Ubiquitination reactions of cyclin A by APC / CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5 .

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Ubiquitination reactions of cyclin A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.


Ubiquitination reactions of Nek2A by APC / CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5 .

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Ubiquitination reactions of Nek2A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.


Western blot of Hek293 showing siRNA - mediated depletion of UbcH5 , UbcH10 , or both E2 enzymes together . Tubulin serves a loading control .

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Western blot of Hek293 cells showing the siRNA-mediated depletion of UbcH5, UbcH10, or both E2 enzymes together. Tubulin serves a loading control.


Exemplary still from time courses between NEBD anaphase of eGFP - Nek2A degradation in HEK . were either treated with siGL2 as control or depleted of indicated E2 enzymes . chromosomes are coloured in cyan eGFP - Nek2A in green , with outline of are indicated with dashed yellow lines . Time is given as hh : mm . Scale bar 10 μm .

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Exemplary still images from time courses between NEBD and anaphase of eGFP-Nek2A degradation in HEK cells. Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes. The chromosomes are coloured in cyan and eGFP-Nek2A in green, with the outline of the cells are indicated with dashed yellow lines. Time is given as hh:mm. Scale bar 10 μm.


Degradation profiles of eGFP - cyclin A2 ( top ) eGFP - Nek2A ( bottom ) in HEK during mitosis . timepoint of NEBD is marked at 0 minutes in graphs . Asterisks indicate values that are significantly different from same time point of siGL2 control as determined by a Mann - Whitney U - Mean ± s . d . is shown . number of analysed are N = 66 ( Cyclin A2 siGL2 ) , 52 ( Cyclin A2 siUbcH5 ) , 28 ( Cyclin A2 siUbcH10 ) , 27 ( Cyclin A2 siU5 / siU10 ) , 80 ( Nek2A siGL2 ) , 64 ( Nek2A siUbcH5 ) , 21 ( Nek2A siUbcH10 ) , 32 ( Nek2A siU5 / siU10 ) . data are from at least two biological replicates .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O

Degradation profiles of eGFP-cyclin A2 (top) and eGFP-Nek2A (bottom) in HEK cells during mitosis. The timepoint of NEBD is marked at 0 minutes in the graphs. Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann-Whitney U-test Mean ± s.d. is shown. The number of cells analysed are N = 66 (Cyclin A2 siGL2), 52 (Cyclin A2 siUbcH5), 28 (Cyclin A2 siUbcH10), 27 (Cyclin A2 siU5/siU10), 80 (Nek2A siGL2), 64 (Nek2A siUbcH5), 21 (Nek2A siUbcH10), 32 (Nek2A siU5/siU10). All data are from at least two biological replicates.


I , J . Cumulative numbers of WT Tfcp2l1 _ KO 2i - ESCs ( I ) FBS - ESCs ( J ) . Results are from three independent clones of WT Tfcp2l1 _ KO ESCs .

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I, J. Cumulative cell numbers of WT and Tfcp2l1_KO 2i-ESCs (I) and FBS-ESCs (J). Results are from three independent clones of WT and Tfcp2l1_KO ESCs.


A . Real - time fatty acid oxidation ( FAO ) profile of WT Tfcp2l1 _ KO 2i - ESCs using FAO stress kit in Seahorse XF . Oxygen consumption rate ( OCR ) were measured . Error bars indicate ; n = 5 biological repeats . Palm - BSA , palmitate conjugated BSA , a substrate of FAO ; Oligomycin , an inhibitor of ATP synthase ( V ) ; FCCP , Carbonyl cyanide - 4 ( trifluoromethoxy ) phenylhydrazone , a proton gradient uncoupler ; Rotenone Antimycin , a I inhibitor a III inhibitor , respectively .

A. Real-time fatty acid oxidation (FAO) profile of WT and Tfcp2l1_KO 2i-ESCs using the FAO stress test kit in Seahorse XF. Oxygen consumption rate (OCR) were measured. Error bars indicate SEM; n = 5 biological repeats. Palm-BSA, palmitate conjugated BSA, a substrate of FAO; Oligomycin, an inhibitor of ATP synthase (complex V); FCCP, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, a proton gradient uncoupler; Rotenone and Antimycin, a complex I inhibitor and a complex III inhibitor, respectively.


F . Real - time FAO profile using FAO Stress in Seahorse XF . n = 4 biological repeats ; error bars indicate ; * * , p - value < 0 . 01 ; NS , not significant ; t - .

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F. Real-time FAO profile using the FAO Stress Test in Seahorse XF. n = 4 biological repeats; error bars indicate SEM; **, p-value <0.01; NS, not significant; t-test.


G . of dead measured by live - propidium iodide detecting spontaneous in 2i - ESCs . n = 3 biological repeats ; error bars indicate ; p - values were calculated using two - sample t - . H . Cumulative number of WT , Tfcp2l1 _ KO , Tfcp2l1 _ KO with Cpt1a overexpression ( OE ) 2i - ESCs . Error bars indicate ; n = 3 biological repeats ; p - values were calculated using two - sample t - .

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G. Percentage of dead cells measured by live-cell propidium iodide staining detecting spontaneous cell death in 2i-ESCs. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test. H. Cumulative cell number of WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using two-sample t-test.


C . Relative of metabolites in glycolysis TCA cycle . n = 9 biological repeats ; upper whiskers , maximum - third quartile ; lower whiskers , first quartile - minimum ; boxes , third quartile - first quartile ; central indicates median ; p - values were calculated using two - sample t - .

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C. Relative levels of metabolites in glycolysis and the TCA cycle.n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.


D . Live - propidium iodide to measure in WT , Tfcp2l1 _ KO , Tfcp2l1 _ KO with Cpt1a overexpression ( OE ) 2i - ESCs after 5 mM 2 - DG treatment for 24 hours . n = 3 biological repeats ; error bars indicate ; p - value were calculated using two - sample t - .

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D. Live-cell propidium iodide staining to measure cell death in WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs after 5 mM 2-DG treatment for 24 hours. n=3 biological repeats; error bars indicate SEM; p-value were calculated using two-sample t-test.


F . Relative of metabolites in glutamine metabolism . n = 9 biological repeats ; upper whiskers , maximum - third quartile ; lower whiskers , first quartile - minimum ; boxes , third quartile - first quartile ; central indicates median ; p - values were calculated using two - sample t - .

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F. Relative levels of metabolites in glutamine metabolism. n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.


D . 0 . 5 million of 2i - ESCs with indicated genotypes were seeded treated with 200 nM INK - 128 on day0 . numbers were counted on day2 , 4 , 6 . n = 4 biological repeats ; error bars indicate ; p - values were calculated using two - sample t - .

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D. 0.5 million of 2i-ESCs with indicated genotypes were seeded and treated with 200 nM INK-128 on day0. Cell numbers were counted on day2, 4, and 6. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.


E . Immunoblotting of WT Tfcp2l1 _ KO 2i - ESCs untreated or treated with 200 nM INK - 128 .

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E. Immunoblotting of WT and Tfcp2l1_KO 2i-ESCs untreated or treated with 200 nM INK-128.


F . Real - time PCR showing Cpt1a mRNA in WT Tfcp2l1 _ KO 2i - ESCs in presence absence of 200 nM INK - 128 . n = 3 biological repeats ; error bars indicate ; p - values were calculated using two - sample t - .

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F. Real-time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1_KO 2i-ESCs in the presence and absence of 200 nM INK-128. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.


D . Kaplan - Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK - 128 . n , number of blastocysts of each genotype . p - value was calculated using log rank . E . Kaplan - Meier curve of ex vivo WT blastocysts cultured in M2 medium in presence of 200 nM of INK - 128 with or without 50 µM of etomoxir ( Eto ) / or 100 µM of octanoate , as indicated . p - values were calculated using log rank .

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D. Kaplan-Meier curve of ex vivo blastocysts untreated or treated with 200 nM of INK-128. n, number of blastocysts of each genotype. p-value was calculated using the log rank test. E. Kaplan-Meier curve of ex vivo WT blastocysts cultured in the M2 medium in the presence of 200 nM of INK-128 with or without 50 µM of etomoxir (Eto) and/or 100 µM of octanoate, as indicated. p-values were calculated using the log rank test.


