( B ) Depletion of Beclin ‐ 1 leads to defects in K ‐ MT attachment . siRNA ‐ treated were cooled on ice before fixation to reveal stable k ‐ fibres then stained with anti ‐ α ‐ tubulin ( red ) anti ‐ CREST ( green ) antibodies . Numbers point to magnified areas indicate mode of attachment of k ‐ fibres to kinetochores ( 1 , bi ‐ oriented ; 2 , unattached ; 3 , mono ‐ oriented kinetochores ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; NS , not significant ; siRNA , small interfering .

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(B) Depletion of Beclin‐1 leads to defects in K‐MT attachment. siRNA‐treated cells were cooled on ice before fixation to reveal stable k‐fibres and then stained with anti‐α‐tubulin (red) and anti‐CREST (green) antibodies. Numbers point to magnified areas and indicate the mode of attachment of k‐fibres to kinetochores (1, bi‐oriented; 2, unattached; 3, mono‐oriented kinetochores). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; NS, not significant; siRNA, small interfering RNA.


( A ) CENP ‐ E CREST . Two asterisks indicate significant results , P = 0 . 0053 .

(A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053.


( B ) CENP ‐ F CREST . Three asterisks indicate significant results , P = 0 . 0006 .

(B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006.


( C ) ZW10 CREST . Three asterisks indicate significant results , P = 0 . 0007 .

(C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007.


( D ) MAD2 CREST . Two asterisks indicate significant results , P = 0 . 0032 .

(D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032.


( F ) KNL ‐ 1 CREST . On right , magnified areas of of of interest normalized to of CREST are shown . Graphs represent three independent experiments ( error bars = s . d . ) . Scale bar , 10 μm .

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(F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm.


( G ) Western blot of whole ‐ lysates from control Beclin ‐ 1 siRNA ‐ treated . siRNA , small interfering .

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(G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.


( A ) Immunoprecipitation of endogenous Zwint ‐ 1 in HeLa extracts expressing HA ‐ Beclin ‐ 1 or HA ‐ GFP . Input is 3 . 5 % of immunoprecipitated material .

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(A) Immunoprecipitation of endogenous Zwint‐1 in HeLa cell extracts expressing HA‐Beclin‐1 or HA‐GFP. Input is 3.5% of the immunoprecipitated material.


( B ) GST ‐ Zwint ‐ 1 or GST ‐ beads were used to pull down recombinant purified His6 ‐ Beclin ‐ 1 . ( Left ) Coomassie ‐ blue ‐ stained gel of purified recombinant ( 1 μg ) . ( Right ) A western blot of GST His6 ‐ Beclin ‐ 1 in input bound fractions . His6 ‐ Beclin ‐ 1 in input represents 4 % of amount used for pull ‐ down .

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(B) GST‐Zwint‐1 or GST‐beads were used to pull down recombinant purified His6‐Beclin‐1. (Left) Coomassie‐blue‐stained gel of the purified recombinant proteins (1 μg). (Right) A western blot of GST proteins and His6‐Beclin‐1 in the input and bound fractions. His6‐Beclin‐1 in the input represents 4% of the amount used for the pull‐down.


( C ) Zwint ‐ 1 do not change in Beclin ‐ 1 ‐ depleted . were treated with nocodazole before fixation . On right , magnified kinetochore pairs stained for Zwint ‐ 1 ( red ) CREST ( green ) , of Zwint ‐ 1 normalized to CREST signal from three independent experiments ( error bars = s . d . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Zwint‐1 levels do not change in Beclin‐1‐depleted cells. Cells were treated with nocodazole before fixation. On the right, magnified individual kinetochore pairs stained for Zwint‐1 (red) and CREST (green), and quantification of Zwint‐1 levels normalized to CREST signal from three independent experiments (error bars=s.d.).


( D ) Beclin ‐ 1 is associated with both centrosome mitotic spindle during mitosis . HeLa were permeabilized before fixation immunostained for Beclin ‐ 1 ( green ) , α ‐ tubulin ( red ) ( DAPI , blue ) .

