter allele in silent mat locus is shown in top panel . Log phase containing mat3 - M : : ade6 + allele were subjected to five - fold serial dilution spotted onto YE5S agar plates lacking adenine ( Low Ade ) were incubated for 4 days at 30 ° C . Data are representative of at least three independent biological repeats . Note , microarray indicates that deletion of abo1 + does not influence expression of ade6 + ( or ura4 + ) when these genes are present at their normal genomic loci .

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

E The position of insertion of ade6+ reporter allele in the silent mat locus is shown in the top panel. Log phase cells containing the mat3-M::ade6+ allele were subjected to five-fold serial dilution and spotted onto YE5S agar plates lacking adenine (Low Ade) were incubated for 4 days at 30°C. Data are representative of at least three independent biological repeats. Note, microarray analysis indicates that deletion of abo1+ does not influence the expression of ade6+ (or ura4+) when these genes are present at their normal genomic loci.


A ChIP was performed on wild type ( untagged ) abo1 - GFP resulting analysed by qPCR for centromeric ( dh , dg imr ) repeat sequences . Data are mean of four independent biological repeats error bars represent ± . P - values calculated using a two - unpaired t - indicates that loci are significantly enriched ( P < 0 . 05 ) relative untagged control .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A ChIP analysis was performed on wild type (untagged) and abo1-GFP cells and the resulting DNA analysed by qPCR for centromeric (dh, dg and imr) repeat sequences. Data are the mean of four independent biological repeats and error bars represent ±SEM. P-values calculated using a two-tailed unpaired t-test indicates that all loci are significantly enriched (P < 0.05) relative the untagged control.


B indicated strains expressing GFP - Swi6 were subjected to ChIP . level of centromeric ( dg ) repeat sequences relative to euchromatic control locus , adh1 + was determined by qPCR scaled to a clr4Δ ( - H3 K9me control ) mutant . Data are mean of two independent ChIP experiments error bars represent range of data .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B The indicated strains expressing GFP-Swi6 were subjected to ChIP analysis. The level of centromeric (dg) repeat sequences relative to the euchromatic control locus, adh1+ was determined by qPCR and scaled to a clr4Δ (- H3 K9me control) mutant. Data are the mean of two independent ChIP experiments and error bars represent the range of the data.


C ChIP of histone H3 at centromeric ( dg ) repeat sequences was determined by qPCR . Data are mean of three independent ChIP experiments error bars are ± . P - values were calculated using a two - unpaired t - .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C ChIP analysis of histone H3 levels at centromeric (dg) repeat sequences was determined by qPCR. Data are the mean of three independent ChIP experiments and error bars are ±SEM. P-values were calculated using a two-tailed unpaired t-test.


D . ChIP of Abo1 - GFP at centromeric ( dh imr ) repeat sequences regions in wild type pob3Δ . Data are mean of three independent experiments error bars represent ± . P - values , calculated using a two - unpaired t - , indicated no significant difference ( P > 0 . 05 ) for wild type pob3Δ .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences regions in wild type and pob3Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant difference (P > 0.05) for wild type and pob3Δ cells.


E ChIP of Abo1 - GFP at centromeric ( dh imr ) repeat sequences in wild type pob3Δ . Data are mean of duplicate experiments error bars represent range of data .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

E ChIP analysis of Abo1-GFP at centromeric (dh and imr) repeat sequences in wild type and pob3Δ cells. Data are the mean of duplicate experiments and error bars represent the range of the data.


F ChIP of Abo1 - GFP over dh imr regions in wild type swi6Δ . Data are mean of three independent experiments error bars represent ± . P - values , calculated using a two - unpaired t - , indicated no significant differences ( P > 0 . 05 ) for wild type swi6Δ .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F ChIP analysis of Abo1-GFP over the dh and imr regions in wild type and swi6Δ cells. Data are the mean of three independent experiments and error bars represent ±SEM. P-values, calculated using a two-tailed unpaired t-test, indicated no significant differences (P > 0.05) for wild type and swi6Δ cells.


A was extracted from mid log phase Tf2 mRNA were determined by qRT - PCR , normalised to act1 + mRNA scaled relative to wild type level . Data are mean of three independent biological repeats error bars represent ± .

A RNA was extracted from mid log phase cells and Tf2 mRNA levels were determined by qRT-PCR, normalised to act1+ mRNA and scaled relative to the wild type level. Data are the mean of three independent biological repeats and error bars represent ±SEM.


B Mid log phase with indicated integrated lacZ reporter were subjected to quantitative β - galactosidase assays . Data are mean of three independent biological repeats error bars represent ± . C As for

O O O O O O O O O O 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 Mid log phase cells with the indicated integrated lacZ reporter were subjected to quantitative β-galactosidase assays. Data are the mean of three independent biological repeats and error bars represent ±SEM.C As for B


D ChIP samples from wild type ( untagged ) abo1 - GFP were analysed by qPCR for Tf2 LTR . Data are mean of four independent biological repeats error bars represent ± .

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

D ChIP DNA samples from wild type (untagged) and abo1-GFP cells were analysed by qPCR for Tf2 LTR. Data are the mean of four independent biological repeats and error bars represent ±SEM.


E Normalised cumulative nucleosome ( 150 ± 30 bp ) position frequency profiles for Tf2 LTR retrotransposons aligned at ATG plotted from low MNase ( biorep1 ) high MNase ( biorep 2 ) datasets . P - values were calculated using a two - unpaired t - .

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

E Normalised cumulative nucleosome (150 ± 30 bp) position frequency profiles for Tf2 LTR retrotransposons aligned at the ATG plotted from low MNase (biorep1) and high MNase (biorep 2) datasets.P-values were calculated using a two-tailed unpaired t-test.


A Log phase were subjected to five - fold serial dilution spotted onto rich ( YE5S ) agar or agar supplemented with TBZ ( 15 μg / ml ) incubated for 3 - 5 days 30 ° C . Data are representative of at three independent biological repeats .

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

A Log phase cells were subjected to five-fold serial dilution and spotted onto rich (YE5S) agar or agar supplemented with TBZ (15 μg/ml) and incubated for 3-5 days 30°C. Data are representative of at three independent biological repeats.


B frequency of Ch16 minichromosome loss was assayed by plating onto adenine limiting agar determining frequency of half sectored ( red - ) colonies . At least two independently derived isolates were used for each genotype total number of colonies counted for each genotype is indicated in parenthesis .