( D E ) Growth comparisons of bir1 ∆ - ad2 strains that contain ( 18 strains ) or do not contain ( 28 strains ) confirmed suppressor mutations as measured by of colony growth after serial dilution . mean ( red ) is shown . 10 - fold serial dilutions were made on YPAD plates containing either 0 . 1 % DMSO ( D ) or 10µg / mL of benomyl ( E ) .

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(D and E) Growth comparisons of bir1∆-ad2 strains that contain (18 strains) or do not contain (28 strains) confirmed suppressor mutations as measured by area of colony growth after serial dilution. The mean (red line) is shown. 10-fold serial dilutions were made on YPAD plates containing either 0.1% DMSO (D) or 10µg/mL of benomyl (E).


( A ) Serial dilutions on YPAD media at permissive ( 25 ˚ C ) or restrictive ( 35 ˚ C ) temperatures for ipl1 - 321 mutation . suppressor mutations from Dam1c ( blue ) were only mutants that rescued viability at restrictive .

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(A) Serial dilutions on YPAD media at the permissive (25˚C) or restrictive (35˚C) temperatures for the ipl1-321 mutation. The suppressor mutations from the Dam1c (blue) were the only mutants that rescued viability at the restrictive temperature.


( B ) Top : representative showing Bub3 - mNeonGreen localization to kinetochores ( Nuf2 - mRuby3 ) , indicating spindle assembly checkpoint activation in wild - type yeast . Scale bar is 2µm . Bottom : of proportion of prometaphase with Bub3 - mNeonGreen foci . Only mutations in Dam1 ( blue ) show a significant increase in SAC activity . Data are from at least 5 independent experiments . Means standard deviations are shown .

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(B) Top: representative images showing Bub3-mNeonGreen localization to kinetochores (Nuf2-mRuby3), indicating spindle assembly checkpoint activation in wild-type yeast. Scale bar is 2µm. Bottom: quantification of the proportion of prometaphase cells with Bub3-mNeonGreen foci. Only the mutations in Dam1 complex (blue) show a significant increase in SAC activity. Data are from at least 5 independent experiments. Means and standard deviations are shown.


( B ) Serial dilutions of strains engineered with indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on indicated media are shown . dam1 ( S20D ) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1 .

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(B) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. dam1(S20D) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1.


( B ) Left : representative showing localization of Dad3 - mNeonGreen ( Dam1c member ) Nuf2 - mRuby3 ( kinetochore marker ) in prometaphase . Scale bar is 2µm . Right : of of Dad3 - mNeonGreen at kinetochores / spindles in prometaphase . Each point on graph represents a single measurement of Dad3 - mNeonGreen ; up to 10 measurements per replicate . Data are from 5 independent experiments .

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(B) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in prometaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at kinetochores/spindles in prometaphase cells. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.


( C ) Left : representative showing localization of Dad3 - mNeonGreen ( Dam1c member ) Nuf2 - mRuby3 ( kinetochore marker ) in anaphase . Scale bar is 2µm . Right : of of Dad3 - mNeonGreen at spindle in anaphase . was measured in middle of spindle using a perpendicular - scan . Each point on graph represents a single measurement of Dad3 - mNeonGreen ; up to 10 measurements per replicate . Data are from 5 independent experiments .

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(C) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in anaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at the spindle in anaphase cells. The intensity was measured in the middle of the spindle using a perpendicular line-scan. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.


( D ) of of Dad3 - mNeonGreen at kinetochores in anaphase . Kinetochore position was on Nuf2 - mRuby3 localization . differences in localization at anaphase kinetochores are much weaker than those measured along spindle ( Fig 5C ) . Each point on graph represents a single measurement of Dad3 - mNeonGreen ; up to 10 measurements per replicate . Data are from 5 independent experiments . mean ( red ) is shown .

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(D) Quantification of the intensity of Dad3-mNeonGreen at kinetochores in anaphase cells. Kinetochore position was based on Nuf2-mRuby3 localization. The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle (Fig 5C). Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments. The mean (red line) is shown.


( A ) Serial dilutions of strains engineered with indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on indicated media are shown . met30 - 6 cdc4 - 1 alleles are sensitive . Two independent clones of each of these alleles are shown . serial dilutions were performed at permissive ( 20 ˚ C ) .

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(A) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. The met30-6 and cdc4-1 alleles are temperature sensitive. Two independent clones of each of these alleles are shown. The serial dilutions were performed at the permissive temperature (20˚C).


( B ) amount of mNeonGreen - Sli15 ( CPC member ) located at kinetochores ( Nuf2 - mCherry ) was measured in prometaphase . Sgo1 was depleted by placing it under a galactose - inducible promoter switching to glucose - containing media ( YPAD ) . Representative are on left . Scale bar is 2µm . Averages from 3 independent experiments are shown .

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(B) The amount of mNeonGreen-Sli15 (CPC member) located at kinetochores (Nuf2-mCherry) was measured in prometaphase. Sgo1 was depleted by placing it under a galactose-inducible promoter and switching to glucose-containing media (YPAD). Representative images are on the left. Scale bar is 2µm. Averages from 3 independent experiments are shown.


( C ) Measurements of Aurora B at inner centromere of prometaphase kinetochores . Inner centromeres were identified as regions directly between two centromeres ( ACA ) . from an example WT are shown to right . Scale bar is 5µm long . Each data point is an chromosome . More than 15 for each were measured across two independent experiments . mean ( red ) is shown .

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(C) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores. Inner centromeres were identified as the regions directly between two centromeres (ACA staining). Images from an example WT cell are shown to the right. Scale bar is 5µm long. Each data point is an individual chromosome. More than 15 cells for each cell line were measured across two independent experiments. The mean (red line) is shown.


( B ) Notch signaling effector Hes5 is expressed high in NSCs while Eomes ( Tbr2 ) Btg2 are expressed high in both NSCs BPs at mRNA level . Each dot defines mean lines define Standard Deviation ( SD ) . 3 - 4 biological replicates were collected for each time point .

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(B) Notch signaling effector Hes5 is expressed high in NSCs while Eomes (Tbr2) and Btg2 are expressed high in both NSCs and BPs at the mRNA level. Each dot defines the mean and lines define the Standard Deviation (SD). 3-4 biological replicates were collected for each time point.


( C ) validating known - type specific marker gene expression from sequencing data . ( E ) illustrating novel marker genes identified from sequencing data , as signature genes for NSCs , BPs NBNs . NSCs - Neural , BPs - Basal progenitors , NBNs - Newborn neurons , E - Embryonic day , PN - Post natal , VZ - Ventricular zone , SVZ - Subventricular zone , IZ - Intermediate Zone . Expression values on heatmaps are log2 ( transcripts per million ) .

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(C) Heatmap validating the known cell-type specific marker gene expression from RNA sequencing data. (E) Heatmap illustrating the novel marker genes identified from the RNA sequencing data, as signature genes for NSCs, BPs and NBNs. NSCs- Neural stem cells, BPs-Basal progenitors, NBNs-Newborn neurons, E-Embryonic day, PN-Post natal, VZ-Ventricular zone, SVZ- Subventricular zone, IZ-Intermediate Zone. Expression values on the heatmaps are log2 (transcripts per million).


( D ) Expression profiles of PC1 PC2 along developmental time ( top ) expression profiles of top three genes with highest negative positive projection on PC1 ( first second column ) highest negative positive projection on PC2 ( third fourth column ) for NSCs . Data Information : In D , ( bottom ) , x - axis is embryonic days , y - axis is log2 ( TPM ) .

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(D) Expression profiles of PC1 and PC2 along developmental time (top) and expression profiles of the top three genes with highest negative and positive projection on PC1 (first and second column) and highest negative and positive projection on PC2 (third and fourth column) for NSCs. Data Information: In D, (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( H ) Expression profiles of PC1 PC2 across samples with time from left to right NSCs in green , BPs in red , NBNs in purple ( top ) , gene expression profiles of top three genes with highest negative positive projection on PC1 ( first second column ) highest negative positive projection on PC2 ( third fourth column ) . Data Information : In H , ( bottom ) , x - axis is embryonic days , y - axis is log2 ( TPM ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O

(H) Expression profiles of PC1 and PC2 across all samples with time from left to right and NSCs in green, BPs in red, and NBNs in purple (top), and the gene expression profiles of top three genes with highest negative and positive projection on PC1 (first and second column) and highest negative and positive projection on PC2 (third and fourth column). Data Information: In H, (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( L ) As in panel D but now for BPs . Data Information : In ( bottom ) , x - axis is embryonic days , y - axis is log2 ( TPM ) .

