Immunoblots of WT and non - palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants ( DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine , C - terminal mutant = mutant where the 3 C - terminal palmitoylation sites of DHHC5 have been mutated to alanine residues . ) . WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C - terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C - terminus of DHHC5 is essential for the stability of this interaction .

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Immunoblots of WT and non-palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants (DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine, C-terminal mutant = mutant where the 3 C-terminal palmitoylation sites of DHHC5 have been mutated to alanine residues.). WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C-terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C-terminus of DHHC5 is essential for the stability of this interaction.


Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5 . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and the catalytic mutant DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Quantification of the plasma membrane signal of DHHC5 in each of the images ( A - F ) , the graph displays mean + / - s . e . m . * * * * = p < 0 . 001 , n . s . = not significant , unpaired t - test . N = 35 , DHHC5 + WT Golga7b . N = 41 , DHHC5 + mutant Golga7b . N = 35 , DHHS5 + WT Golga7b . N = 31 , DHHS5 + Golga7b mutant . N = 29 , C - terminal mutant + WT Golga7b . N = 30 , C - terminal mutant + mutant Golga7b . Co expression of palmitoylation - deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane . The majority of the DHHC5 C - terminal palmitoylation - deficient mutant is localised to the plasma membrane in a Golga7b independent manner . All N numbers refer to the number of cells imaged and quantified , all experiments repeated 4 times . Data information : All images show ectopic protein expression only . All scale bars 10µm .

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Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5. Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and the catalytic mutant DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Quantification of the plasma membrane signal of DHHC5 in each of the images (A-F), the graph displays mean +/- s.e.m. ****=p<0.001, n.s. = not significant, unpaired t-test. N=35, DHHC5 + WT Golga7b. N=41, DHHC5 + mutant Golga7b. N=35, DHHS5 + WT Golga7b. N=31, DHHS5 + Golga7b mutant. N=29, C-terminal mutant + WT Golga7b. N=30, C-terminal mutant + mutant Golga7b. Co expression of palmitoylation-deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane. The majority of the DHHC5 C-terminal palmitoylation-deficient mutant is localised to the plasma membrane in a Golga7b independent manner. All N numbers refer to the number of cells imaged and quantified, all experiments repeated 4 times. Data information: All images show ectopic protein expression only. All scale bars 10µm.


Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged WT Golga7b . Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged mutant Golga7b .

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Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged WT Golga7b. Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged mutant Golga7b.


Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non - targeting siRNA . Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA .

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Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non-targeting siRNA. Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA.


Quantification of plasma membrane signal of endogenous DHHC5 when co - expressed with WT or mutant Golga7b or without any transfected Golga7b . * * * * = P < 0 . 001 , t - test , endogenous + WT Golga7b n = 37 , endogenous + mutant Golga7b n = 23 , endogenous n = 42 . Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell , while WT Golga7b stabilises DHHC5 at the plasma membrane .

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Quantification of plasma membrane signal of endogenous DHHC5 when co-expressed with WT or mutant Golga7b or without any transfected Golga7b. ****=P<0.001, t-test, endogenous + WT Golga7b n=37, endogenous + mutant Golga7b n=23, endogenous n=42. Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell, while WT Golga7b stabilises DHHC5 at the plasma membrane.


Quantification of the plasma membrane signal of DHHC5 in ( C and D ) , * * * * = p < 0 . 001 t - test , control siRNA n = 23 , Golga7b siRNA n = 23 .

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Quantification of the plasma membrane signal of DHHC5 in (C and D), ****=p<0.001 t-test, control siRNA n=23, Golga7b siRNA n=23.


FLAG - DHHC5 immunoprecipitations were performed using lysates from Hela cells co - expressing either wild - type ( WT ) or palmitoylation deficient mutant ( m ) Golga7b . The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b , quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 .

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FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.


Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG - DHHC5 is expressed alone or co - expressed with either WT or mutant Golga7b . IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation .

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Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG-DHHC5 is expressed alone or co-expressed with either WT or mutant Golga7b. IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation.


Volcano plot of DHHC5 interactors from AP - MS analysis from HeLa cells co - expressing FLAG - DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin - mediated endocytosis , cell : cell adhesion and known substrates of DHHC5 .

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Volcano plot of DHHC5 interactors from AP-MS analysis from HeLa cells co-expressing FLAG-DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin-mediated endocytosis, cell:cell adhesion and known substrates of DHHC5.