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

B The frequency of Ch16 minichromosome loss was assayed by plating cells onto adenine limiting agar and determining the frequency of half sectored (red-white) colonies. At least two independently derived strain isolates were used for each genotype and the total number of colonies counted for each genotype is indicated in parenthesis.


C Heterothallic ( h90 ) strains were cultured on nitrogen limiting ( EMMG ) medium for 3 days at 25 ° C to induce mating meiosis . resulting asci were stained with DAPI visualised by microscopy . Data are representative of three biological

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

C Heterothallic (h90) strains were cultured on nitrogen limiting (EMMG) medium for 3 days at 25°C to induce mating and meiosis. The resulting asci were stained with DAPI and visualised by fluorescence microscopy. Data are representative of three biological repeats


D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4 - 5 days at 30 ° C . Data are mean of three independent biological repeats error bars are ± . P - value was calculated using a two - unpaired t - .

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

D Spore viability was measured by dissecting spores onto YE5S agar followed by incubation for 4-5 days at 30°C. Data are the mean of three independent biological repeats and error bars are ±SEM. P-value was calculated using a two-tailed unpaired t-test.


H & amp ; amp ; E of adult head sections of ( A ) repo > GFP . nls ( B ) repo > Shits1 at 29 ° C for 3 days . Lamina degeneration was identified as vacuoles in ( B ) . Cryosection of adult ( C ) repo > H2B - RFP exhibited expression of red ( RFP ) in glia ( epithelialglialnucleus : arrowhead ) ( D ) repo > Shits1 at 28 ° C for 12 days . Vacuoles were identified in lamina neuropile ( D ) . Epithelial ( arrowhead ) ( E ) , marginal ( arrow ) distal satellite ( arrowhead ) glianuclei ( F ) are labeled by HisCl - GAL4 NP2109 - GAL4 , respectively . Note that HisCl - GAL4 is not expressed in epithelialglia . ( G , H ) Weak lamina vacuolization was identified in HisCl > Shits1 ( G , 0 . 92 % ) NP2109 > Shits1 ( H , 1 . 46 % ) at 29 ° C for 14 days . ( I , J ) A single MARCMglia clone ( GFP , green ) expressed Shits1 at 21 ° C ( I ) 29 ° C ( J ) . ( J ) A vacuole occurred within a glial clone . DAPI ( ) stains nuclei ( C , D , G - J ) . ( K ) of vacuole in lamina of repo > Shits1flies at 28 ° C progressively increased . n indicated in each column . P - values were calculated using one - way ANOVA with Bonferroni ' s post - . ( L ) When repo > Shits1flies were shifted to 28 ° C for 12 days then shifted to 17 ° C for 9 additional days , vacuolization was not alleviated . P - values were calculated using one - way ANOVA with Tukey ' s post - . n indicated in each column .

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

H&amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.


( A - E ) Horizontal sections of adult head lamina cartridge . ( A ) In repo > GFP . nls flies , two lamina neurons L1 , L2 axons ( L ) R1 - 6 axons ( blue ) were surrounded by electron - dense cytoplasm of epithelial glia ( G ) . section was examined at three different depths , of a single cartridge is not significantly different at different depths . ( B - D ) repo > Shits1 adults maintained at 29 ° C for 4 days . Axons were enlarged but intact . A large number of small medium vacuoles were identified in epithelial glia . ( C ) Small vacuoles in glia that contained a double - structure ( arrowhead ) . ( C ' ) Higher magnification of boxed in ( C ) . ( D ) A double - autophagosome - like structure ( arrowhead ) in glialcytoplasm . ( E ) In repots > DERDN adults maintained at 29 ° C for 2 days , large vacuoles were identified in between lamina cartridges . N : glialnuclei . ( E ' ) Higher magnification of boxed in ( E ) , which shows autophagosome - like vesicles ( arrowhead ) in gliacytoplasm .

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

(A-E) Horizontal sections of the adult head lamina cartridge. (A) In repo>GFP.nls flies, two lamina neurons L1, L2 axons (L) and R1-6 axons (blue area) were surrounded by the electron-dense cytoplasm of the epithelial glia (G). The section was examined at three different depths, and the size of a single cartridge is not significantly different at different depths. (B-D) repo>Shits1 adults maintained at 29°C for 4 days. Axons were enlarged but intact. A large number of small and medium vacuoles were identified in the epithelial glia. (C) Small vacuoles in the glia that contained a double-membrane structure (arrowhead). (C') Higher magnification of the boxed area in (C). (D) A double-membrane autophagosome-like structure (arrowhead) in the glialcytoplasm. (E) In repots>DERDN adults maintained at 29°C for 2 days, large vacuoles were identified in between the lamina cartridges. N: glialnuclei. (E') Higher magnification of the boxed area in (E), which shows autophagosome-like vesicles (arrowhead) in the gliacytoplasm.


( A ) Rh1 > lacZ + H2B - RFP exhibited nuclearRFP expression in R1 - 6 photoreceptors ( red ) . ( B ) Rh1 > lacZ + Shits1 maintained at 29 ° C for 14 days resulted in lamina vacuolization . ( C ) Pan - Rh7 > H2B - RFP exhibited expression in R7 ( arrow ) R8 ( arrowhead ) ( red ) . ( D ) Pan - Rh7 > Shits1 incubated at 29 ° C for 14 days did not exhibit lamina degeneration . ( E ) Lamina monopolar ( L ) neurons ( red ) were selectively labeled by Ln - GAL4 driven H2B - RFP with repo - GAL80 . ( F ) Ln > Shits1 , repoGAL80 shifted to 29 ° C for 14 days did not exhibit lamina degeneration . ( G ) R1 - 6 photoreceptors were killed in Rh1ts > lacZ + Hid shifted to 29 ° C for 14 days . Rh1ts indicates tubGAL80ts ; Rh1 - GAL4 . Degeneration was induced in lamina in addition to retina . ( H ) rdgC306 / + mutant exposed to constant for 14 days exhibited degeneration in retina , as well as lamina . DAPI : nuclei ( in A - H ) . ( I , K ) Rh1 > lacZ + Shits1 at 29 ° C for 4 days exhibited vacuoles in electron dense cytoplasm of epithelialglia or near glianucleus . Arrow : vacuole with internal debris . Arrowhead : large vacuole . ( J ) Lamina cartridge of Rh1 > lacZ + H2B - RFP . N indicates glialnuclei . ( L ) of vacuole in lamina at 29 ° C for 14 days was examined . P - values were calculated using one - way ANOVA with Tukey ' s post - . Scale bar : 20 μm .