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(D) Beclin‐1 is associated with both the centrosome and the mitotic spindle during mitosis. HeLa cells were permeabilized before fixation and immunostained for Beclin‐1 (green), α‐tubulin (red) and DNA (DAPI, blue).


( E ) Beclin ‐ 1 is retained on k ‐ fibres . HeLa were cooled on ice before permeabilization fixation , stained with anti ‐ α ‐ tubulin ( red ) anti ‐ Beclin ‐ 1 ( green ) antibodies .

(E) Beclin‐1 is retained on the k‐fibres. HeLa cells were cooled on ice before permeabilization and fixation, and stained with anti‐α‐tubulin (red) and anti‐Beclin‐1 (green) antibodies.


( F ) Beclin ‐ 1 forms discrete foci in vicinity of outer kinetochore in a microtubule ‐ dependent manner . Untreated ( left ) nocodazole ‐ treated ( right ) were pre ‐ extracted fixed before labelling with Beclin ‐ 1 ( green ) Zwint ‐ 1 ( red ) antibodies .

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(F) Beclin‐1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule‐dependent manner. Untreated cells (left) and nocodazole‐treated cells (right) were pre‐extracted and fixed before labelling with Beclin‐1 (green) and Zwint‐1 (red) antibodies.


( G ) ‐ tagged Cherry ‐ Beclin ‐ 1 forms foci in vicinity of outer kinetochore . HeLa transfected with indicated constructs were pre ‐ extracted fixed before labelling with Zwint ‐ 1 ( green ) antibody . Magnified areas are shown on right .

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(G) The fluorescent‐tagged Cherry‐Beclin‐1 forms foci in the vicinity of the outer kinetochore. HeLa cells transfected with indicated constructs were pre‐extracted and fixed before labelling with Zwint‐1 (green) antibody. Magnified areas are shown on the right.


( H ) Beclin ‐ 1 localization close to kinetochore does not depend on ZW10 . treated with Control , Beclin ‐ 1 ZW10 siRNA were pre ‐ extracted fixed before labelling with Beclin ‐ 1 ( green ) , CREST ( blue ) ZW10 ( red ) antibodies . was visualized with DAPI . ( C - H ) Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; , deoxyribonucleic acid ; GFP , green ; GST , glutathione S ‐ transferase ; HA , haemagglutinin .

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(H) Beclin‐1 localization close to the kinetochore does not depend on the ZW10 protein. Cells treated with Control, Beclin‐1 and ZW10 siRNA were pre‐extracted and fixed before labelling with Beclin‐1 (green), CREST (blue) and ZW10 (red) antibodies. DNA was visualized with DAPI. (C-H) Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin.


Mabs - laden wet - spun yarns showing progressive myogenic differentiation over culturing time . As can be noted , Mabs evolves from a round shape ( single at early time points ) into a more elongated one ( generally within first 3 - 5 days of culture ) , eventually forming long - range myotubes . For sake of clarity , samples used for bright field investigation were prepared out of few layers of to favor recognition of organization .

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Mabs-laden wet-spun yarns showing progressive myogenic differentiation over culturing time. As can be noted, Mabs morphology evolves from a round shape (single cells at early time points) into a more elongated one (generally within the first 3-5 days of culture), eventually forming long-range myotubes. For the sake of clarity, samples used for bright field investigation were prepared out of few layers of fibers to favor the recognition of cell organization.


A ) Immunocompromised mouse leg showing ablated TA ( control ) implanted TA ( graft ) at day 0 ; inset showing muscle mass removed next to implanted Mabs loaded constructs shaped to fill TA lodge . B ) Implanted myo - substitute 20 days after grafting .

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A) Immunocompromised mouse leg showing ablated TA (control) and implanted TA (graft) at day 0; inset showing muscle mass removed next to the implanted Mabs loaded constructs shaped to fill the TA lodge. B) Implanted myo-substitute 20 days after grafting.


D ) Graphical representation of scored TA ( n = 6 ) . dashed indicates range of native TA mass depending primarily on mouse , statistical significance was analyzed by Anova ( P < 0 . 05 was considered significant : * = 0 , 0306 ) .