Validation of novel DHHC5 interactors by co - IP : immunoblots of Dsg2from Hela cells co - expressing DHHC5 and either Wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of clathrin heavy chain from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of ZO - 1 protein from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * * = p < 0 . 01 ratio paired t - test , n = 3 .

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Validation of novel DHHC5 interactors by co-IP: immunoblots of Dsg2from Hela cells co-expressing DHHC5 and either Wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of clathrin heavy chain from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of ZO-1 protein from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, **=p<0.01 ratio paired t-test, n=3.


30 minute DMSO treatment of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) . 30 minute Dynasore treatment ( 25µM ) of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) .

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30 minute DMSO treatment of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged). 30 minute Dynasore treatment (25µM) of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).


Non - targeting control siRNA treatment ( 10nM ) of HeLa cells expressing mutant Golga7b ( HA tagged ) . Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment ( 10nM ) of HeLa cells that were subsequently transfected with mutant Golga7b ( HA tagged ) . Images are detecting endogenous DHHC5 and the HA tagged Golga7b .

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Non-targeting control siRNA treatment (10nM) of HeLa cells expressing mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment (10nM) of HeLa cells that were subsequently transfected with mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b.


Quantification of plasma membrane signal of DHHC5 in ( A and B ) . * * * * = p < 0 . 001 , unpaired t - test . N = 37 , + Dynasore . N = 28 , + DMSO .

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Quantification of plasma membrane signal of DHHC5 in (A and B). ****=p<0.001, unpaired t-test. N=37, +Dynasore. N=28, +DMSO.


Quantification of plasma membrane signal of DHHC5 in ( C and D ) . * * * * = p < 0 . 001 , unpaired t - test . All scale bars 10μm .

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Quantification of plasma membrane signal of DHHC5 in (C and D). ****=p<0.001, unpaired t-test. All scale bars 10μm.


Biotin endocytosis assays of HeLa cells with endogenous DHHC5 , over - expression of DHHC5 alone , co - expression of DHHC5 and WT Golga7b , co - expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C - terminal mutant . Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5 . The lack of signal in the pull - down of the DHHC5 C - terminal mutant or when DHHC5 and WT Golga7b are co - expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions . The robust signal in the pull - down of endocytosed proteins from cells co - expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition . N = 3 , * = p < 0 . 05 , paired t test "

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Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test"


ABE purifications from HeLa cells treated with 10nM non - targeting siRNA ( nt siRNA ) or DHHC5 siRNA were immunoblotted for Dsg2 . Loss of the band in the + HA pull - down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5 .

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ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5.


Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments .

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Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.


Confocal images of DHHC5 , Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . See Appendix Figure S6 for quantification of data in panels B - D .

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Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D.


Immunoblots and quantification of Dsg2 pulled - down in cell surface biotinylation assays in nt siRNA , DHHC5 siRNA or Golga7b siRNA treated conditions . Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input . A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA . * =

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Immunoblots and quantification of Dsg2 pulled-down in cell surface biotinylation assays in nt siRNA, DHHC5 siRNA or Golga7b siRNA treated conditions. Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input. A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA. *=


Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium ( 5µM calcium ) conditions , with quantification of the intensity of the DSG2 signal in the pulldown normalised to input . There is a significant reduction in the Dsg2 signal in the + HA pull - down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition . * = p < 0 . 05 , paired t - test , N = 4 , error bars represent s . e . m . All scale bars 10μm .

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Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.


10x brightfield images of A431 cells treated with non - targeting siRNA at time 0 ( left ) and after 48 hours ( right ) of 5 ng / ml EGF treatment . 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . Quantification of cell scattering of A431 cells , showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed .

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10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5 ng/ml EGF treatment. 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed.


10x brightfield images of A431 cells treated with non - targeting siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . Quantification of cell scattering of A431 cells , showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion , was observed .

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10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion, was observed.


Plot showing the resistance of A431 cells from a trans - epithelial electrical resistance assay treated with either DHHC5 siRNA , Golga7b siRNA or non - targeting siRNA . A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion .

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Plot showing the resistance of A431 cells from a trans-epithelial electrical resistance assay treated with either DHHC5 siRNA, Golga7b siRNA or non-targeting siRNA. A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion.


B . Chromatin accessibility and p300 binding at differential ATAC - seq peaks visualized using K - means clustering .

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B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering.


B . Boxplots showing differential gene expression ( AC versus DMZ ) and associated ATAC - seq signals ( Two biological replicates ) . The central band within the boxplot represents the median and the whiskers show 1 . 5 times the range between the first and third quartile ( IQR ) .