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

(A) Rh1>lacZ+H2B-RFP exhibited nuclearRFP expression in the R1-6 photoreceptors (red). (B) Rh1>lacZ+Shits1 maintained at 29°C for 14 days resulted in lamina vacuolization. (C) Pan-Rh7>H2B-RFP exhibited expression in R7 (arrow) and R8 (arrowhead) (red). (D) Pan-Rh7>Shits1 incubated at 29°C for 14 days did not exhibit lamina degeneration. (E) Lamina monopolar (L) neurons (red) were selectively labeled by the Ln-GAL4 driven H2B-RFP with repo-GAL80. (F) Ln>Shits1, repoGAL80 shifted to 29°C for 14 days did not exhibit lamina degeneration. (G) R1-6 photoreceptors were killed in Rh1ts>lacZ+Hid shifted to 29°C for 14 days. Rh1ts indicates tubGAL80ts; Rh1-GAL4. Degeneration was induced in the lamina in addition to the retina. (H) rdgC306/+ mutant exposed to constant light for 14 days exhibited degeneration in the retina, as well as the lamina. DAPI: nuclei (white in A-H). (I, K) Rh1>lacZ+Shits1 at 29°C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus. Arrow: vacuole with internal debris. Arrowhead: large vacuole. (J) Lamina cartridge of Rh1>lacZ+H2B-RFP. N indicates glialnuclei. (L) The percentage of the vacuole area in the lamina at 29°C for 14 days was examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( A ) repots > Shits1 + H2B - RFP , ( B ) repots > Shits1 + Egfrλtop ( C ) repots > Shits1 + RlSem incubated for 12 days at 28 ° C . ( D ) repots > DERDN for 7 days at 28 ° C . ( E ) Epithelial glia MARCM clone ( F ) Egfrco mutant glia MARCM clone ( labeled with Tomato , green ) . penetrance is indicated as number of samples with vacuoles over total number of samples . DAPI : nuclei ( in A - F ) . ( G ) of vacuole in lamina neuropile of ( A - D ) . P - values were calculated using one - way ANOVA with Dunnett ' s post - . ( H ) of vacuole in lamina of repots > Shits1 flies in indicated genetic background . adults were shifted to 28 ° C for 12 days . P - values were calculated using one - way ANOVA with Tukey ' s post - . Scale bar : 20 μm .

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

(A) repots>Shits1+H2B-RFP, (B) repots>Shits1+Egfrλtop and (C) repots>Shits1+RlSem incubated for 12 days at 28°C. (D) repots>DERDN for 7 days at 28°C. (E) Epithelial glia MARCM clone and (F) Egfrco mutant glia MARCM clone (labeled with Tomato, green). The penetrance is indicated as the number of samples with vacuoles over the total number of samples. DAPI: nuclei (white in A-F). (G) Percentage of vacuole area in the lamina neuropile of (A-D). The P-values were calculated using one-way ANOVA with Dunnett's post-test. (H) Percentage of vacuole area in the lamina of repots>Shits1 flies in the indicated genetic background. The adults were shifted to 28°C for 12 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


( A , A ' ) Anti - Spitz ( red ) immunostaining of w1118 adult head . Spitz can be detected in retina lamina . ( B , B ' ) full - transmembrane form of Spitz - GFP ( mSpi , green ) expressed in retina in GMR > mSpiGFP flies was predominantly identified in photoreceptor soma terminally localized in lamina cartridge . mSpitz - GFP requires processing by Rho Star to become a secreted form . Overexpressed mSpitzGFP has been demonstrated to be retained in perinuclear ER even in presence of endogenous Rho / Star [ 56 ] ( C ) Knockdown of both EGFR ligands Spi Krn in R1 - 6 photoreceptors in Rh1 > Dcr2 + Spi - RNAi + Krn - RNAi ( D ) blockade of Spi processing in Rh1 > lacZ + iRhom exhibited lamina degeneration after shifting to 29 ° C for 14 days . ( E - E ' ) Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28 ° C for 12 days . clone is labeled by DsRed ( red ) . ( F ) Spi secretion is inhibited in Rh1 > lacZ + Rab11S25N . ( G ) percentages of vacuole areas of ( C , D , F ) in lamina at 29 ° C for 14 days were examined . P - values were calculated using one - way ANOVA with Tukey ' s post - . DAPI : nuclei ( in A ' , B ' , C - F ) . Scale bar : 20 μm .

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

(A, A') Anti-Spitz (red) immunostaining of w1118 adult head. Spitz can be detected in the retina and lamina. (B, B') The full-length transmembrane form of Spitz-GFP (mSpi, green) expressed in the retina in GMR>mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge. mSpitz-GFP requires processing by Rho and Star to become a secreted form. Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho/Star [56] (C) Knockdown of both EGFR ligands Spi and Krn in R1-6 photoreceptors in Rh1>Dcr2+Spi-RNAi+Krn-RNAi and (D) blockade of Spi processing in Rh1>lacZ+iRhom exhibited lamina degeneration after shifting to 29°C for 14 days. (E-E') Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28°C for 12 days. The clone is labeled by DsRed (red). (F) Spi secretion is inhibited in Rh1>lacZ+Rab11S25N. (G) The percentages of the vacuole areas of (C, D, F) in lamina at 29°C for 14 days were examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. DAPI: nuclei (white in A', B', C-F). Scale bar: 20 μm.


( A , A ' ) Anti - Spitz detected Spitz ( magenta ) colocalizing ( arrowhead ) with epithelialgliacytoplasm marked by anti - Black ( green ) . Scale bar : 5 μm . ( B - B ' ) EGFR reporter pointed - lacZ ( pnt - lacZ ) exhibited expression in epithelial ( arrow ) marginal glia ( arrowhead ) . ( C , C ' ) Dominant - negative form of EGFR ( DERDN ) expressed in glia at 28 ° C for 3 days inhibited pnt - lacZ expression . ( D , D ' ) pnt - laZ expression was lost in Shits - expressing glia at 28 ° C for 3 days . ( E , E ' ) knockdown of Spi in R1 - 6 photoreceptors at 28 ° C for 12 days also inhibited pnt - lacZ expression in glia . penetrances of ( B - E ) are shown in upper right corner of each panel . DAPI : nuclei ( in B ' - E ' ) . Scale bar : 20 μm .