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-TISSUE O O O O O O O O O O O

(L) As in panel D but now for BPs. Data Information: In (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( P ) As in panels D L but now for NBNs . Data Information : In P ( bottom ) , x - axis is embryonic days , y - axis is log2 ( TPM ) .

O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O

(P) As in panels D and L but now for NBNs. Data Information: In P (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( B ) illustrating dynamics of expression of cortical layering markers in NSCs at population level . In B , , heatmaps are on z - score of log2 ( TPM ) expression values .

O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(B) Heatmap illustrating the dynamics of expression of cortical layering markers in NSCs at population level. In B, , heatmaps are based on z-score of log2(TPM) expression values.


( C ) Examples of expression dynamics of deep layer markers Tbr1 , Ctip2 upper layer markers Satb2 , Cux2 in NSCs , BPs NBNs , profiles at population level ( left ) single level ( right ) . Each dot defines mean lines define SD . 3 - 4 biological replicates were collected for each time point .

O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O 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) Examples of expression dynamics of deep layer markers Tbr1, Ctip2 and upper layer markers Satb2, Cux2 in NSCs, BPs and NBNs, profiles at population level (left) and single cell level (right). Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( D ) Experimental validation of NSCs isolated at E13 . 5 using Hes5 : : GFP transgenic , showing no detectable for Tbr1 , Ctip2 Satb2 . NSCs do express Brn2 ( Pou3f2 ) in vitro in vivo at level . Scale bar = 20μm .

O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O

(D) Experimental validation of NSCs isolated at E13.5 using Hes5::GFP transgenic embryos, showing no detectable protein for Tbr1, Ctip2 and Satb2. NSCs do express Brn2 (Pou3f2) in vitro and in vivo at protein level. Scale bar = 20μm.


( E ) illustrating dynamics of expression of cortical layering markers in BPs at population level . ( G ) illustrating dynamics of expression of cortical layering markers in NBNs at population level . heatmaps are on z - score of log2 ( TPM ) expression values .

O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(E) Heatmap illustrating the dynamics of expression of cortical layering markers in BPs at population level. (G) Heatmap illustrating the dynamics of expression of cortical layering markers in NBNs at population level. heatmaps are based on z-score of log2(TPM) expression values.


( F ) Experimental validation of BPs isolated at E16 . 5 using Tbr2 : : GFP transgenic , showing no detectable for Tbr1 , Ctip2 Satb2 . Scale bar = 20μm . ( H ) Experimental validation of NBNs isolated at E16 . 5 using Tbr2 : : GFP transgenic , showing expression for Tbr1 , Ctip2 , Satb2 Brn2 ( Pou3f2 ) . Scale bar = 20μm .

O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O

(F) Experimental validation of BPs isolated at E16.5 using Tbr2::GFP transgenic embryos, showing no detectable protein for Tbr1, Ctip2 and Satb2. Scale bar = 20μm. (H) Experimental validation of NBNs isolated at E16.5 using Tbr2::GFP transgenic embryos, showing protein expression for Tbr1, Ctip2, Satb2 and Brn2(Pou3f2). Scale bar = 20μm .


( D ) Expression profile of selected target or modulator of key signaling pathways : BMP , Wnt , Shh Notch signaling . Each dot defines mean lines define SD . 3 - 4 biological replicates were collected for each time point .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 profile of selected target or modulator of key signaling pathways: BMP, Wnt, Shh and Notch signaling. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( B ) Expression profile of selected maintenance markers Hes1 , Hey1 Id4 . Each dot defines mean lines define SD . 3 - 4 biological replicates were collected for each time point . ( C ) Expression profile of selected neurogenic markers Neurog2 , Neurod2 , Neurod6 . Each dot defines mean lines define SD . 3 - 4 biological replicates were collected for each time point . ( D ) Expression profile of selected gliogenic markers Olig1 , Olig2 Id1 . Each dot defines mean lines define SD . 3 - 4 biological replicates were collected for each time point .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD 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-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD 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-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(B) Expression profile of selected stem cell maintenance markers Hes1, Hey1 and Id4. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point. (C) Expression profile of selected neurogenic markers Neurog2, Neurod2, and Neurod6. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point. (D) Expression profile of selected gliogenic markers Olig1, Olig2 and Id1. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


C , D Growth rates as measured by change in optical density ( OD595 ) over time for wild type ( C ) Torin1 - resistant ( D ) upon 5 µM of Torin1 treatment ( at T = 0 min ) . Respective strains were treated with DMSO vehicle alone to control for potential effects of solvent on growth . Logarithmic values of OD595 increase are plotted against time in minutes . Data aggregated from 3 independent experiments with error bars representing standard deviations ( SD ) . Linear regressions were fitted using curve fitting function on Prism9 . Ratios in rate of mass doubling ( as calculated from gradient of regression lines detailed in Methods ) of Torin1 treated to DMSO control were 0 . 34 in wild type ( C ) 0 . 97 for Torin1 - resistant ( D ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

C, D Growth rates as measured by the change in optical density (OD595) over time for wild type (C) and Torin1-resistant (D) cells upon 5 µM of Torin1 treatment (at T = 0 min). Respective strains were treated with the DMSO vehicle alone to control for potential effects of the solvent on cell growth. Logarithmic values of the OD595 increase are plotted against time in minutes. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD). Linear regressions were fitted using the curve fitting function on Prism9. Ratios in the rate of mass doubling (as calculated from gradient of regression lines detailed in Methods) of Torin1 treated cells to DMSO control were 0.34 in wild type (C) and 0.97 for Torin1-resistant (D) cells.


E Rate of synthesis as measured by amount of HPG incorporation after 20 mins of differing time intervals ( 0 - 60 mins ) of 5 µM Torin1 for wild type ( green ) tor1 ∆ ( yellow ) . Values are normalised to T = 0 minutes . Exponential one - phase decay non - linear regressions were fitted using curve fitting function on Prism9 . Data aggregated from 3 independent experiments with error bars representing standard deviations ( SD ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 Rate of protein synthesis as measured by the amount of HPG incorporation after 20 mins of differing time intervals (0 - 60 mins) of 5 µM Torin1 for wild type (green) and tor1∆ (yellow). Values are normalised to T = 0 minutes. Exponential one-phase decay non-linear regressions were fitted using the curve fitting function on Prism9. Data aggregated from 3 independent experiments with error bars representing the standard deviations (SD).


C - F Phosphorylation changes of conserved phosphosites , S235 S236 , on Rps601 ( C ) Rps602 ( D - F ) , over 85 minutes of Torin1 ( 5 µM ) treatment wild type ( green ) Torin1 - resistant ( brown ) . M - values ( in brackets ) represent different multiplicities of same phosphosites . Lines were fitted with curve fitting function on Prism9 using non - linear regressions exponential one - phase decay ( C , D ) or plateau followed by one - phase decay ( E , F ) functions respectively . Grey dashed lines indicate 2 - fold threshold .

O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phosphorylation changes of the conserved phosphosites, S235 and S236, on Rps601(C) and Rps602 (D - F), over 85 minutes of Torin1 (5 µM) treatment wild type (green) and Torin1-resistant (brown) cells. M-values (in brackets) represent the different multiplicities of the same phosphosites. Lines were fitted with the curve fitting function on Prism9 using the the non-linear regressions exponential one-phase decay (C, D) or plateau followed by one-phase decay (E, F) functions respectively. Grey dashed lines indicate 2-fold threshold.


A Relative expression of 3 S6 kinase homologues , Psk1 , Sck1 Sck2 , plotted against time of Torin1 addition . Respective are represented by different coloured lines as detailed in legend .

O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

A Relative protein expression levels of the 3 S6 kinase homologues, Psk1, Sck1 and Sck2, plotted against time of Torin1 addition. Respective proteins are represented by the different coloured lines as detailed in the legend.


B Relative phosphorylation level of T632 phosphosite identified on Sck1 in 40 - minute time course study . Phosphorylation of both wild type ( green ) tor1 ∆ ( yellow ) are presented relative to value for wild type at T = 0 minutes . Grey dashed indicates 2 - fold threshold .