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B. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC-seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR).


F . Genomic tracks showing aggregated accessibility of single - cell ATAC - seq clusters at the t ( tbxt ) , tfap2a , gsc , ctcf , lhx1 and sox11 loci .

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F. Genomic tracks showing aggregated accessibility of single-cell ATAC-seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.


C . Feature maps , showing the expression of selected genes in single cells ( cf . Appendix Fig . S2 ) . Color scale represents the gene expression value ( log1p - transformed counts per 10 , 000 unique reads ) for each cell for a given gene , from low ( grey ) to high ( orange ) .

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C. Feature maps, showing the expression of selected genes in single cells (cf. Appendix Fig. S2). Color scale represents the gene expression value (log1p-transformed counts per 10,000 unique reads) for each cell for a given gene, from low (grey) to high (orange).


A . Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1 , Foxb1 - Eomes , Eomes , Irx3 , Irx3 - Otx2 , Otx2 and Lhx8 .

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A. Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1, Foxb1-Eomes, Eomes, Irx3, Irx3-Otx2, Otx2 and Lhx8.


C . Fold enrichment of genes with zygotically defined ( ZyD ) H3K4me3 at their promoter in AC with transcription factor overexpression . Asterisk indicates hypergeometric p - value < = 0 . 01 .

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C. Fold enrichment of genes with zygotically defined (ZyD) H3K4me3 at their promoter in AC with transcription factor overexpression. Asterisk indicates hypergeometric p-value <= 0.01.


( A , B ) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice . Brain sections were prepared 1 day after BrdU injection . The white box area is magnified in the lower panels : DAPI ( blue ) , Ki67 ( green ) , and BrdU ( red ) . Arrow heads indicate BrdU and Ki - 67 double positive cells in the SGZ .

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(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ.


( C , D ) Differentiating cells are identified by staining with BrdU and DCX . Brain sections were prepared 10 days after BrdU injection .

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(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.


( E , F ) Maturation of progenitor cells are assessed by staining with BrdU and NeuN . Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness , and a total of 24 sections were obtained from one hippocampus . Scale bars : 100 μm in the large panel , and 10 μm in the magnified images . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.


( A ) miR - 124 expression is altered in DSCR1 mutants . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 01 .

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(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.


( B ) Promoter activity of miR - 124 in Neuro2 A cells with DSCR1 reduction or overexpression . Firefly luciferase reporter under the control of miR - 124 promoter is measured . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.


( C ) Bisulfite sequencing analyses of the miR - 124 promoter . Each CpG site is indicated , and the methylation status of two different regions are shown . Open and filled circles represent unmethylated and methylated CpG sites , respectively . The percentage of methylated CpGs among total number of CpGs is also shown .

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(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.


( D ) Neuro2A cells containing DSCR1shRNA show increased activity of miR - 124 promoter measured . The miR - 124 promoter was inserted in front of the luciferase reporter . Site - directed mutation of two methylation sites ( 31 and 58 ) in the promoter shows the luciferase activity similar to that of DSCR1 reduction . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( E ) Overexpression of DSCR1 reduced the luciferase activity , while removing all methylation sites in the miR - 124 promoter increases it . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( A ) DSCR1 binds to TET1 introns . The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA .

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(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.


( B ) DSCR1 missing RNA recognition motif ( △ RRM ) does not bind to the TET1 intron 8 .

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(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.


( C , D ) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4 . DSCR1 reduction increases the activity of this luciferase construct , while DSCR1 overexpression decreases its activity . In contrast , altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 . * * * P < 0 . 001 .

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(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.


( E ) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants . TET1 is increased in DSCR1 knockout , while it is decreased in DSCR1 transgenic mice . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animal for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.


( B ) 3 weeks after lentivirus injection , neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region . The white arrow indicates Sox2 and GFP double positive cells . Scale bar , 10 μm . ( C ) The number of GFP and Sox2 double positive cells is restored to that of wild type . Scale bar : 10μm . Values are shown as mean ± SEM and tested for statistical significance by paired t - test . N = 3 animal for each condition , * P < 0 . 05 .

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(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.


( A ) Genotype of Ts65Dn / DSCR1 + / - mouse was confirmed by detecting markers for Ts65Dn and DSCR1

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(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO


( B ) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2 . 5 folds compared to that of wild type . However , Ts65Dn / DSCR1 + / - mouse showed normal level of DSCR1 mRNA transcript . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.