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

(A, A') Anti-Spitz detected Spitz (magenta) colocalizing (arrowhead) with epithelialgliacytoplasm marked by anti-Black (green). Scale bar: 5 μm. (B-B') EGFR reporter pointed-lacZ (pnt-lacZ) exhibited expression in the epithelial (arrow) and marginal glia (arrowhead). (C, C') Dominant-negative form of EGFR (DERDN) expressed in glia at 28°C for 3 days inhibited pnt-lacZ expression. (D, D') pnt-laZ expression was lost in Shits-expressing glia at 28°C for 3 days. (E, E') The knockdown of Spi in R1-6 photoreceptors at 28°C for 12 days also inhibited pnt-lacZ expression in the glia. The penetrances of (B-E) are shown in the upper right corner of each panel. DAPI: nuclei (white in B'-E'). Scale bar: 20 μm.


( A - C ) Immunostaining of TUNEL ( red ) , active Caspase - 3 ( magenta , A ' - C ' ) Repo ( green , A " - C " ) . ( A ) GMR - wIR ; repots > lacZ . ( B ) GMR - wIR ; repots > Shits1 . ( C ) GMR - wIR ; repots > DERDN . GMR - wIR was used to reduce autofluorescence of eye pigments . Scale bar : 20 μm . ( D - F ) in vivo sensor of caspase activity ( Apoliner ) indicated there was no active caspase activity in glia . ( D ) repots > Apoliner + lacZ . ( E ) repots > Apoliner + Shits1 . ( F ) repots > Apoliner + DERDN . adults were shifted to 28 ° C for 5 days . nls - GFP ( green , D - F ) ; merge of GFP ( green ) mRFP ( red ) in ( D ' - F ' ) ; merge of nls - GFP DAPI ( magenta ) in ( D " - F " ) . Scale bar : 10 μm . ( G ) of vacuole in lamina in repots > Shits1flies that coexpressed anti - apoptotic factors P35 Diap1 . adults were shifted to 28 ° C for 21 days . P - values were calculated using one - way ANOVA with Tukey ' s post - . ( H ) number of epithelialglia was not reduced in vacuolated lamina . numbers of epithelial glia in lamina of repo > H2B - RFP repo > H2B - RFP + Shits1 adults incubated at 28 ° C for 14 days were examined by counting RFP at epithelial layer from entire Z - stacks of confocal . P - values were calculated via Mann - Whitney .

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

(A-C) Immunostaining of TUNEL assay (red), active Caspase-3 (magenta, A'-C') and Repo (green, A"-C"). (A) GMR-wIR; repots>lacZ. (B) GMR-wIR; repots>Shits1. (C) GMR-wIR; repots>DERDN. GMR-wIR was used to reduce the autofluorescence of the eye pigments. Scale bar: 20 μm. (D-F) The in vivo fluorescent sensor of caspase activity (Apoliner) indicated there was no active caspase activity in the glia. (D) repots>Apoliner+lacZ. (E) repots>Apoliner+Shits1. (F) repots>Apoliner+DERDN. All adults were shifted to 28°C for 5 days. nls-GFP (green, D-F); merge of GFP (green) and mRFP (red) in (D'-F'); merge of nls-GFP and DAPI (magenta) in (D"-F"). Scale bar: 10 μm. (G) Percentage of vacuole area in the lamina in repots>Shits1flies that coexpressed the anti-apoptotic factors P35 and Diap1. The adults were shifted to 28°C for 21 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. (H) The cell number of the epithelialglia was not reduced in the vacuolated lamina. The numbers of epithelial glia in the lamina of repo>H2B-RFP and repo>H2B-RFP+Shits1 female adults incubated at 28°C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z-stacks of confocal images. P-values were calculated via Mann-Whitney tests.


In repots > GFP - LC3 + DERDN shifted to 28 ° C , GFP signal ( green ) was weak on day 1 ( A - A " ) progressively increased on days 2 ( B - B " ) 3 ( C - C " ) colocalized with Ref ( 2 ) P ( magenta ) . ( D ) Ref ( 2 ) P ( magenta ) accumulated within Egfrco mutant MARCM clone ( green ) . double - tagged GFP - mCherry - Atg8a is used to distinguish autophagosomes ( GFP mCherry , yellow ) autolysosomes ( mCherry , red ) in autophagic flux . ( E ) In repots > GFP - mCherry - Atg8a + dAtg1 , autophagosomes ( arrowhead ) autolysosomes ( arrow ) were induced in glia with normal autophagic flux . ( F ) repots > GFP - mCherry - Atg8a . ( G ) repots > GFP - mCherry - Atg8a + Shits1 . Epithelialglial nuclei are indicated ( arrow ) . ( H ) repots > GFP - mCherry - Atg8a + DERDN . adults were shifted to 28 ° C for 3 days . GFP : green ( E - H ) ; mCherry : red ( E ' - H ' ) ; merge ( E " - H " ) . Scale bar : 10 μm .

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

In repots>GFP-LC3+DERDN shifted to 28°C, the GFP signal (green) was weak on day 1 (A-A") and progressively increased on days 2 (B-B") and 3 (C-C") and colocalized with Ref(2)P (magenta). (D) Ref(2)P (magenta) accumulated within the Egfrco mutant MARCM clone (green). The double-tagged GFP-mCherry-Atg8a is used to distinguish the autophagosomes (GFP and mCherry, yellow) and autolysosomes (mCherry, red) in autophagic flux. (E) In repots>GFP-mCherry-Atg8a+dAtg1, autophagosomes (arrowhead) and autolysosomes (arrow) were induced in the glia with normal autophagic flux. (F) repots>GFP-mCherry-Atg8a. (G) repots>GFP-mCherry-Atg8a+Shits1. Epithelialglial nuclei are indicated (arrow). (H) repots>GFP-mCherry-Atg8a+DERDN. The adults were shifted to 28°C for 3 days. GFP: green (E-H); mCherry: red (E'-H'); merge (E"-H"). Scale bar: 10 μm.


( A - C ) repots > GFP - LAMP1 . ( D - F ) repots > GFP - LAMP1 + Shits1 . ( G - I ) repots > GFP - LAMP1 + DERDN . Adults were incubated at 28 ° C for 12 h ( A , D , G ) , 24 h ( B , E , H ) , 48 h ( C , F , I ) , respectively . GFP - LAMP1 signal ( green ) was induced at 12 h progressively enhanced in ( D - F ) ( G - I ) . ( J ) repots > Rab7 - mCherry + H2B - RFP . ( K ) repots > Rab7 - mCherry + Shits1 . ( L ) repots > Rab7 - mCherry + DERDN . adults were shifted to 28 ° C for 2 days . Rab7 - mCherry ( red ) were increased in ( K , L ) . DAPI : nuclei ( in J ' - L ' ) . Scale bar : 20 μm .