O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O 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 Relative phosphorylation level of the T632 phosphosite identified on Sck1 in the 40-minute time course study. Phosphorylation levels of both wild type (green) and tor1∆ (yellow) are presented relative to the value for wild type cells at T = 0 minutes. Grey dashed line indicates 2-fold threshold.


A , B Phosphosites identified on ( A ) Tif212 ( B ) Erf1 respectively showing 2 - fold phosphorylation change within 20 minutes of Torin1 ( 5 µM ) addition . Both wild type ( green ) tor1 ∆ ( yellow ) phosphorylation values are relative to starting phosphorylation before Torin1 addition in wild type ( T = 0 minutes ) . Grey dashed lines indicate 2 - fold threshold .

O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

A, B Phosphosites identified on (A) Tif212 and (B) Erf1 respectively showing 2-fold phosphorylation change within 20 minutes of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T=0 minutes). Grey dashed lines indicate 2-fold threshold.


E , F Changes in rates of synthesis of tif471 _ 18A mutant compared to wild type control ( tif471 + ) upon Torin1 ( 5 µM ) treatment . Residual rates of synthesis after 60 minutes of Torin1 treatment was 17 % for tif471 _ 18A phosphomutant 4 . 6 % for tif471 + control relative to starting in respective strains . two graphs represent untransformed ( E ) log2 transformed ( F ) rates respectively . Error bars represent standard deviation ( SD ) of data aggregated from 3 independent experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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, F Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471+) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471+ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log2 transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.


A . Spindle in a fixed HeLa expressing PRC1 - GFP ( green ) mRFP - CENP - B ( magenta ) oriented horizontally with respect to imaging plane , as shown in scheme . of different z - slices ( central plane of spindle z = 0 , two below , z = - 4 µm z = - 2 µm , above , z = + 3 µm z = + 5 µm ) , maximum projection of a z - stack ( max z ) , 3D projections ( 3D ) with 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown . Additional z - slices of this spindle are shown in Fig EV1A . B . Spindle in a fixed HeLa expressing PRC1 - GFP ( green ) mRFP - CENP - B ( magenta ) oriented vertically with respect to imaging plane , as shown in scheme . Legend as in A . Additional z - slices of this spindle are shown in Fig EV1B . C . Correlation between number of PRC1 - labeled bundles number of pairs of sister kinetochores counted throughout horizontal ( black ) vertical ( blue ) spindles of fixed HeLa in metaphase . Data points represent spindles , lines show linear fits . D . Pie charts showing fraction of PRC1 - labeled bundles associated with kinetochore pairs ( blue ) , PRC1 - labled bundles not associated with kinetochores ( green ) kinetochores not associated with PRC1 bundles ( magenta ) in horizontal ( top row ) vertical spindles ( bottom row ) from with up to 70 chromosomes ( left column ) more than 70 chromosomes ( right column ) . Horizontal spindles contained a total of 1786 kinetochore - PRC1 pairs ( n = 29 ) vertical spindles 1133 kinetochore - PRC1 pairs ( n = 16 ) . E , F . Spindle spindle width as a function of number of kinetochore pairs coupled with PRC1 - labeled bundles . G . Number of kinetochore pairs coupled with PRC1 - labeled bundles per unit of cross section of central part of horizontal ( black ) vertical ( blue ) spindles as a function of number of kinetochore pairs coupled with PRC1 - labeled bundles in spindle .

O O B-SUBCELLULAR O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O 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-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O 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-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O

A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z=0, two images below, z=-4 µm and z=-2 µm, and above, z=+3 µm and z=+5 µm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A.B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in A. Additional z-slices of this spindle are shown in Fig EV1B.C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits.D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1786 kinetochore-PRC1 pairs (n=29 cells) and vertical spindles 1133 kinetochore-PRC1 pairs (n=16 cells).E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles.G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle.


A . Central plane of spindle in a live HeLa expressing PRC1 - GFP ( green ) mRFP - CENP - B ( magenta ) oriented vertically with respect to imaging plane . B . Examples of trajectories , with respect to spindle ' s center of mass , of PRC1 - GFP ( green ) corresponding mRFP - CENP - B ( magenta ) signals from spindle in A that moved together for at least 200 s . Dots represent starting points of trajectories , t = 0 s . Trajectories finish at t = 200 s . Grey circle represents center of mass of spindle . C . Pie chart showing fraction of PRC1 - labeled sister kinetochores that moved together for at least 60 s ( blue , n = 226 pairs in 5 ) . A small fraction of PRC1 - labeled ( green , n = 32 in 5 ) kinetochores ( magenta , n = 16 in 5 ) did not move together .

O O O O O B-SUBCELLULAR O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

A. Central plane of the spindle in a live HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane.B. Examples of trajectories, with respect to the spindle's center of the mass, of individual PRC1-GFP (green) and corresponding mRFP-CENP-B (magenta) signals from the spindle in A that moved together for at least 200 s. Dots represent starting points of trajectories, t=0 s. Trajectories finish at t=200 s. Grey circle represents the center of mass of the spindle.C. Pie chart showing the fraction of PRC1-labeled fibers and sister kinetochores that moved together for at least 60 s (blue, n=226 pairs in 5 cells). A small fraction of PRC1-labeled fibers (green, n=32 in 5 cells) and kinetochores (magenta, n=16 in 5 cells) did not move together.


D . Correlation coefficients between trajectories ( with respect to spindle ' s center of mass ) of a kinetochore pair trajectories of PRC1 - labeled fiber coupled to kinetochore pair ( green ) , nearest neighbor PRC1 - labeled fiber ( blue ) , next nearest neighbor ( black ) a randomly chosen PRC1 - labeled fiber ( grey ) ; n , number of trajectories from 3 .

O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

D. Correlation coefficients between trajectories (with respect to the spindle's center of the mass) of a kinetochore pair and the trajectories of the PRC1-labeled fiber coupled to the kinetochore pair (green), the nearest neighbor PRC1-labeled fiber (blue), the next nearest neighbor (black) and a randomly chosen PRC1-labeled fiber (grey); n, the number of trajectories from 3 cells.


E . Examples of scenarios observed in dynamic interplay of PRC1 - labeled bundles ( green ) kinetochores ( magenta ) from spindle in A . Row 1 : PRC1 - labeled bundle kinetochore move together until they separate at 130 s . Row 2 : kinetochore is free of any PRC1 - labeled bundle until it appears at 130 s . Row 3 : kinetochore PRC1 - labeled bundle move together . Row 4 : kinetochore moves with PRC1 - labeled bundle which merges with neighboring bundle at 169 s . Row 5 : two kinetochores move together with a single PRC1 - labeled bundle .

O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O

E. Examples of scenarios observed in dynamic interplay of PRC1-labeled bundles (green) and kinetochores (magenta) from the spindle in A. Row 1: PRC1-labeled bundle and kinetochore move together until they separate at 130 s. Row 2: kinetochore is free of any PRC1-labeled bundle until it appears at 130 s. Row 3: kinetochore and PRC1-labeled bundle move together. Row 4: kinetochore moves with PRC1-labeled bundle which merges with neighboring bundle at 169 s. Row 5: two kinetochores move together with a single PRC1-labeled bundle.


A . Top row : MG132 arrested HeLa stably expressing tubulin - GFP ( green ) immunostained for endogenous PRC1 ( AlexaFluor555 shown in magenta ) in 5 z - . Enlargements of boxed region ( top : merge , middle : GFP , bottom : AlexaFluor555 ) below first image show bridging fiber determined as connecting two ends of sister k - in green channel localization of endogenous PRC1 ( magenta ) . Corresponding maximum projections are shown to right ( max z , left : merge , middle : GFP , right : AlexaFluor555 ) . B . of tubulin - GFP bridging ( green bar ) immunostained for endogenous PRC1 ( magenta bar ) .

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A. Top row: MG132 arrested HeLa cells stably expressing tubulin-GFP (green) and immunostained for endogenous PRC1 (AlexaFluor555 shown in magenta) in 5 individual z-images. Enlargements of the boxed region (top: merge, middle: GFP, bottom: AlexaFluor555) below the first image show the bridging fiber determined as the line connecting two ends of the sister k-fibers in green channel and localization of endogenous PRC1 (magenta). Corresponding maximum projections are shown to the right (max z, left: merge, middle: GFP, right: AlexaFluor555).B. Quantification of tubulin-GFP bridging fibers (green bar) immunostained for endogenous PRC1 (magenta bar).