As expected , the levels of TET1 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the TET1 mRNA expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

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As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


As expected , the levels of miR - 124 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the miR - 124 expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

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As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


( E , F ) The number proliferating progenitor neurons identified by BrdU and Ki - 67 double staining is restored in the SGZ of Ts65Dn / DSCR1 + / - mouse compared to that of Ts65Dn mouse . The white box area is magnified in the lower panels : DAPI ( blue ) , Ki67 ( green ) , and BrdU ( red ) . Arrow heads indicate BrdU and Ki - 67 double positive cells in the SGZ . Scale bars : 100 μm in the large image and 10 μm in magnified image . Each hippocampal section was 40 μm in thickness , and total 6 hippocampi were used for analysis . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) animals , * P < 0 . 05 .

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(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.


( G - J ) Ts65Dn has learning and memory defects , but Ts65Dn / DSCR1 + / - mouse clearly rescues learning and memory defects to control levels . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 8 ( control ) , 5 ( Ts65Dn ) , 10 ( Ts65Dn / DSCR1 + / - ) animals , * P < 0 . 001 ( G , H ) . * P < 0 . 05 ( I ) , * * P < 0 . 01 ( I ) . ( J ) Open - field analysis shows no defects in movement of tested animals . Values are shown as mean ± SEM , N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) animals .

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(G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.


( b ) Suppression of Htt103Q toxicity in yeast by Gpx1 , Hyr1 ( Gpx3 ) and mGpx1 . The viability of the parental strain , Y258 , and of cells overexpressing yeast GPxs was determined using spotting assays . Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose ( GAL ) to induce the expression of Htt103Q and the indicated ORF .

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(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.


a ) Total GPx activity was measured in mGpx1 - overexpressing ( mGpx1 ) or control ( WT ) cell lines with ( induced ) or without ( uninduced ) Htt103Q expression . Overexpression of mGpx1 increases total GPx activity in PC12 cells ( * * * * P 0 . 0001 compared to uninduced controls ; n = 7 per condition ) .

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a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition).


( b ) Quantification of caspase - 3 / 7 activation in response to Htt103Q expression in cells overexpressing mGpx1 . Overexpression of mGpx1 reduces Htt103Q - mediated caspase - 3 / 7 activation ( * * * * P 0 . 0001 ) . Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q ( n = 12 ( WT ) ; n = 9 ( monomeric red fluorescent protein ( mRFP ) ) ; n = 8 ( mGpx1 ) per condition ) .

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(b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition).


( c ) Effect of the GPx mimetic ebselen on Htt103Q - mediated caspase activation . A single 10 μM dose of ebselen significantly reduced caspase - 3 / 7 activation in Htt103Q - expressing cells ( * * * * P 0 . 0001 ) . Values represent the ratio of caspase - 3 / 7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q . Statistical analysis was performed using an unpaired , two - tailed Mann - Whitney test ( n = 12 per condition ) .

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(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).


( d ) mGpx1 overexpression ( * * * * P 0 . 0001 ) and ebselen treatment ( * * * * P 0 . 0001 ) significantly reduce Htt103Q - mediated ROS production ( n = 9 per condition ) . Unless stated otherwise , all statistical analyses were performed using one - way analysis of variance ( ANOVA ) with post - hoc tests . NS , not significant . * * P 0 . 01 . All data are shown as the mean ± s . e . m .

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

(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.


( a ) Pseudopupil images from wild - type flies , Huntington ' s disease flies and Huntington ' s disease flies overexpressing mGpx1 at day 10 . In wild - type flies , seven rhabdomeres are visible per ommatidium . Scale bar , ∼ 10

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

(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM


( b ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with and without pan - neural mGpx1 overexpression at days 1 ( n = 14 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) , 7 ( n = 7 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) and 10 ( n = 8 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) after eclosion . Significant effects of genotype ( F1 , 50 = 82 . 3 , P = 3 . 82 × 10 − 12 ) and age ( F2 , 50 = 94 . 3 , P = 1 . 06 × 10 − 17 ) were observed , as well as a significant interaction between genotype and age ( F2 , 50 = 7 . 76 , P = 0 . 00116 ) , indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies . Post - hoc Newman - Keuls comparisons showed significant rescue of neurodegeneration in mGpx1 - expressing flies at days 1 ( * P 0 . 05 ) , 7 ( * * * P 0 . 001 ) and 10 ( * * * P 0 . 001 ) . Browne - Forsythe analyses for equality of variance revealed a significant inequality of variance ( P = 0 . 02 ) due to a single group ( Htt93Q + mGpx1 , day 1 ) . The nonparametric Mann - Whitney test confirmed a difference at day 1 between the two groups ( P = 0 . 03 ) , and between Htt93Q + mGpx1 flies at days 1 and 7 ( P 0 . 0001 ) .