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

(A-C) repots>GFP-LAMP1. (D-F) repots>GFP-LAMP1+Shits1. (G-I) repots>GFP-LAMP1+DERDN. Adults were incubated at 28°C for 12 h (A, D, G), 24 h (B, E, H), and 48 h (C, F, I), respectively. The GFP-LAMP1 signal (green) was induced at 12 h and progressively enhanced in (D-F) and (G-I). (J) repots>Rab7-mCherry+H2B-RFP. (K) repots>Rab7-mCherry+Shits1. (L) repots>Rab7-mCherry+DERDN. The adults were shifted to 28°C for 2 days. Rab7-mCherry puncta (red) were increased in (K, L). DAPI: nuclei (white in J'-L'). Scale bar: 20 μm.


( A ) repots > Rab5S43N . ( B ) repots > Rab7T22N . ( C ) repots > Rab11S25N . ( D - H ) MARCM clones labeled by RFP or GFP ( green ) of control ( D ) , Rab52 ( E ) , α - Ada3 ( F ) , HrsD28 ( G ) , Rab7KO ( H ) , dor8 ( I ) carΔ146 ( J ) . penetrance ( number of samples with vacuole over number of samples examined ) is indicated in each panel . Adults of genotypes were incubated at 28 ° C for 14 days . DAPI : nuclei ( in A - J ) . Scale bar : 20 μm . ( K ) of vacuole in ( A - C ) was summarized . Adults of these genotypes were incubated at 28 ° C for 12 days . P - values were calculated using Kruskal - Wallis with Dunn ' s post - .

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

(A) repots>Rab5S43N. (B) repots>Rab7T22N. (C) repots>Rab11S25N. (D-H) MARCM clones labeled by RFP or GFP (green) of control (D), Rab52 (E), α-Ada3 (F), HrsD28 (G), Rab7KO (H), dor8 (I) and carΔ146 (J). The penetrance (number of samples with vacuole over the number of samples examined) is indicated in each panel. Adults of all genotypes were incubated at 28°C for 14 days. DAPI: nuclei (white in A-J). Scale bar: 20 μm. (K) The percentage of the vacuole area in (A-C) was summarized. Adults of these genotypes were incubated at 28°C for 12 days. P-values were calculated using Kruskal-Wallis with Dunn's post-tests.


repots > DERDN was coexpressed with ( A ) H2B - RFP , ( B ) Dorwt , ( C ) Dor - RNAi , ( D ) H2B - RFP , ( E ) Car , ( F ) Car - RNAi , ( G ) lacZ , ( H ) Car - RNAi + Dor - RNAi ( I ) Car + Dorwt . autophagy reporter Ref ( 2 ) P ( green ) was stained ( G ' - H ' ) . Scale bar : 20 μm . ( J , K , L ) percentages of vacuole areas in lamina of ( A - C , D - F , G - I ) were summarized , respectively . adults were shifted to 28 ° C for 7 days . DAPI : nuclei ( in A - I ) . P - values were calculated using Kruskal - Wallis with Dunn ' s post - .

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

repots>DERDN was coexpressed with (A) H2B-RFP, (B) Dorwt, (C) Dor-RNAi, (D) H2B-RFP, (E) Car, (F) Car-RNAi, (G) lacZ, (H) Car-RNAi+Dor-RNAi and (I) Car+Dorwt. The autophagy reporter Ref(2)P (green) was stained (G'-H'). Scale bar: 20 μm. (J, K, L) The percentages of the vacuole areas in the lamina of (A-C, D-F, G-I) were summarized, respectively. The adults were shifted to 28°C for 7 days. DAPI: nuclei (white in A-I). All P-values were calculated using Kruskal-Wallis tests with Dunn's post-tests.


A . Typical trace collected over 10 seconds .

A. Typical fluorescence intensity trace collected over 10 seconds.


B . Typical normalized FCS curves of Smc3 - GFP at different cycle phases . Dotted lines indicate fitting of one - component anomalous diffusion model to respective FCS curve .

O O O O O O O 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. Typical normalized FCS curves of Smc3-GFP at different cell cycle phases. Dotted lines indicate fitting of the one-component anomalous diffusion model to the respective FCS curve.


C - F . Diffusion times of Smc3 - GFP , Scc3 - GFP , Scc1 - GFP at G1 phase ( C ) , S phase ( D ) , G2 phase ( E ) , mitosis ( F ) . Data shown as mean ± s . e . m . from at least three independent experiments . * : p < 0 . 01 , # : p < 10 - 13 with Mann - Whitney .

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

C-F. Diffusion times of Smc3-GFP, Scc3-GFP, and Scc1-GFP at the G1 phase (C), S phase (D), G2 phase (E), and mitosis (F). Data shown as mean ± s.e.m. from at least three independent experiments. *: p<0.01, #: p<10-13 with the Mann-Whitney test.


Representative normalized FCS curve of Scc1 - GFP at different cycle phases . Dotted lines indicate fitting of one - component anomalous diffusion models to respective FCS curve .

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

Representative normalized FCS curve of Scc1-GFP at different cell cycle phases. Dotted lines indicate fitting of one-component anomalous diffusion models to the respective FCS curve.


diffusion times of Scc1 - GFP at S phase ( B ) were measured in strains yIO664 ( wild - type ) yEB005 ( WPL1 ∆ ) , in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) yAM946 ( ECO1 - AID ) . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

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

The diffusion times of Scc1-GFP at S phase (B) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.


diffusion times of Scc1 - GFP at G2 phase ( C ) M phase ( D ) were measured in strains yIO664 ( wild - type ) yEB005 ( WPL1 ∆ ) , in auxin - depleted yEB002 ( PDS5 - AID ) , yAM085 ( SCC3 - AID ) yAM946 ( ECO1 - AID ) . Data are shown as mean ± s . e . m . from at least three independent experiments . * : p < 10 - 5 , * * : p < 10 - 13 .

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

The diffusion times of Scc1-GFP at G2 phase (C) and M phase (D) were measured in strains yIO664 (wild-type) and yEB005 (WPL1∆), and in auxin-depleted yEB002 (PDS5-AID), yAM085 (SCC3-AID) and yAM946 (ECO1-AID) cells. Data are shown as mean ± s.e.m. from at least three independent experiments. *: p< 10-5, **: p< 10-13.


A . Typical fitted photon counting histograms of GFP GFP - GFP in live yeast .