D) Graphical representation of the scored TA weight (n=6). The dashed area indicates the range of native TA mass depending primarily on mouse weight and age, statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *=0,0306).


Cross longitudinal sections of ablated ( control ) implanted ( graft ) TA were marked with LacZ immunoassayed for MHC ( red ) , LAM ( green ) . Cross - longitudinal sections were immunostained against MHC LAM overlaid to LacZ labeled . Dashed indicates enlarged view displaying sarcomeres ( asterisks ) in combination with LacZ positive nuclei ( arrowheads ) .

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Cross and longitudinal sections of ablated (control) and implanted (graft) TA were marked with LacZ and immunoassayed for MHC (red), LAM (green). Cross- and longitudinal sections were immunostained against MHC and LAM and overlaid to LacZ labeled images. Dashed area indicates the enlarged view displaying sarcomeres (asterisks) in combination with LacZ positive nuclei (arrowheads).


D , E ) Vessel density assessment by means of , statistical significance was analyzed by Anova ( P < 0 . 05 was considered significant : * = 0 , 0019 ) .

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D, E) Vessel density assessment by means of fluorescence, statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *=0,0019).


A ) Immunofluorescence against NeuF ( green ) MHC ( red ) revealing reconstructed muscle innervation ( asterisks ) . B ) Superimposed image in ( a ) over LacZ stained image .

A) Immunofluorescence against NeuF (green) and MHC (red) revealing reconstructed muscle innervation (asterisks). B) Superimposed image in (a) over LacZ stained image.


C ) Syn BTX immunolabelled sections showing neuromuscular junction formation ( arrowheads ) , inset enlarged view of dashed box .

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C) Syn and BTX immunolabelled sections showing neuromuscular junction formation (arrowheads), inset enlarged view of dashed box.


Wet - spun yarn containing human myogenic precursors showed progressive differentiation over time ( arrows in day 3 ) ; dashed box enlargement reveals still undifferentiated after 15 days of culture ( Upper panel ) . Middle lower panel showing MHC immunostaining ( red ) performed on microfiber yarns bulk structure loaded with human myoblasts upon 15 days of culture , revealing a significant difference in terms of myotube organization between two hydrogel systems , with wet - spun constructs greatly outperforming bulk gels . Such difference is quantified in polar plots . Nuclei were counterstained by DAPI ( Blue ) .

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Wet-spun yarn containing human myogenic precursors showed progressive cell differentiation over time (arrows in day 3); dashed box enlargement reveals still undifferentiated cells after 15 days of culture (Upper panel). Middle and lower panel showing MHC immunostaining (red) performed on microfiber yarns and bulk structure loaded with human myoblasts upon 15 days of culture, revealing a significant difference in terms of myotube organization between the two hydrogel systems, with the wet-spun constructs greatly outperforming the bulk gels. Such difference is quantified in the polar plots. Nuclei were counterstained by DAPI (Blue).


( A ) Transcription at GAL3 is visualized by addition of PP7 loops at 5 ' of GAL3 in budding yeast . Example trace of quantified of transcription site . Traces are binarized to determine on ( active ) off ( inactive ) times .

(A) Transcription at GAL3 is visualized by addition of PP7 loops at the 5' of GAL3 in budding yeast. Example trace of the quantified fluorescence intensity of the transcription site. Traces are binarized to determine on (active) and off (inactive) times.


( B ) Histogram of GAL3 on time ( burst duration ) for with wt mutated UAS , respectively , reveals shorter on times for mutated UAS . Errors indicate SE of 3 experiments , n = 324 UASwt , n = 250 UASmut . ( C ) Histogram of GAL3 off times . Errors indicate SE of 3 experiments .

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(B) Histogram of GAL3 on time (burst duration) for cells with wt and mutated UAS, respectively, reveals shorter on times for mutated UAS. Errors indicate SE of 3 experiments, n = 324 cells UASwt, n = 250 cells UASmut. (C) Histogram of GAL3 off times. Errors indicate SE of 3 experiments.