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

(b) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).


( c ) Total locomotor activity was assessed as the average number of beam crossings per 30 - min bin per fly . Htt93Qex1 - expressing flies ( n = 29 ) have reduced locomotor activity compared to controls ( * * * P 0 . 001 ; n = 30 ( WT ( w118 ) ) ; n = 32 ( mGpx1 ) ) , which is restored by mGpx1 ( * * P 0 . 01 ; n = 32 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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(c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( d ) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7 . Flies expressing Htt93Qex1 driven by Pdf - GAL4 ( n = 16 brain hemispheres ) showed loss of s - LNVs compared to Canton S ( CS ) control flies ( n = 13 ) , which was ameliorated by coexpression of mGpx1 ( * * * * P 0 . 0001 ; n = 16 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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 O O O O O O O O O

(d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( e ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with ebselen or vehicle ( DMSO ; n = 10 ) treatment at day 7 after eclosion and in newly emerged day 0 adult flies ( untreated control ; n = 10 ) . Significant treatment effects were observed , both with the 100 μM ( * * P 0 . 01 ; n = 9 ) and 300 μM ( * * * P 0 . 001 ; n = 9 ) doses of ebselen . All data are shown as the mean ± s . e . m .

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 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

(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.


PC12 cells were treated with either 10 μM ebselen or 10 mM N - acetylcysteine ( NAc ) for 24 h , with the addition of 400 nM bafilomycin A1 for the final 4 h . Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h . ( a ) Autophagy was determined by measuring the amount of LC3 - II by immunoblot analysis . ( b ) Densitometric analysis of the bands was performed using ImageJ , and the amounts of LC3 - II are expressed relative to the untreated control . All statistical analyses were performed using one - way ANOVA with post - hoc tests ( * * * * P 0 . 0001 ) . All data are shown as the mean ± s . e . m . ; n = 7 per condition .

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

PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.


A , Genome browser images of ChIP - seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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

A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


C , Mean ChIP - seq signal for USP22 , GCN5 , and ATXN7L3 surrounding gene promoters from B . Nonparametric Mann - Whitney analysis of 7 bins ( grey shade ) surrounding maximal peak in Thaps treated conditions are reported .

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C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.


A , HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs . Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours . Scale bar = 400 µm . B , Quantification of apoptotic populations as indicated by Cas3 / 7 fluorescence from A . Three independent experiments are represented as mean ± SEM , with significance calculated by Two - way ANOVA between conditions over time ( f ( 3 ) = 50 . 530 , p < 0 . 001 ) ; significant pairwise comparisons by Tukey ' s HSD post - hoc assessment are indicated by * . C

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

A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C


A , HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22 . Cells were harvested and whole - cell lysate was subjected to immunoblotting with the indicated antibodies .

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

A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.


A , Genome browser images of ChIP - seq for Pol II and Ub - H2B before and after induction of ER stress , with and without USP22 knockdown , at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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

A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


B , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , USP22 - Bound genes . mRNA analyses of select Bound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 C , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , SAGA - Unbound genes . Nonparametric Mann - Whitney analyses of 7 bins ( grey shade ) surrounding TSS ( TSS ) and the midpoint of the profiles ( Body ) in Thaps treated conditions are reported . mRNA analysis of select Unbound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 005 .

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

B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.


C , ChIP - qPCR for total RNA Pol II , pSer5 Pol II , and pSer2 Pol II at ERP70 , GRP78 , and a gene desert ( Prom = gene promoter , Coding = downstream coding region - see Appendix Table S1 for primer sequences ) . Three independent experiments are represented as mean ± SEM . with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.


H , An aliquot of cells from D ‑ G were harvested for whole - cell lysate and subjected to immunoblotting with the indicated antibodies .

O O 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, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.


B , HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22 . Cells were harvested , fractionated , and fractions were subjected to immunoblotting with the indicated antibodies .

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

B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.


C , In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24 . HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22 . Lysates were generated using buffer containing protease cocktail inhibitor , pan - DUB inhibitor PR619 , and the JAMM protease inhibitor o - phenanthroline . Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested ( lanes 1 and 2 ) or digested with USP2 to strip polyubiquitin ( lanes 3 and 4 ) and reduce target proteins to unit length . Digestion reactions were subjected to immunoblotting with the indicated antibodies .