C . show pole - to - pole tracking ( curve ) of tubulin signal ( top , green ) , which was used to measure endogenous PRC1 signal along same contour ( middle , magenta ) , corresponding scheme ( bottom ) from a HeLa expressing tubulin - GFP immunostained for PRC1 . of same spindle without ( left ) with tracked contour ( right ) are shown . graph shows normalized pole - to - pole profiles ( each profile was scaled so that one pole is at x = 0 other at x = 1 ) of endogenous PRC1 ( grey lines ) acquired in tubulin - GFP HeLa immunostained for PRC1 . Black shows mean value .

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C. Images show pole-to-pole tracking (white curve) of tubulin signal (top, green), which was used to measure the endogenous PRC1 signal along the same contour (middle, magenta), and the corresponding scheme (bottom) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (each intensity profile was scaled so that one pole is at x=0 and the other at x=1) of endogenous PRC1 (grey lines) acquired in tubulin-GFP HeLa cells immunostained for PRC1. Black line shows the mean value.


D . show pole - to - pole tracking ( curve ) of PRC1 - GFP signal ( top , merge ; middle , GFP ; bottom , scheme ) from a HeLa expressing PRC1 - GFP mRFP - CENP - B . of same spindle without ( left ) with tracked contour ( right ) are shown . graph shows normalized pole - to - pole profiles ( legend as in C ) of PRC1 - GFP acquired in HeLa expressing PRC1 - GFP mRFP - CENP - B .

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D. Images show pole-to-pole tracking (white curve) of PRC1-GFP signal (top, merge; middle, GFP; bottom, scheme) from a HeLa cell expressing PRC1-GFP and mRFP-CENP-B. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (legend as in C) of PRC1-GFP acquired in HeLa cells expressing PRC1-GFP and mRFP-CENP-B.


E . Example of measurement of immunostained PRC1 signal ( LPRC1 ) in HeLa expressing tubulin - GFP immunostained for PRC1 .

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E. Example of length measurement of the immunostained PRC1 signal (LPRC1) in HeLa cell expressing tubulin-GFP and immunostained for PRC1.


F . Comparison of LPRC1 between PRC1 - GFP signal ( green , corresponds to PRC1 fused to GFP from a PRC1 - GFP ) PRC1 - AlexaFluor555 ( magenta , corresponds to endogenous immunostained PRC1 ) ( p = 0 . 10 ) .

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F. Comparison of the length LPRC1 between PRC1-GFP signal (green, corresponds to the PRC1 fused to GFP from a PRC1-GFP cell line) and PRC1-AlexaFluor555 (magenta, corresponds to the endogenous immunostained PRC1) (p=0.10).


G . Cross section signal of immunostained endogenous PRC1 ( magenta ) from a HeLa expressing tubulin - GFP immunostained for PRC1 . under peak is defined as Icross , measured at position of blue as in scheme . Horizontal lines mark background signal ( black ) , vertical lines delimit ( grey ) where signal was measured .

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G. Cross section signal intensity of the immunostained endogenous PRC1 (magenta line) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Area under the peak is defined as Icross, measured at the position of the blue line as in scheme. Horizontal lines mark the background signal (black line), vertical lines delimit the area (grey) where the signal was measured.


A . of HeLa stably expressing tubulin - GFP ( green ) transiently mRFP - CENP - B ( magenta ) treated with control siRNA ( left ) siRNA targeting PRC1 ( right ) . Enlargements of boxed region , shown to right of image of whole spindle , are focused on kinetochores bridging fiber ( top : merge ; middle : GFP , bottom : scheme ) .

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A. Images of HeLa cells stably expressing tubulin-GFP (green) and transiently mRFP-CENP-B (magenta) treated with control siRNA (left images) and siRNA targeting PRC1 (right images). Enlargements of the boxed region, shown to the right of the image of the whole spindle, are focused on kinetochores and bridging fiber (top: merge; middle: GFP, bottom: scheme).


B . Western blot showing PRC1 from unlabeled , tubulin - GFP HeLa PRC1 - GFP HeLa . Detailed measurements are in Expanded View 4 .

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B. Western blot showing PRC1 protein from unlabeled cells, tubulin-GFP HeLa cell line and PRC1-GFP HeLa cell line. Detailed measurements are in Expanded View 4.


C . Mean of signal intensities of bridging fiber ( Ib , measured at position of a blue as in scheme ) sum of bridging k - fiber ( Ibk , measured at position of orange as in scheme ) , measured in control ( grey bar ) PRC1 siRNA treated ( black bar ) ( p = 0 . 0003 ) . D . Mean interkinetochore ( dk , see scheme ) measured in control ( grey bar ) PRC1 siRNA treated ( black bar ) ( p = 0 . 0001 ) .

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C. Mean ratio of signal intensities of the bridging fiber (Ib, measured at the position of a blue line as in scheme) and sum of the bridging and k-fiber (Ibk, measured at the position of the orange line as in scheme), measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0003).D. Mean interkinetochore distance (dk, see scheme) measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0001).


E . of spindles of HeLa expressing tubulin - GFP immunostained for PRC1 obtained from control ( top ) PRC1 siRNA treated ( bottom ) . were imaged with same imaging parameters . Only AlexaFluor555 channel is shown .

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E. Images of individual spindles of HeLa cells expressing tubulin-GFP and immunostained for PRC1 obtained from control cells (top) and PRC1 siRNA treated cells (bottom). All cells were imaged with the same imaging parameters. Only AlexaFluor555 channel is shown.


F . Mean signal I of immunostained PRC1 in control ( grey bar ) PRC1 siRNA treated ( black bar ) in HeLa expressing tubulin - GFP immunostained for PRC1 ( p = 0 . 0001 ) .

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F. Mean signal intensity I of the immunostained PRC1 in control cells (grey bar) and PRC1 siRNA treated cells (black bar) in HeLa cells expressing tubulin-GFP and immunostained for PRC1 (p=0.0001).


G . of HeLa stably expressing PRC1 - GFP treated with control ( left image ) siRNA targeting PRC1 ( right image ) immunostained for PRC1 ( magenta ) . Graph showing of immunostained PRC1 signal , LPRC1 ( p = 0 . 5167 ) , signal , I ( p = 0 . 0003 ) Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa expressing PRC1 - GFP immunostained for PRC1 . H . of HeLa stably expressing PRC1 - GFP ( green ) treated with control ( left image ) siRNA targeting PRC1 ( right image ) immunostained for PRC1 . Graph showing of PRC1 - GFP signal , LPRC1 ( p = 0 . 3407 ) , signal , I ( p = 0 . 0001 ) , Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa expressing PRC1 - GFP immunostained for PRC1 .

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G. Images of HeLa cells stably expressing PRC1-GFP treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1 (magenta). Graph showing the length of the immunostained PRC1 signal, LPRC1 (p=0.5167), signal intensity, I (p=0.0003) and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.H. Images of HeLa cells stably expressing PRC1-GFP (green) treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1. Graph showing the length of the PRC1-GFP signal, LPRC1 (p=0.3407), signal intensity, I (p=0.0001), and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.


( B ) qRT - PCR using primers overlapping exons 15 16 permitted to quantify non ‐ skipped α1S form : 18 ± 6 % ( * * P ⩽ 0 . 001 , n = 19 ) 10 ± 7 % ( * * P ⩽ 0 . 001 n = 19 ) ( black bars ) of total α1S mRNA ( grey bars ) at 2 6 months post injection in TA of AAV1 ‐ ( U7 ‐ ESE ) AAV1 ‐ ( U7 ‐ SA ) : ΔDHPR or AAV1 ‐ ( U7 ‐ Ctrl ) : c as a control , respectively .

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(B) qRT-PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non‐skipped α1S form: 18±6% (**P⩽0.001, n=19) and 10±7% (**P⩽0.001 n=19) (black bars) of the total α1S mRNA (grey bars) at 2 and 6 months post injection in TA of AAV1‐(U7‐ESE) and AAV1‐(U7‐SA):ΔDHPR or AAV1‐(U7‐Ctrl): c as a control, respectively.