( D ) were UAS was mutated to a lower affinity UAS .

(D) cells were the UAS was mutated to a lower affinity UAS.


( E ) . Average on time for UASwt UASmut from exponential fit . Errors indicate SD of fit . ( F ) Average off time for UASwt UASmut from exponential fit . Errors indicate SD of fit .

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(E). Average on time for UASwt and UASmut from exponential fit. Errors indicate SD of fit. (F) Average off time for UASwt and UASmut from exponential fit. Errors indicate SD of fit.


( G ) Example traces of quantified at transcription site for 5 ' PP7 3 ' MS2 at GAL3 .

(G) Example traces of quantified fluorescence intensity at the transcription site for 5'PP7 and 3'MS2 at GAL3.


( H ) Cross correlation of PP7 MS2 signals shows a peak at 37 . 9s ± 1 . 8s delay , n = 137 . Shaded errors indicate .

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(H) Cross correlation of the PP7 and MS2 signals shows a peak at 37.9s±1.8s delay, n = 137 cells. Shaded area and errors indicate SEM.


( I ) Distribution of number of nascent RNAs at transcription site determined by smFISH . distribution of UASwt does not fit a Poisson distribution ( grey ) , supporting that GAL3 is not constitutively transcribed , but is transcribed in bursts . Example image in shown , yellow arrows indicate TSs . Scale bars : 5 μm . n = 11 , 839 . ( J ) for with UASmut . distribution of UASmut fits a Poisson distribution , indicating that GAL3 - UASmut is transcribed with random initiation of polymerases , similar to constitutive genes . n = 10 , 616 .

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

(I) Distribution of number of nascent RNAs at the transcription site determined by smFISH. The distribution of UASwt does not fit a Poisson distribution (grey line), supporting that GAL3 is not constitutively transcribed, but is transcribed in bursts. Example image in shown, yellow arrows indicate TSs. Scale bars: 5 μm. n = 11,839 cells. (J) for cells with UASmut. The distribution of UASmut fits a Poisson distribution, indicating that GAL3-UASmut is transcribed with random initiation of individual polymerases, similar to constitutive genes. n = 10,616 cells.


( A ) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt ( green ) UASmut ( blue ) motifs . Gal4 occupancy on UASwt Cy3 / Cy5 was determined by measuring induced enhancement ( PIFE ) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore ) .

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(A) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt (green) and UASmut (blue) motifs. Gal4 occupancy on UASwt Cy3/Cy5 DNA was determined by measuring protein induced fluorescence enhancement (PIFE) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore).


( B ) Titrating unlabeled UASwt or UASmut competitor reduces UASwt Cy3 / Cy5 occupancy . IC50UASwt = 4 . 0 ± 0 . 6 nM , IC50UASmut = 17 . 3 ± 3 . 5 nM . n = 3 . Error bars indicate SD .

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(B) Titrating unlabeled UASwt or UASmut competitor DNA reduces UASwt Cy3/Cy5 DNA occupancy. IC50UASwt = 4.0 ± 0.6 nM, IC50UASmut = 17.3 ± 3.5 nM. n = 3. Error bars indicate SD.


( C ) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal shows 4 . 3 ± 1 . 1x difference in KD from naked 6 . 8 ± 1 . 7x from nucleosomal . n = 3 . Error bars indicate SD .

(C) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal DNA shows 4.3 ± 1.1x difference in KD from naked DNA and 6.8 ± 1.7x from nucleosomal DNA. n = 3. Error bars indicate SD.


( D ) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal . Gal4 binds to site 8 bp into nucleosome . A FRET pair in entry / exit region provides readout on binding events ( one fluorophore on , one on histone ) . In absence of Gal4 , nucleosomes are in high FRET state . A Gal4 binding event traps nucleosome in low FRET state .

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(D) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal DNA. Gal4 binds to site 8 bp into nucleosome. A FRET pair in the entry/exit region provides readout on binding events (one fluorophore on DNA, one on histone). In the absence of Gal4, nucleosomes are in the high FRET state. A Gal4 binding event traps nucleosome in low FRET state.