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

C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.


A , Genome browser image of ChIP - seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40 . H3K4me1 and H3K27ac tracks are from the ENCODE Consortium ( GEO accessions GSM945858 and GSM945853 ) . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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

A, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


B - F Live confocal images of ddaC clones expressing mCD8 : : GFP driven by ppk - Gal4 at WP stage or 16 h APF . l ( 3 ) 810 and l ( 3 ) 924 mutant ddaC clones ( C , E ) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF . ( D , F ) Introduction of one genomic construct of msps , HN267 / g - msps , rescued dendrite pruning defects in msps810 and msps924 clones . Red arrowheads point to the ddaC somas .

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

B-F Live confocal images of ddaC clones expressing mCD8::GFP driven by ppk-Gal4 at WP stage or 16 h APF. l(3)810 and l(3)924 mutant ddaC clones (C, E) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF. (D, F) Introduction of one genomic construct of msps, HN267/g-msps, rescued dendrite pruning defects in msps810 and msps924 clones. Red arrowheads point to the ddaC somas.


G Percentages of ddaC clones showing severing defects at 16 h APF .

G Percentages of ddaC clones showing severing defects at 16 h APF.


H Quantitative analysis of unpruned dendrite lengths at 16 h APF .

H Quantitative analysis of unpruned dendrite lengths at 16 h APF.


A Pan - neuronal driver elav - Gal4 was used to co - express GFP - Msps and TACC in postmitotic neurons . elav - Gal4 driven mCD8 : : GFP expression was used as a control . TACC was pulled down by GFP - Msps but not by mCD8 : : GFP . Alpha - tubulin was used as a loading and probing control . Neither mCD8 : : GFP nor GFP - Msps could pull down alpha - tubulin .

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

A Pan-neuronal driver elav-Gal4 was used to co-express GFP-Msps and TACC in postmitotic neurons. elav-Gal4 driven mCD8::GFP expression was used as a control. TACC was pulled down by GFP-Msps but not by mCD8::GFP. Alpha-tubulin was used as a loading and probing control. Neither mCD8::GFP nor GFP-Msps could pull down alpha-tubulin.


B Endogenous Msps proteins were co - immunoprecipitated with Venus - TACC by GFP beads but not by control IgG beads . Alpha - tubulin was used as loading and probing control .

O O B-MEASURED_VAR 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

B Endogenous Msps proteins were co-immunoprecipitated with Venus-TACC by GFP beads but not by control IgG beads. Alpha-tubulin was used as loading and probing control.


C Endogenous Msps or TACC was pulled down by their respective antibodies , and the other protein was simultaneously detected in the immunoprecipitated contents . Alpha - tubulin was used as loading and probing control .

O O B-MEASURED_VAR 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

C Endogenous Msps or TACC was pulled down by their respective antibodies, and the other protein was simultaneously detected in the immunoprecipitated contents. Alpha-tubulin was used as loading and probing control.


D - G Confocal images of ddaC neurons expressing control RNAi ( D ) , msps RNAi ( E ) , tacc RNAi ( F ) and αTub84B RNAi ( G ) that were immunostained for TACC at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at the top right corner .

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D-G Confocal images of ddaC neurons expressing control RNAi (D), msps RNAi (E), tacc RNAi (F) and αTub84B RNAi (G) that were immunostained for TACC at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas .

H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas.


I - L Confocal images of ddaC neurons expressing control RNAi ( I ) , msps RNAi ( J ) , tacc RNAi ( K ) and αTub84B RNAi ( L ) that were immunostained for Msps at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at the top right corner .

I-L Confocal images of ddaC neurons expressing control RNAi (I), msps RNAi (J), tacc RNAi (K) and αTub84B RNAi (L) that were immunostained for Msps at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas .

M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas.


N , O Protein expression of Msps and TACC in brain tissues . ( N ) Larval brains of msps810 / mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α - Msps and α - TACC antibody . Actin blotting was used as loading control . The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae .

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

N, O Protein expression of Msps and TACC in brain tissues. (N) Larval brains of msps810/mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading control. The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae.


O Protein expression of Msps and TACC in brain tissues . ( O ) Adult brains of tacc59 / tacc74 transheterozygotes were immunoblotted and probed with α - Msps and α - TACC antibody . Actin blotting was used as loading and probing control . The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes .

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

O Protein expression of Msps and TACC in brain tissues. (O) Adult brains of tacc59/ tacc74 transheterozygotes were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading and probing control. The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes.