A. Typical fitted photon counting histograms of GFP and GFP-GFP in live yeast cells.


B . molecular brightness of different GFP tagged constructs was obtained with GFP monomer GFP - GFP dimer as brightness standard . of yIO664 ( SCC1 - GFP ) were analyzed throughout cycle by PCH . To examine effects of Wpl1 , Pds5 , Eco1 on cohesin dimerization , PCH was repeated in yEB005 ( SCC1 - GFP WPL1 ∆ ) , yEB002 ( SCC1 - GFP PDS5 - AID ) , yAM946 ( SCC1 - GFP ECO1 - AID ) in which Pds5 - AID / Eco1 - AID was depleted with auxin .

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

B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin.


C . PCH of yAM085 ( SMC3 - GFP ) throughout cycle .

C. PCH analysis of strain yAM085 (SMC3-GFP) throughout the cell cycle.


D . PCH of strains yAM953 ( SCC1 - GFP SMC3 - GFP ) yAP941 ( SCC1 - GFP SCC3 - GFP ) containing two GFP tagged subunits . Data shown as boxplot ( + data overlap ) with values of minimum , 5 % percentile , 25 % percentile , median , 75 % percentile , maximum , mean ( center square marks ) from at least three independent experiments .

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

D. PCH analysis of strains yAM953 (SCC1-GFP SMC3-GFP) and yAP941 (SCC1-GFP SCC3-GFP) containing two GFP tagged subunits. Data shown as boxplot (+data overlap) with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) from at least three independent experiments.


( A ) AP radiograph demonstrates characteristic findings of ATD in R05 - 365A proband . Note shortened humeri ( closed arrowhead ) elongated clavicles ( arrow ) . ( B ) Radiographs of Cmh001543 - 01 proband showing similar findings .

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

(A) AP radiograph demonstrates characteristic findings of ATD in the R05-365A proband. Note the shortened humeri (closed arrowhead) and elongated clavicles (arrow). (B) Radiographs of the Cmh001543-01 proband showing similar findings.


( A ) Sanger trace showing homozygosity for c . 469C > T mutation , predicting a stop codon in exon 6 of GRK2 ( arrow ) .

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

(A) Sanger sequence trace showing homozygosity for the c.469C>T mutation, predicting a stop codon in exon 6 of GRK2 (arrow).


( B ) Exome showing biallelic changes c . 1348 _ 1349del c . 555 + 1G > A . Red arrow points to place of mutation . Orange green boxes indicate reference nucleotide its substitution , respectively .

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

(B) Exome analysis showing the biallelic changes c. 1348_1349del and c.555 +1G>A. Red arrow points to the place of mutation. Orange and green boxes indicate the reference nucleotide and its substitution, respectively.


( D ) Expression of GRK2 as measured by RT - PCR in three replicates each of control R05 - 365A fibroblasts , demonstrating absence of GRK2 transcript in ATD . GAPDH served as a positive control .

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

(D) Expression of GRK2 as measured by RT-PCR in three replicates each of control and R05-365A fibroblasts, demonstrating absence of GRK2 transcript in ATD cells. GAPDH served as a positive control.


( E ) Two control ATD fibroblast samples were immunoblotted for GRK2 . Note absence of GRK2 in ATD . Actin served as a loading control . data shown are representative of three independent experiments .

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(E) Two control and ATD fibroblast samples were immunoblotted for GRK2. Note the absence of GRK2 protein in the ATD cells. Actin served as a loading control. The data shown are representative of three independent experiments.


( A ) GRK2 immunohistochemistry in a control 17 - week gestational distal femur growth plate demonstrating widespread expression , particularly in bone ( B ) , proliferating zone of growth plate ( P ) , perichondrium ( arrowheads ) . Non - reactive antibody was used as a control ( Ab control ) . R , reserve zone . H , hypertrophic zone . Scale bars , 100 μm .

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(A) GRK2 immunohistochemistry in a control 17-week gestational age distal femur growth plate demonstrating widespread expression, particularly in the bone (B), proliferating zone of the growth plate (P), and the perichondrium (arrowheads). Non-reactive antibody was used as a control (Ab control). R, reserve zone. H, hypertrophic zone. Scale bars, 100 μm.


( B ) Picrosirius red of femoral growth plates of a control ATD R05 - 365A . control growth plate shows normal architecture with clearly distinguishable reserve ( R ) , proliferating ( P ) hypertrophic ( H ) zones . A profound disruption of growth plate architecture is obvious in GRK2 - / - growth plate , which shows irregularly - shaped reserve zone chondrocytes , a lack of discernable proliferating chondrocytes a significantly under - developed hypertrophic zone . Scale bars , 100 μm .

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(B) Picrosirius red staining of the femoral growth plates of a control and the ATD R05-365A fetus. The control growth plate shows normal architecture with clearly distinguishable reserve (R), proliferating (P) and hypertrophic (H) zones. A profound disruption of growth plate architecture is obvious in the GRK2-/- growth plate, which shows irregularly-shaped reserve zone chondrocytes, a lack of discernable proliferating chondrocytes and a significantly under-developed hypertrophic zone. Scale bars, 100 μm.


( C ) Shortened long bones in chicken wings injected with CMPD101 . right limb bud was injected with CMPD101 , left one was left as a control . Note shortened humerus ( H ) , ulna ( U ) radius ( R ) in CMPD101 - injected wing . Mean ± . Mann - Whitney U ; number of is indicated . Scale bar , 150 μm .

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(C) Shortened long bones in the chicken wings injected with CMPD101. The right limb bud was injected with CMPD101, the left one was left as a control. Note the shortened humerus (H), ulna (U) and radius (R) in the CMPD101-injected wing. Mean ± SEM. Mann-Whitney U test; number of animals is indicated. Scale bar, 150 μm.


( D ) Alcian blue of femoral growth plates of a control ATD R05 - 365A . Note significantly lesser blue of R05 - 365A growth plate stained by alcian blue pH 1 . 0 , suggesting impaired sulfation of extracellular matrix . Scale bars , 200 μm .

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(D) Alcian blue staining of the femoral growth plates of a control and the ATD R05-365A fetus. Note the significantly lesser blue staining of the R05-365A growth plate stained by alcian blue pH 1.0, suggesting impaired sulfation of the extracellular matrix. Scale bars, 200 μm.


( E ) Alcian blue of chicken limb bud micromasses treated with GRK2 inhibitor CMPD101 . Note gradual inhibition of cartilage nodules with increasing of CMPD101 , quantified below . Mean ± . Mann - Whitney U ; number of micromasses is indicated . A representative experiment of four ( pH 2 . 5 ) three ( pH 1 . 0 ) is shown . Scale bar , 100 μm . ( F ) Alcian blue of micromasses generated from Grk2 + / + Grk2 - / - NIH3T3 . Note mildly weaker by alcian blue pH 2 . 5 nearly absent sulfation stained by alcian blue pH 1 . 0 in Grk2 - / - micromasses . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U ; number of micromasses is indicated . Scale bar , 1 mm .