( E ) Gal4 affinity to nucleosomes containing UASwt or UASmut sequences gives S1 / 2 of 7 . 2 ± 0 . 8 nM 48 . 9 ± 10 . 8 nM , respectively . n = 3 . Error bars indicate SD .

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(E) Gal4 affinity to nucleosomes containing UASwt or UASmut sequences gives S1/2 of 7.2 ± 0.8 nM and 48.9 ± 10.8 nM, respectively. n = 3. Error bars indicate SD.


( F ) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal at two different Gal4 concentrations . States are determined using HMM fit . ( G ) Same as F but for UASmut .

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(F) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal DNA at two different Gal4 concentrations. States are determined using HMM fit. (G) Same as F but for UASmut.


( H ) Binding - rates of Gal4 are dependent , but are similar for UASwt UASmut : kon UASwt = 0 . 011 ± 0 . 002 s - 1 nM - 1 , kon UASmut = 0 . 009 ± 0 . 001 s - 1 nM - 1 . n = 2 for , except n = 4 for UASwt 10 nM Gal4 n = 3 for UASwt 20 nM Gal4 . Error bars indicate SD .

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(H) Binding-rates of Gal4 are concentration dependent, but are similar for UASwt and UASmut: kon UASwt = 0.011 ± 0.002 s-1 nM-1, kon UASmut = 0.009 ± 0.001 s-1 nM-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD.


( I ) dissociation - rate of Gal4 at UASwt is ~ 5 - fold slower compared to UASmut : koff UASwt = 0 . 20 ± 0 . 01 s - 1 , koff UASmut = 0 . 92 ± 0 . 05 s - 1 . n = 2 for , except n = 4 for UASwt 10 nM Gal4 n = 3 for UASwt 20 nM Gal4 . Error bars indicate SD .

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(I) The dissociation-rate of Gal4 at UASwt is ~5-fold slower compared to the UASmut: koff UASwt = 0.20 ± 0.01 s-1, koff UASmut = 0.92 ± 0.05 s-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD.


( J ) Histogram of Gal4 dwell time to nucleosomal containing UASwt UASmut sequences . n = 11 for UASwt , n = 8 for UASmut . Error bars indicate SE .

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(J) Histogram of Gal4 dwell time to nucleosomal DNA containing UASwt and UASmut sequences. n = 11 for UASwt, n = 8 for UASmut. Error bars indicate SE.


( K ) Scatter plot showing average number of nascent at TS ( from smFISH ) vs affinity of Gal4 to different UAS sequences in nucleosomal ( from in vitro measurements ) . In vivo transcription correlate with in vitro affinity of Gal4 , but transcription saturates above wildtype . UAS sequences are shown in box . For mean number of nascent from smFISH , n = 2 for UASconsensus , UAS - 2C , UAS - 8T n = 8 for UASwt , UASmut errors indicate . For in vitro affinity measurements , n = 3 errors indicate SD .

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(K) Scatter plot showing the average number of nascent RNA at TS (from smFISH) vs affinity of Gal4 to different UAS sequences in nucleosomal DNA (from in vitro measurements). In vivo transcription levels correlate with in vitro affinity of Gal4, but transcription saturates above wildtype sequence. UAS sequences are shown in the box. For mean number of nascent RNA from smFISH, n = 2 for UASconsensus, UAS-2C, UAS-8T and n = 8 for UASwt, UASmut and errors indicate SEM. For in vitro affinity measurements, n = 3 and errors indicate SD.


Profiles of nucleosome midpoint positions by MNase - seq experiments . Samples were digested with indicated MNase concentrations in both raffinose ( raf ) galactose ( gal ) containing media . Midpoints of nucleosomes are smoothed by 31 bp . In galactose stable nucleosomes move away from Gal4UAS , creating space for an additional fragile nucleosome ( indicated by arrow ) .

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Profiles of nucleosome midpoint positions by MNase-seq experiments. Samples were digested with the indicated MNase concentrations in both raffinose (raf) and galactose (gal) containing media. Midpoints of nucleosomes are smoothed by 31 bp. In galactose the stable nucleosomes move away from the Gal4UAS, creating space for an additional fragile nucleosome (indicated by arrow).