A - C Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . While expression of control RNAi in ddaC neurons ( A ) did not affect the dendrite morphogenesis nor the pruning processes , knockdown of tacc using RNAi # 1 ( B ) or tacc59 / tacc74 transheterozygous mutant ( C ) severely perturbed the dendrite arborization as well as pruning . Red arrowheads point to the ddaC somas .

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

A-C Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. While expression of control RNAi in ddaC neurons (A) did not affect the dendrite morphogenesis nor the pruning processes, knockdown of tacc using RNAi #1 (B) or tacc59/tacc74 transheterozygous mutant (C) severely perturbed the dendrite arborization as well as pruning. Red arrowheads point to the ddaC somas.


D Percentages of ddaC neurons showing severing defects in ( A - C ) at 16 h APF .

D Percentages of ddaC neurons showing severing defects in (A-C) at 16 h APF.


E Quantitative analysis of unpruned dendrite lengths in ( A - C ) at 16 h APF .

E Quantitative analysis of unpruned dendrite lengths in (A-C) at 16 h APF.


F - H Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown of tacc and msps via co - expressing tacc RNAi and msps RNAi ( H ) in the ddaC neurons did not enhance the pruning defects , compared to those neurons co - expressing control RNAi and msps RNAi ( F ) . Red arrowheads point to the ddaC somas .

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

F-H Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown of tacc and msps via co-expressing tacc RNAi and msps RNAi (H) in the ddaC neurons did not enhance the pruning defects, compared to those neurons co-expressing control RNAi and msps RNAi (F). Red arrowheads point to the ddaC somas.


I Percentages of ddaC neurons showing severing defects in ( F - H ) at 16 h APF .

I Percentages of ddaC neurons showing severing defects in (F-H) at 16 h APF.


J Quantitative analysis of unpruned dendrite lengths in ( F - H ) at 16 h APF .

J Quantitative analysis of unpruned dendrite lengths in (F-H) at 16 h APF.


Confocal images of ddaC neurons expressing UAS - mCD8 : : GFP , UAS - Nod - lacZ or UAS - Kin - lacZ and immunostained for β - galactosidase at wL3 stages . ( A - C ) Wild - type ddaC neurons ( A ) displayed normal distribution of Nod - lacZ signals in dendrites . In contrast , in msps810 mutants ( B ) and tacc59 / tacc74 mutants ( C ) , ddaC neurons exhibited altered Nod - lacZ distribution patterns , with highly enriched stainings in the somas and decreased signals in the dendrites .

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

Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites.


Confocal images of ddaC neurons ( D - F ) Kin - lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons ( D ) , whereas part of the Kin - lacZ were mislocalized to dendrites in msps RNAi ( E ) and tacc RNAi ( F ) ddaC neurons . Asterisks indicate the location of ddaC somas . White arrows indicate the location of axons . Curly brackets mark the dendritic regions where fluorescence intensity of Nod - lacZ was measured . White arrowheads point to dendritically localized Kin - lacZ signals .

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

Confocal images of ddaC neurons (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals.


G Percentages of ddaC neurons showing defective Nod - lacZ distribution in mutants or RNAi - expressing neurons .

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G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons.


H Quantitative analysis of normalized Nod - lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma .

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

H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma.


I Percentages of ddaC neurons showing defective Kin - lacZ distribution .

I Percentages of ddaC neurons showing defective Kin-lacZ distribution.


A - F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates the direction towards the somas , and vertical arrow indicates the time . EB1 : : GFP expression was driven under Gal44 - 77 . In the control ddaC neurons ( A , D ) , EB1 comets move predominantly in retrograde direction . EB1 comets were largely diminished in msps810 / mspsP mutant neurons ( B ) . Interestingly , in msps810 / mspsP18 mutants ( C ) , tacc RNAi ( E ) and tacc59 / tacc74 mutant ( F ) , increased anterograde comets were detected in ddaC dendrites .

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

A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites.


G - J Quantitative analysis of the percentages of anterograde EB1 : : GFP comets , the track length , the number ( per 30 µm ) and the velocity of the comets in each neuron imaged .

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

G-J Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.


A - F Confocal images of ddaC neurons expressing UAS - mCD8 : : GFP ( Green channel ) , UAS - Nod - lacZ and immunostained for β - galactosidase at wL3 stages . Simultaneous knockdown of klp10A and msps ( B ) significantly restored Nod - lacZ localization in the dendrites , compared to msps and control RNAi ( A ) . ddaC neurons with colchicine treatment ( D ) or with Kat - 60 overexpression ( F ) exhibited perturbed Nod - lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites . Asterisks indicate the location of ddaC somas . White arrows indicate the location of axons . Curly brackets mark the dendritic regions where fluorescence intensity of Nod - lacZ was measured .