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(E) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101. Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101, quantified below. Mean ± SEM. Mann-Whitney U test; number of micromasses is indicated. A representative experiment of four (pH 2.5) and three (pH 1.0) is shown. Scale bar, 100 μm. (F) Alcian blue staining of the micromasses generated from Grk2+/+ and Grk2-/- NIH3T3 cells. Note the mildly weaker staining by alcian blue pH 2.5 and the nearly absent sulfation stained by alcian blue pH 1.0 in the Grk2-/- micromasses. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of micromasses is indicated. Scale bar, 1 mm.


( A , B ) Defective GLI3 processing in GRK2 - / - fibroblasts . were serum - starved to induce cilia before treatment with Hh pathway activator SAG ( Smoothened agonist ) , whole lysates were immunoblotted for GLI3 . Note SAG - mediated downregulation of both full - ( FL ) repressor ( R ) GLI3 in control fibroblasts , demonstrating normal processing . There was no effect of SAG on GLI3 processing in GRK2 - / - . A representative experiment of four is shown . ( B ) Quantitation of GLI3FL to GLI3R using densitometry . Mean ± . Mann - Whitney U ; number of biological replicates is indicated .

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(A,B) Defective GLI3 processing in GRK2-/- fibroblasts. Cells were serum-starved to induce cilia before treatment with the Hh pathway activator SAG (Smoothened agonist), and the whole cell lysates were immunoblotted for GLI3. Note the SAG-mediated downregulation of both full-length (FL) and repressor (R) GLI3 in control fibroblasts, demonstrating normal processing. There was no effect of SAG on GLI3 processing in GRK2-/- cells. A representative experiment of four is shown. (B) Quantitation of the GLI3FL to GLI3R ratio using densitometry. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.


( C ) Defective SAG response in GRK2 - / - fibroblasts . were treated with SAG for 24 hours . Transcript of GLI3 targets GLI1 PTCH1 were determined by qRT - PCR ; GAPDH was used for normalization . Note SAG - mediated induction of GLI1 PTCH1 expression in controls but not in GRK2 - / - . Mean ± . Mann - Whitney U ; number of biological replicates is indicated .

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(C) Defective SAG response in GRK2-/- fibroblasts. Cells were treated with SAG for 24 hours. Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT-PCR; GAPDH was used for normalization. Note the SAG-mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.


( D ) Under - phosphorylation of Smoothened ( SMO ) in GRK2 - / - fibroblasts . Lysates of SMO - transfected were resolved by phospho ( p ) - shift PAGE immunoblotted for SMO . portion of pSMO analyzed by densitometry is shown . GRK2 add - back demonstrates a rescue of pSMO in GRK2 - / - fibroblasts . Mean ± . Mann - Whitney U ; number of biological replicates is indicated .

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(D) Under-phosphorylation of Smoothened (SMO) in GRK2-/- fibroblasts. Lysates of SMO-transfected cells were resolved by the phospho(p)-shift PAGE and immunoblotted for SMO. The portion of pSMO analyzed by densitometry is shown. GRK2 add-back demonstrates a rescue of the pSMO in GRK2-/- fibroblasts. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.


Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . were treated with SAG immunostained for SMO , ARL13B pericentrin . Note failure in SMO accumulation in cilia of GRK2 - / - ( arrow ) . Scale bar , 2 µm ( E ) .

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Defective SMO accumulation in cilia of GRK2-/- fibroblasts. Cells were treated with SAG and immunostained for SMO, ARL13B and pericentrin. Note the failure in SMO accumulation in cilia of GRK2-/- cells (arrow). Scale bar, 2 µm (E).


Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . of SMO - positive cilia was calculated plotted . Mean ± . Welch ´ s t - ; number of biological experiments total numbers of analyzed cilia are indicated ( F ) .

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Defective SMO accumulation in cilia of GRK2-/- fibroblasts. The percentage of SMO-positive cilia was calculated and plotted. Mean ± SEM. Welch´s t-test; number of biological experiments and the total numbers of analyzed cilia are indicated (F).


Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . of ciliary SMO was analyzed plotted . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U ; number of biological experiments total numbers of analyzed cilia are indicated ( G ) .

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Defective SMO accumulation in cilia of GRK2-/- fibroblasts. The intensity of ciliary SMO was analyzed and plotted. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated (G).


Defective SMO cilia accumulation inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 12 ( A , B ) ( A , B ) Cilia were stained by ARL13B SMO antibodies , of ciliary SMO was analyzed plotted . Note impaired SMO accumulation in cilia caused by GRK2 inhibition ( black arrow ) . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U ; number of biological experiments total numbers of analyzed cilia are indicated . Scale bar , 1 µm .

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Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 12 (A, B) (A,B) Cilia were stained by ARL13B and SMO antibodies, and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO accumulation in cilia caused by GRK2 inhibition (black arrow). Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.


Defective SMO cilia accumulation inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors . Control human R00 - 082 chondrocytes were serum starved in presence of a GRK2 inhibitor , either CMPD101 ( C ) or paroxetine ( P ) , for 24 hours before they were treated with SAG for additional 24 hours ( C D ) . ( C ) lysates were immunoblotted for GLI3 processing GLI1 upregulation ; actin served as a loading control . GLI3FL GLI3R , full - repressor GLI3 variants , respectively . ( D ) of GLI3 GLI1 by densitometry . Note impaired GLI3 processing no GLI1 upregulation in treated with CMPD101 or paroxetine . Mean ± . Mann - Whitney U ( GLI3 ) Welch ´ s t - ( GLI1 ) ; number of biological experiments is indicated .

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Defective SMO cilia accumulation and inhibited Hh signaling in chondrocytes treated with GRK2 inhibitors. Control human R00-082 chondrocytes were serum starved in the presence of a GRK2 inhibitor, either CMPD101 (C) or paroxetine (P), for 24 hours before they were treated with SAG for additional 24 hours (C D). (C) Cell lysates were immunoblotted for GLI3 processing and GLI1 upregulation; actin levels served as a loading control. GLI3FL and GLI3R, full-length and repressor GLI3 variants, respectively. (D) Quantification of GLI3 and GLI1 levels by densitometry. Note the impaired GLI3 processing and no GLI1 upregulation in cells treated with CMPD101 or paroxetine. Mean ± SEM. Mann-Whitney U test (GLI3 ratio) and Welch´s t-test (GLI1 levels); number of biological experiments is indicated.