( C ) Six months post injection , lysates from TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were immuno ‐ blotted for α1S or α - actin for four mice . Graph depicts mean ± s . e . m . of relative expression of α1S subunit determined by densitrometry nomalized to α - actin expression for each muscle . Results were expressed in of α1S subunit in TAΔDHPR normalized to TACtrl for each mice , * * P ⩽ 0 . 001 , n = 4 .

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(C) Six months post injection, lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for α1S or α-actin for four mice. Graph depicts mean±s.e.m. of relative expression of α1S subunit determined by densitrometry and nomalized to the α-actin expression for each muscle. Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice, **P⩽0.001, n=4.


( D ) Longitudinal cryo ‐ sections from TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were stained with anti ‐ α1S subunit ( red ) anti ‐ laminin ( green ) antibodies , nuclei were visualized by Dapi ( blue ) imaged by confocal microscopy . Bars represent 20 μm .

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(D) Longitudinal cryo‐sections from TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red) and anti‐laminin (green) antibodies, nuclei were visualized by Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( A ) Six months post injection , TAs from eight mice were dissected weighed .

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(A) Six months post injection, TAs from eight mice were dissected and weighed.


( B ) internal diameters ( shortest diameter ) from fibres throughout total muscle section were recorded analysed . from five different were examined . bar graph presents mean ± s . e . m . of number of myofibres by fibre diameter class for TAΔDHPR ( black ) TACtrl ( grey ) .

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(B) The internal diameters (shortest diameter) from all fibres throughout the total muscle section were recorded and analysed. Muscles from five different animals were examined. The bar graph presents mean±s.e.m. of the number of myofibres by fibre diameter class for TAΔDHPR (black) and TACtrl(grey).


( C ) Transversal sections of TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were stained with haematoxylin eosin , bars represent 100 μm .

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(C) Transversal sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with haematoxylin and eosin, bars represent 100 μm.


( D ) To quantify fibrosis , transversal sections of total muscle were stained with Red Sirius quantified using Histolab Software ( marked in blue ) , data were normalized with total surface of each muscle ( orange surrounding sections ) , bars represent 500 μm . ( E ) is presented in bar graph showed 4 . 1 ± 0 . 1 fold increase of fibrosis in TAΔDHPR ( ΔDHPR ) compared to TACtrl ( c ) ( * * P0 . 001 , n = 4 ) .

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(D) To quantify fibrosis, transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software (marked in blue), data were normalized with total surface of each muscle (orange line surrounding the sections), bars represent 500 μm. (E) Quantification is presented in bar graph and showed 4.1±0.1 fold increase of fibrosis in TAΔDHPR (ΔDHPR) compared to TACtrl (c) (**P0.001, n=4).


( A ) mRNA from TAΔDHPR TACtrl tissues were extracted nNOS expression was quantified by qRT - PCR . Results are expressed as mean ± s . e . m . , * * P0 . 001 , n = 4 .

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(A) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT-PCR. Results are expressed as mean±s.e.m., **P0.001, n=4.


( B ) Transversal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were stained with anti ‐ nNOS ( red ) , anti ‐ laminin ( green ) antibodies , nuclei with Dapi ( blue ) imaged by confocal microscopy . Bars represent 20 μm .

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(B) Transversal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐nNOS (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( C ) tissues from extracts were analysed by western blot with FoxO3a ‐ P antibody normalized with α ‐ actin antibody . Graph depicts mean ± s . e . m . of relative expression of FoxO3a ‐ P determined by densitometry nomalized to α ‐ actin expression for each muscle , * P ⩽ 0 . 005 , n = 3 .

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(C) The tissues from extracts were analysed by western blot with FoxO3a‐P antibody and normalized with α‐actin antibody. Graph depicts mean±s.e.m. of relative expression of FoxO3a‐P determined by densitometry and nomalized to the α‐actin expression for each muscle, *P⩽0.005, n=3.


( D ) Longitudinal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were stained with anti ‐ FoxO3a ( red ) , anti ‐ laminin ( green ) antibodies , nuclei with Dapi ( blue ) imaged by confocal microscopy . Bars represent 20 μm .

(D) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐FoxO3a (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( E ) mRNA from TAΔDHPR TACtrl tissues were extracted regulation of autophagy genes expression was followed by qRT - PCR . Bnip3 , CathepsinL , LC3 PI3KIII expression ( noted in red ) were significantly increased in TAΔDHPR compared with controlateral TACtrl , * * P0 . 001 , n = 4 .

O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O

(E) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT-PCR. Bnip3, CathepsinL, LC3 and PI3KIII expression (noted in red) were significantly increased in TAΔDHPR compared with the controlateral TACtrl, **P0.001, n=4.


( A ) Ultrathin sections were imaged by electron microscopy , M , mitochondria ; C , collagen fibres ; T , T ‐ tubule . Arrows show double ‐ vesicules called auphagosomes .

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(A) Ultrathin sections were imaged by electron microscopy, M, mitochondria; C, collagen fibres; T, T‐tubule. Arrows show double‐membrane vesicules called auphagosomes.


( B ) Transversal cryo ‐ sections were stained with anti ‐ LC3b ( red ) , anti ‐ dystrophin ( green ) antibodies , nuclei with Dapi ( blue ) imaged by confocal microscopy . Upper panel : TACtrl ( Ctrl ) lower panel : TAΔDHPR ( ΔDHPR ) . Bars represent 20 μm .

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(B) Transversal cryo‐sections were stained with anti‐LC3b (red), anti‐dystrophin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Upper panel: TACtrl (Ctrl) and lower panel: TAΔDHPR (ΔDHPR). Bars represent 20 μm.


( C ) Myofibres isolated from control ( Ctrl ) or 6 months post ‐ injected FDB ( ΔDHPR ) were processed for immuno ‐ labelling for P62 ( green ) LC3b ( red ) imaged by confocal microscopy . Scale bars , 10 μm .

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(C) Myofibres isolated from control (Ctrl) or 6 months post‐injected FDB (ΔDHPR) muscles were processed for immuno‐fluorescent labelling for P62 (green) and LC3b (red) and imaged by confocal microscopy. Scale bars, 10 μm.


( A ) Ultrathin sections of TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were imaged by electron microscopy . Cis , terminal cisternae ; T , tubule ; SR , sarcoplasmic reticulum . Bars : 500 nm .

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(A) Ultrathin sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were imaged by electron microscopy. Cis, terminal cisternae; T, tubule; SR, sarcoplasmic reticulum. Bars: 500 nm.


( B ) Myofibres isolated from Ctrl or ΔDHPR FDB 6 months post injection were processed for immuno ‐ labelling for RyR1 ( green ) , SERCA ( red ) , Dapi ( blue ) imaged by confocal microscopy . Scale bars , 10 μm .

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(B) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for RyR1 (green), SERCA (red), Dapi (blue) and imaged by confocal microscopy. Scale bars, 10 μm.


( A ) Lysates from TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were immuno ‐ blotted for RyR1 or α ‐ actin . ( n = 4 ) . Graph depicts mean ± s . e . m . of relative expression of RyR1 determined by densitometry nomalized to α ‐ actin expression for each muscle . Results were expressed in of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice , * P ⩽ 0 . 005 , n = 4 .

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(A) Lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for RyR1 or α‐actin. (n=4). Graph depicts mean±s.e.m. of relative expression of RyR1 determined by densitometry and nomalized to the α‐actin expression for each muscle. Results were expressed in protein levels of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice, *P⩽0.005, n=4.


( B ) Longitudinal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) TACtrl ( c ) were stained with anti ‐ α1S subunit ( red ) , anti ‐ RyR1 ( green ) antibodies imaged by confocal microscopy . Bars = 20 μm .

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(B) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red), anti‐RyR1 (green) antibodies and imaged by confocal microscopy. Bars=20 μm.


Localization of α 1S subunit . Whole skeletal muscle fibres enzymatically isolated from Ctrl ( A - D ) or ΔDHPR ( E - H ) FDB 6 months post injection were processed for immuno ‐ labelling for α1 S subunit ( red ) laminin ( green ) . Scale bars , 10 μm ; Arrows indicate α1 S subunit expression on sarcolemma ; antibody labelling was visualized by serial confocal microscopy represented as movies of optical sections ( Supplementary Movies S5A S5B ) . ( D , H ) Present projections of confocal Z ‐ series ( step between each frame is 1 μm ) along XZ YZ planes as indicated .