( A ) Simultaneous imaging of Gal4 binding kinetics in vivo using single - molecule tracking imaging of GAL10 target gene . Gal4 is tagged with a HALO - tag , which covalently binds to dye JF646 . Transcription of target gene GAL10 is visualized by PP7 - loops .

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(A) Simultaneous imaging of Gal4 binding kinetics in vivo using single-molecule tracking and RNA imaging of the GAL10 target gene. Gal4 is tagged with a HALO-tag, which covalently binds to the dye JF646. Transcription of the target gene GAL10 is visualized by PP7-loops.


( B ) Example image of a , showing GAL10 TS ( arrow , left panel ) , single molecules of Gal4 ( arrows , middle panel ) an overlay ( right panel ) . Scale bar : 2 μm .

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(B) Example image of a cell, showing the GAL10 TS (arrow, left panel), single molecules of Gal4 (arrows, middle panel) and an overlay (right panel). Scale bar: 2 μm.


( C ) Example kymograph of a . Upper panel ( PP7 - GAL10 ) shows position of TS over time . Middle panel ( Gal4 - HALO ) show tracks of Gal4 . Data is in , colored lines show analyzed tracks . Lower panel shows overlay , showing colocalization of some Gal4 tracks to GAL10 TS . Scale bar : 1 μm .

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(C) Example kymograph of a cell. Upper panel (PP7-GAL10) shows the position of the TS over time. Middle panel (Gal4-HALO) show tracks of Gal4. Data is in white, colored lines show analyzed tracks. Lower panel shows overlay, showing colocalization of some the Gal4 tracks to the GAL10 TS. Scale bar: 1 μm.


( D ) Survival probability of duration of Gal4 tracks ( after displacement thresholding ) at 200 ms interval from grown in raffinose ( n = 258 tracks in 30 ) or galactose ( n = 346 tracks in 25 ) . Lines show bi - exponential fit , indicating 2 Gal4 populations . Inset shows data in semi - logarithmic plot . ( E ) Residence time of fast slow component of fits from ( D ) . slow component changes between conditions . Error bars indicate 95 % CI .

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(D) Survival probability of the duration of Gal4 tracks (after displacement thresholding) at 200 ms interval from cells grown in raffinose (n = 258 tracks in 30 cells) or galactose (n = 346 tracks in 25 cells). Lines show bi-exponential fit, indicating 2 Gal4 populations. Inset shows data in semi-logarithmic plot. (E) Residence time of the fast and slow component of the fits from (D). The slow component changes between conditions. Error bars indicate 95% CI.


( F ) Survival probability of Gal4 residence times that colocalize with GAL10 TS ( < 250nm ) , showing that Gal4 with long residence time colocalizes with GAL10 . Data was taken at 1s interval shows overlap between 267 Gal4 tracks 83 GAL10 tracks , representing 458 bound molecules . shows exponential fit , with mean of 12 . 3s ± 0 . 83s . Errors indicate 95 % CI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival probability of Gal4 residence times that colocalize with GAL10 TS (<250nm), showing that Gal4 with long residence time colocalizes with GAL10. Data was taken at 1s interval and shows overlap between 267 Gal4 tracks and 83 GAL10 tracks, representing 458 bound molecules. Line shows exponential fit, with mean of 12.3s ± 0.83s. Errors indicate 95% CI.


( A ) Schematic of 3D orbital tracking of GAL10 TS . Scale bar : 2 μm .

(A) Schematic of 3D orbital tracking of GAL10 TS. Scale bar: 2 μm.


( B ) Example trace of Gal4 binding GAL10 transcription in same .

(B) Example trace of Gal4 binding and GAL10 transcription in the same cell.


( C ) Autocorrelation of Gal4 . exponential fit shows an average dwell time of 34 . 8 ± 0 . 5s . n = 19 traces . Shaded errors indicate .