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

A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.


G - L Live confocal images of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown klp10A and msps ( H ) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps , control RNAi neurons . Wild type larvae fed with colchicine ( J ) showed pruning defects in comparison with those feed with DMSO ( I ) . ddaC neurons with Kat - 60 overexpression ( L ) exhibited abnormal dendrite arborization and pruning defects . Red arrowheads point to the ddaC somas .

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

G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.


M , N Quantitative analysis of normalized Nod - lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively .

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

M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively.


O Percentages of ddaC neurons showing severing defects at 16 h APF .

O Percentages of ddaC neurons showing severing defects at 16 h APF.


P Quantitative analysis of unpruned dendrite lengths at 16 h APF .

P Quantitative analysis of unpruned dendrite lengths at 16 h APF.


- F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates the direction towards the somas , and vertical arrow indicates the time . ( B ) RNAi knockdown of klp10A in msps810 / mspsP18 mutants restored the retrograde movement pattern of EB1 comets . MT depolymerization or severing induced by Colchicine treatment ( D ) or overexpression of Kat - 60 ( F ) led to mixed MT orientation in ddaC neurons .

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

-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.


G - J Quantitative analysis of the percentages of anterograde EB1 comets , the track length , number ( per 30 µm ) and the velocity of EB1 comets in each neuron imaged .

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 B-MEASURED_VAR O O O O O O

G-J Quantitative analysis of the percentages of anterograde EB1 comets, the track length, number (per 30 µm) and the velocity of EB1 comets in each neuron imaged.


( A ) 2D STED micrographs of RPE1 cells co - stained with the indicated antibodies . Scale bar : 200 nm .

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) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies. Scale bar: 200 nm.


( B ) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1 / ODF2 fluorescence ratio in the indicated cell lines and genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed for each condition , a . u . = arbitrary units . Unpaired Mann - Whitney test ( * * * * P < 0 . 0001 ) .

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

(B) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/ODF2 fluorescence ratio in the indicated cell lines and genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed for each condition, a.u. = arbitrary units. Unpaired Mann-Whitney test (****P < 0.0001).


( C ) Representative fluorescence micrographs from the indicated cell lines co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(C) Representative fluorescence micrographs from the indicated cell lines co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Co - localization between ODF2 and PIDD1 ( n = 11 ) , FBF1 and PIDD1 ( n = 21 ) , and ANKRD26 and PIDD1 ( n = 17 ) assessed on images The value represents the fraction PIDD1 objects touching either ODF2 , FBF1 or ANKRD26 objects .

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(D) Co-localization between ODF2 and PIDD1 (n = 11), FBF1 and PIDD1 (n = 21), and ANKRD26 and PIDD1 (n = 17) assessed on images The value represents the fraction PIDD1 objects touching either ODF2, FBF1 or ANKRD26 objects.


( E ) 2D STED micrographs of RPE1 cells co - stained with the indicated antibodies , scale bar : 200 nm .

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(E) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies, scale bar: 200 nm.


( F ) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co-stained with indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( G ) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1 / CEP128 fluorescence ratio in RPE1 of the indicated genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed from as many individual cells for each condition , a . u . = arbitrary units . Statistical significance was assessed by Kruskal - Wallis test , comparing each sample to the wild type ( * * * * P < 0 . 0001 ) .

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

(G) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/CEP128 fluorescence ratio in RPE1 of the indicated genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed from as many individual cells for each condition, a.u. = arbitrary units. Statistical significance was assessed by Kruskal-Wallis test, comparing each sample to the wild type (****P < 0.0001).


( B ) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Cells were co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(B) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( C ) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . Kruskal - Wallis test ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( D ) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many individual cells ; a . u . = arbitrary units . Kruskal - Wallis test ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

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

(D) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( E ) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes , either transduced with the indicated lentiviral vectors or left untransduced ( mock ) . Cells were co - stained with the indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

(E) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h . A fraction of DHCB treated cells were released into fresh medium for other 24h ( release ) . Samples were subjected to immunoblotting ; n = 3 independent experiments .

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(A) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h. A fraction of DHCB treated cells were released into fresh medium for other 24h (release). Samples were subjected to immunoblotting; n = 3 independent experiments.