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Localization of α 1S subunit. Whole skeletal muscle fibres enzymatically isolated from Ctrl (A-D) or ΔDHPR (E-H) FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for α1 S subunit (red) and laminin (green). Scale bars, 10 μm; Arrows indicate α1 S subunit expression on sarcolemma; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections (Supplementary Movies S5A and S5B). (D, H) Present projections of confocal Z‐series (step between each frame is 1 μm) along XZ and YZ planes as indicated.


A Atf7ip KO show increased expression of SETDB1 - regulated ERVs provirus reporter , MSCV - GFP as evidenced by RT - qPCR . expression was normalized to Hprt expression is shown relative to level in WT . Data are mean ± SD ; n = 3 , technical replicates .

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A Atf7ip KO cells show increased expression of SETDB1-regulated ERVs and the provirus reporter, MSCV-GFP as evidenced by RT-qPCR analysis. RNA expression was normalized to Hprt expression and is shown relative to the level in WT cells. Data are mean ± SD; n=3, technical replicates.


B Atf7ip KO mESCs ( TT # 2 - 12 ) show decreased H3K9me3 at LTR of SETDB1 - regulated reporter ERVs , as evidenced by Native ChIP followed by qPCR . Gapdh was used as a negative control . Data are mean ± ; n = 5 , independent experiments . * P < 0 . 05 * * P < 0 . 01 by paired t - .

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B Atf7ip KO mESCs (TT#2-12) show decreased H3K9me3 at the LTR of the SETDB1-regulated reporter and the ERVs, as evidenced by Native ChIP followed by qPCR analysis. Gapdh was used as a negative control. Data are mean ± SEM; n=5, independent experiments. *P < 0.05 and **P < 0.01 by paired t-test.


C SETDB1 amount is mostly maintained in Atf7ip KO mESCs . Endogenous SETDB1 - ATF7IP interaction is validated by anti - ATF7IP antibody co - IP experiment with anti - ATF7IP antibody .

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C SETDB1 cellular amount is mostly maintained in Atf7ip KO mESCs. Endogenous SETDB1-ATF7IP interaction is validated by anti-ATF7IP antibody co-IP experiment with anti-ATF7IP antibody.


A IF shows preferential localization of SETDB1 in Atf7ip KO mESCs . Representative projected are shown , quantitative analyses are shown in B - D . Scale bar : 10 µm . B SETDB1 signal in nucleus that was determined by DAPI was calculated . mean from three independent experiments is shown as a bar - graph with jittered points indicating average % of each experiment . Over 100 were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - . C , D SETDB1 foci in nucleus that was determined by DAPI were calculated . Violin plot for SETDB1 ' s foci numbers from analysed in three independent experiments is shown in c . mean from three independent experiments is shown as a bar with jittered points indicating average number of each experiment in D . Over 100 were analysed per sample per experiment . * P < 0 . 05 by unpaired Student ' s t - .

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A IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown, and the quantitative analyses are shown in B-D. Scale bar: 10 µm. B SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test. C, D SETDB1 foci in the nucleus that was determined by DAPI staining were calculated. Violin plot for SETDB1's foci numbers from all the cells analysed in three independent experiments is shown in c. The mean from the three independent experiments is shown as a bar with jittered points indicating the average number of each experiment in D. Over 100 cells were analysed per sample per experiment. *P < 0.05 by unpaired Student's t-test.


E , IF shows preferential localization of SETDB1 in Atf7ip KO mESCs . Representative projected are shown . Arrows indicate region used for - plot . were treated with 10 ng / mL LMB or mock for 5 h before . Scale bar : 10 µm . F - plot for E . Relative intensities of SETDB1 signal were measured by Image J . G quantitative for E is shown . SETDB1 signal in nucleus that was determined by DAPI was calculated . mean from three independent experiments is shown as a bar - graph with jittered points indicating average % of each experiment . Over 100 were analysed per sample per experiment . NS : P > 0 . 05 , * * P < 0 . 01 versus " Mock , WT " group by Dunnett ' s .

O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, IF analysis shows preferential cytoplasmic localization of SETDB1 in Atf7ip KO mESCs. Representative projected images are shown. Arrows indicate the region used for line-plot analysis. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis. Scale bar: 10 µm. F The line-plot analysis for E. Relative intensities of SETDB1 signal were measured by Image J. G The quantitative analysis for E is shown. SETDB1 signal in the nucleus that was determined by DAPI staining was calculated. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 100 cells were analysed per sample per experiment. NS: P > 0.05, **P < 0.01 versus "Mock, WT" group by Dunnett's test.


A ATF7IP KO HEK293T shows endogenous SETDB1 localization phenotype . Endogenous SETDB1 in HEK293T shows cytoplasm + localization profile . Scale bar : 10 µm .

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A ATF7IP KO HEK293T cells shows endogenous SETDB1 cytoplasmic localization phenotype. Endogenous SETDB1 in HEK293T cells shows cytoplasm+nuclear localization profile. Scale bar: 10 µm.


B Over - production of exogenous mouse SETDB1 induces endogenous SETDB1 localization in both WT ATF7IP KO HEK293T . representative data of two independent experiments is shown . Scale bar : 10 µm .

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B Over-production of exogenous mouse SETDB1 induces endogenous SETDB1 nuclear localization in both WT and ATF7IP KO HEK293T cells. The representative data of two independent experiments is shown. Scale bar: 10 µm.


C LMB treatment enhances SETDB1 accumulation in WT HEK293T , but no significant impact for ATF7IP KO HEK293T . were treated with 10ng / ml of LMB for 5 h then analysed with immunofluorescent . representative data of three independent experiments is shown . Scale bar : 10 µm . D SETDB1 signal in nucleus shown in C was calculated . mean from three independent experiments is shown as a bar - graph ± ; n = 3 . Over 50 were analysed per sample per experiment . * * P < 0 . 01 by Student ' s t - ( two - sided ) .

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C LMB treatment enhances SETDB1 nuclear accumulation in WT HEK293T cells, but no significant impact for ATF7IP KO HEK293T cells. Cells were treated with 10ng/ml of LMB for 5 h and then analysed with immunofluorescent staining. The representative data of three independent experiments is shown. Scale bar: 10 µm. D SETDB1 signal in the nucleus shown in C was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. **P < 0.01 by Student's t-test (two-sided test).


E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative accumulation in ATF7IP KO HEK293T . representative data of three independent experiments is shown . Scale bar : 10 µm . F SETDB1 signal in nucleus shown in E was calculated . mean from three independent experiments is shown as a bar - graph ± ; n = 3 . Over 50 were analysed per sample per experiment . Statistics comparison was only shown between without with drug treatment in WT ATF7IP KO . * P < 0 . 05 * * P < 0 . 01 by Dunnett ' s method .

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E 5 h of proteasome inhibitor bortezomib treatment do not have much impact on SETDB1 relative nuclear accumulation in ATF7IP KO HEK293T cells. The representative data of three independent experiments is shown. Scale bar: 10 µm. F SETDB1 signal in the nucleus shown in E was calculated. The mean from three independent experiments is shown as a bar-graph ± SEM; n=3. Over 50 cells were analysed per sample per experiment. Statistics comparison was only shown between without and with drug treatment in WT and ATF7IP KO cells. *P < 0.05 and **P < 0.01 by Dunnett's method.


A HEK293T were transfected with V5 - SETDB1 either empty or 3xF - ATF7IP expressing vector . Over 50 transfected analysed per sample were analyzed . representative data of two independent experiments is shown . Scale bar : 10 µm .

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A HEK293T cells were transfected with V5-SETDB1 and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells analysed per sample were analyzed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


B HEK293T were transfected with V5 - SETDB1 d1 mutant either empty or 3xF - ATF7IP expressing vector . Over 50 transfected per sample were analysed . representative data of two independent experiments was shown . Scale bar : 10 µm . yellow arrowheads indicate that show ( ) - localization of introduced exogenous mSETDB1 .