Defective SMO cilia accumulation in chondrocytes with downregulated GRK2 . ( E ) Doxycycline ( DOX ) - inducible GRK2 downregulation in control human R00 - 082 chondrocytes , tested by western blot , normalized to actin plotted below . Mean ± . Welch ´ s t - ; number of biological experiments is indicated . shScr , scramble shRNA .

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Defective SMO cilia accumulation in chondrocytes with downregulated GRK2. (E) Doxycycline (DOX)-inducible GRK2 downregulation in control human R00-082 chondrocytes, tested by western blot, normalized to actin and plotted below. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated. shScr, scramble shRNA.


( F ) pre - treated with DOX for three days were serum starved , SAG treated , stained analyzed as in ( A , B ) , of ciliary SMO was analyzed plotted . Note impaired SMO translocation to cilia caused by GRK2 loss . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U ; number of biological experiments total numbers of analyzed cilia are indicated . Scale bar , 1 µm .

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(F) Cells pre-treated with DOX for three days were serum starved, SAG treated, stained and analyzed as in (A,B), and the intensity of ciliary SMO was analyzed and plotted. Note the impaired SMO translocation to cilia caused by GRK2 loss. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. Scale bar, 1 µm.


( A ) Control GRK2 - / - fibroblasts were transfected with TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , effect of Wnt3A on TOPflash transcriptional activation was determined by dual - luciferase . Note impaired response to Wnt3A in GRK2 - / - . Mean ± . Welch ´ s t - ; number of biological experiments is indicated .

(A) Control and GRK2-/- fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual-luciferase assay. Note the impaired response to Wnt3A in GRK2-/- cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.


( B Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - pT1572 - LRP6 specific antibodies , reduced LRP6 DVL2 expression , increased steady state phosphorylation of DVL2 . A representative experiment of four is shown . # , nonspecific band .

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(B Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band.


C ) Low phosphorylation ( p ) of Wnt3A co - receptor LRP6 as determined by western blot with pS1490 - pT1572 - LRP6 specific antibodies , reduced LRP6 DVL2 expression , increased steady state phosphorylation of DVL2 . ( C ) Densitometry of western blot results demonstrating decreased of LRP6 in GRK2 - / - , lower response of GRK2 - / - to Wnt3A by LRP6 phosphorylation at S1490 , tendency towards similar defect at T1572 . Densitometry also showed less of total DVL2 , increased steady state pDVL2 in GRK2 - / - . Mean ± . Mann - Whitney U ; number of biological experiments is indicated .

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C) Low phosphorylation (p) of Wnt3A co-receptor LRP6 as determined by western blot with pS1490- and pT1572-LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. (C) Densitometry of western blot results demonstrating decreased levels of LRP6 in GRK2-/- cells, lower response of GRK2-/- cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency towards the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


( D ) Loss of interaction between Frizzled 4 ( FZD4 ) ß - Arrestin 2 ( ARRB2 ) in GRK2 - / - fibroblasts . Proximity ligation ( PLA ) between expressed FZD4 - GFP ARRB2 - Flag showed significantly reduced proximity events between two in GRK2 - / - . dashed lines outline transfected . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U ; number of biological experiments total numbers of analyzed are indicated . Scale bar , 10 µm .

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(D) Loss of interaction between Frizzled 4 (FZD4) and ß-Arrestin 2 (ARRB2) in GRK2-/- fibroblasts. Proximity ligation assay (PLA) between expressed FZD4-GFP and ARRB2-Flag showed significantly reduced proximity events between the two proteins in GRK2-/- cells. White dashed lines outline the transfected cells. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 µm.


( A ) Control ( + / + ) Grk2 - / - NIH3T3 were transfected with TOPflash Firefly ( F ) luciferase vector together with a control Renilla ( R ) luciferase vector , treated with Wnt3A ( 100 ng / ml ) for 24 hours subjected to dual - luciferase . Note impaired response to Wnt3A in Grk2 - / - . Mean ± . Mann - Whitney U ; number of biological experiments is indicated .

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(A) Control (+/+) and Grk2-/- NIH3T3 cells were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, treated with Wnt3A (100 ng/ml) for 24 hours and subjected to dual-luciferase assay. Note the impaired response to Wnt3A in Grk2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


( B ) Control ( + / + ) Grk2 - / - NIH3T3 were treated with 40 ng / ml Wnt3A for 1 hour . Note lower LRP6 expression inhibited Wnt3A - induced LRP6 phosphorylation in Grk2 - / - NIH3T3 . Mean ± . Welch ´ s t - ; number of biological experiments is indicated .

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(B) Control (+/+) and Grk2-/- NIH3T3 cells were treated with 40 ng/ml Wnt3A for 1 hour. Note the lower LRP6 expression and inhibited Wnt3A-induced LRP6 phosphorylation in the Grk2-/- NIH3T3 cells. Mean ± SEM. Welch´s t-test; number of biological experiments is indicated.


Loss of LRP6 phosphorylation in chondrocytes treated with GRK2 inhibitor CMPD101 . Control human R00 - 082 chondrocytes ( C ) were treated with 20 µM CMPD101 overnight then treated with 100 ng / ml Wnt3A for 1 hour . Note less LRP6 phosphorylation ( pS1490 - pT1572 - LRP6 ) in treated with CMPD101 . Mean ± . Mann - Whitney U ; number of biological experiments is indicated .

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Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. Control human R00-082 chondrocytes (C) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.


Loss of LRP6 phosphorylation in chondrocytes treated with GRK2 inhibitor CMPD101 . rat chondrosarcoma ( RCS ) ( D ) were treated with 20 µM CMPD101 overnight then treated with 100 ng / ml Wnt3A for 1 hour . Note less LRP6 phosphorylation ( pS1490 - pT1572 - LRP6 ) in treated with CMPD101 . Mean ± . Mann - Whitney U ; number of biological experiments is indicated .

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Loss of LRP6 phosphorylation in chondrocytes treated with the GRK2 inhibitor CMPD101. rat chondrosarcoma (RCS) cells (D) were treated with 20 µM CMPD101 overnight and then treated with 100 ng/ml Wnt3A for 1 hour. Note the less LRP6 phosphorylation (pS1490- and pT1572-LRP6) in cells treated with CMPD101. Mean ± SEM. Mann-Whitney U test; number of biological experiments is indicated.