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B HEK293T cells were transfected with V5-SETDB1 d1 mutant and either empty or 3xF-ATF7IP expressing vector. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments was shown. Scale bar: 10 µm. The yellow arrowheads indicate the cells that show nuclear (and cytoplasmic)-localization of introduced exogenous mSETDB1.


A Co - transfection with ATF7IP augments K885 ubiquitination of SETDB1 . HEK293T were transfected After transfection , V5 - SETDB1 was immune - purified with anti - V5 antibody used for WB .

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A Co-transfection with ATF7IP augments the K885 ubiquitination of SETDB1. HEK293T cells were transfected After the transfection, V5-SETDB1 was immune-purified with anti-V5 antibody and used for WB analysis.


B ATF7IP regulates ubiquitination of SETDB1 in mESCs . Endogenous SETDB1 was immune - purified from WT or Atf7ip KO mESCs used for WB .

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B ATF7IP regulates the ubiquitination of SETDB1 in mESCs. Endogenous SETDB1 was immune-purified from WT or Atf7ip KO mESCs cells and used for WB analysis.


C LMB treatment increased relative of upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs . were treated with 10 ng / mL LMB or mock for 5 h before .

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C LMB treatment increased the relative intensity of the upper ubiquitinated upper band of SETDB1 in Atf7ip KO mESCs. Cells were treated with 10 ng/mL LMB or mock for 5 h before analysis.


D HEK293T were transfected with either 3xF - or 3xF - NLS - SETDB1 . Over 50 transfected per sample were analysed . representative data of two independent experiments is shown . Scale bar : 10 µm .

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D HEK293T cells were transfected with either 3xF- or 3xF-NLS-SETDB1. Over 50 transfected cells per sample were analysed. The representative data of two independent experiments is shown. Scale bar: 10 µm.


E localization itself promotes ubiquitination of SETDB1 . HEK293T were transfected with 3xFLAG - or 3xFLAG - NLS - SETDB1 . After FLAG - IP , purified SETDB1s were analyzed by WB .

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E Nuclear localization itself promotes the ubiquitination of SETDB1. HEK293T cells were transfected with 3xFLAG- or 3xFLAG-NLS-SETDB1. After FLAG-IP, the purified SETDB1s were analyzed by WB.


F , G IF was performed with Atf7ip KO mESCs stably expressing 3xF - or 3xF - NLS - SETDB1 . Representative are shown in F , quantitative is shown in G . Scale bar : 10 µm . Addition of NLS increases localization of SETDB1 in Atf7ip KO mESCs . mean from three independent experiments is shown as a bar - graph with jittered points indicating average % of each experiment . Over 50 were analysed per sample per experiment . * * * P < 0 . 001 by unpaired Student ' s t - .

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F, G IF analysis was performed with Atf7ip KO mESCs stably expressing 3xF- or 3xF-NLS-SETDB1. Representative images are shown in F, and the quantitative analysis is shown in G. Scale bar: 10 µm. Addition of NLS increases nuclear localization of SETDB1 in Atf7ip KO mESCs. The mean from three independent experiments is shown as a bar-graph with jittered points indicating the average % intensity of each experiment. Over 50 cells were analysed per sample per experiment. ***P < 0.001 by unpaired Student's t-test.


H localization promotes ubiquitination of SETDB1 in Atf7ip KO mESCs . Atf7ip KO mESCs expressing 3xF - or 3xF - NLS - SETDB were used for FLAG - IP , purified SETDB1s were analyzed by WB .

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H Nuclear localization promotes the ubiquitination of SETDB1 in Atf7ip KO mESCs. Atf7ip KO mESCs expressing 3xF- or 3xF-NLS-SETDB were used for FLAG-IP, and the purified SETDB1s were analyzed by WB.


I , J . Normal ciliary formation of Rabl2 - / - MEFs . MEFs derived from E12 . 5 mouse were serum - starved for 48 h to induce cilia ( I ) . Statistical results ( J ) were from three independent experiments . Ciliogenesis of at least 200 was scored in each experiment condition . Ciliary lengths of 80 were measured in each experiment condition results of same genotypes were pooled together . K , L . Rabl2 deficiency did not repress ciliary localization of IFT - B . Ciliated MEFs were immunostained for Ift81 Ift57 , two IFT - B subunits ( K ; see Fig 4F for diagram of IFT - B ) . Relative of ciliary Ift81 ( L ) , presented in arbitrary unit , was obtained by normalizing to that of corresponding Ac - tub . Four MEF strains prepared respectively from two per genotype were used for . 50 cilia were quantified from each MEF results of same genotypes were pooled together .

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I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together. K, L. Rabl2 deficiency did not repress ciliary localization of IFT-B. Ciliated MEFs were immunostained for Ift81 and Ift57, two IFT-B subunits (K; see Fig 4F for diagram of IFT-B). Relative intensity of ciliary Ift81 (L), presented in arbitrary unit, was obtained by normalizing to that of corresponding Ac-tub. Four MEF strains prepared respectively from two embryos per genotype were used for quantification. 50 cilia were quantified from each MEF strain and results of the same genotypes were pooled together.


I , J . Normal ciliary formation of Rabl2 - / - MEFs . MEFs derived from E12 . 5 mouse were serum - starved for 48 h to induce cilia ( I ) . Statistical results ( J ) were from three independent experiments . Ciliogenesis of at least 200 was scored in each experiment condition . Ciliary lengths of 80 were measured in each experiment condition results of same genotypes were pooled together .

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I, J. Normal ciliary formation of Rabl2-/- MEFs. MEFs derived from E12.5 mouse embryos were serum-starved for 48 h to induce primary cilia (I). Statistical results (J) were from three independent experiments. Ciliogenesis of at least 200 cells was scored in each experiment and condition. Ciliary lengths of 80 cells were measured in each experiment and condition and the results of the same genotypes were pooled together.


D , E . Increased Gli3 FL / R ratios in E11 . 5 hind limb buds E10 . 5 mouse . Relative band intensities of full - ( FL ) repressor ( R ) forms of Gli3 were quantified ( red ) . FL / R ratios were from four different sets of limb bud preparations ( D ) three different sets of preparations ( E ) .

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D, E. Increased Gli3 FL/R ratios in E11.5 hind limb buds and E10.5 mouse embryos. Relative band intensities of the full-length (FL) and the repressor (R) forms of Gli3 were quantified (red). FL/R ratios were from four different sets of limb bud preparations (D) and three different sets of embryo preparations (E).


Rabl2Q80L inhibited Hh signaling . MEFs from Rabl2 - / - E12 . 5 infected with lentivirus to express GFP , GFP - Rabl2 or GFP - Rabl2Q80L were serum - starved for 24 h treated with DMSO ( mock ) or 100 nM SAG for an additional 24 h . Uninfected MEFs from wildtype littermate served as positive control . samples were immunostained to examine ciliary Gli2 arrowheads ( G ) point to ciliary tips . results were from three independent experiments , each using MEFs from different Rabl2 - deficient . Gli2 intensities , presented in arbitrary unit , were measured from at least 22 cilia in each experiment condition pooled together .

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Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunostained to examine ciliary Gli2 The white arrowheads (G) point to ciliary tips. Quantification results were from three independent experiments, each using MEFs from different Rabl2-deficient embryos. Gli2 intensities, presented in arbitrary unit, were measured from at least 22 cilia in each experiment and condition and pooled together.


H . Rabl2Q80L inhibited Hh signaling . MEFs from Rabl2 - / - E12 . 5 infected with lentivirus to express GFP , GFP - Rabl2 or GFP - Rabl2Q80L were serum - starved for 24 h treated with DMSO ( mock ) or 100 nM SAG for an additional 24 h . Uninfected MEFs from wildtype littermate served as positive control . samples were immunoblotted to examine of Gli1 Gli3 In samples for immunoblotting ( H ) , GFP - positive occupied 82 . 3 % ( GFP ) , 87 . 3 % ( GFP - Rabl2 ) , 81 . 2 % ( GFP - Rabl2Q80L ) , respectively , when 260 in each sample were scored . Relative band intensities are shown in red .

O O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

H. Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting (H), GFP-positive cells occupied 82.3% (GFP), 87.3% (GFP-Rabl2), and 81.2% (GFP-Rabl2Q80L), respectively, when 260 cells in each sample were scored. Relative band intensities are shown in red.