(C) Autocorrelation of Gal4. The exponential fit shows an average dwell time of 34.8 ± 0.5s. n = 19 traces. Shaded area and errors indicate SEM.


( D ) Autocorrelation of GAL10 . fit of 2 linear lines reveals a burst duration of 152 . 4 ± 2 . 3s . n = 19 traces . Shaded errors indicate .

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(D) Autocorrelation of GAL10. The fit of 2 linear lines reveals a burst duration of 152.4 ± 2.3s. n = 19 traces. Shaded area and errors indicate SEM.


( E ) Cross correlations for Gal4 - GAL10 ( blue ) Gal4 - RNR2 ( grey , negative control ) . Asymmetry positive temporal shift of cross correlation function is consistent with a 79 . 5 ± 0 . 2 second delay between middle of Gal4 GAL10 signals ( n = 19 traces ) , which is not seen in negative control ( n = 8 traces ) . Shaded areas errors indicate .

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(E) Cross correlations for Gal4-GAL10 RNA (blue) and Gal4-RNR2 RNA (grey, negative control). Asymmetry and positive temporal shift of the cross correlation function is consistent with a 79.5 ± 0.2 second delay between the middle of Gal4 and GAL10 signals (n = 19 traces), which is not seen in the negative control (n = 8 traces). Shaded areas and errors indicate SEM.


( F ) Schematic of different signal durations of Gal4 binding GAL10 transcription . Gal4 binding overlaps with GAL10 transcription for 14 . 1s .

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(F) Schematic of the different signal durations of Gal4 binding and GAL10 transcription. Gal4 binding overlaps with the GAL10 transcription for 14.1s.


( A ) Example trace of PP7 - GAL10 transcription in 2 % galactose .

(A) Example trace of PP7-GAL10 transcription in 2% galactose.


( B ) Boxplot of onset of first transcription event in different doses of galactose . box indicates quartiles , horizontal inside box indicates median , whiskers indicates 1 . 5 times interquartile range of box . Data points outside whiskers are indicated with circles ( 2 % : n = 86 , 0 . 2 % : n = 79 , 0 . 02 % : n = 84 ) .

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) Boxplot of the onset of the first transcription event in different doses of galactose. The box indicates quartiles, the horizontal line inside the box indicates the median, the whiskers indicates 1.5 times the interquartile range of the box. Data points outside the whiskers are indicated with circles (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells).


( C ) autocorrelation was used to interpret burst duration burst frequency changes . Burst duration is measured by intersect of two linear fits . When burst duration is constant , amplitude is inversely related to burst frequency . ( D ) Burst duration from autocorrelation is constant across 4 different galactose concentrations ( 2 % : n = 86 , 0 . 2 % : n = 79 , 0 . 02 % : n = 84 , 0 . 004 % : n = 53 ) . Error bars indicate . ( E ) Amplitude of autocorrelation decreases ( burst frequency increases ) at higher doses of galactose . ( 2 % : n = 86 , 0 . 2 % : n = 79 , 0 . 02 % : n = 84 , 0 . 004 % : n = 53 ) . Error bars indicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

(C) The autocorrelation was used to interpret the burst duration and burst frequency changes. Burst duration is measured by the intersect of the two linear fits. When burst duration is constant, the amplitude is inversely related to the burst frequency. (D) Burst duration from autocorrelation is constant across 4 different galactose concentrations (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. (E) Amplitude of autocorrelation decreases (burst frequency increases) at higher doses of galactose. (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM.


( F ) Example traces of two single exposed to two different doses of galactose . ( G , H ) Same - dose response shows same burst duration ( G ) but lower amplitude ( H ) at higher galactose , indicating that galactose regulate burst frequency ( n = 13 ) . Error bars indicate .

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

(F) Example traces of two single cells exposed to two different doses of galactose. (G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM.


A , B . Depletion of Ccdc108 markedly affects bead movement by decreasing velocity beads were applied to dorsal surface of Xenopus . Bead tracks every 500 ms over 2 s are shown ( A ) . Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants , which can be fully rescued by co - injecting with Xenopus ccdc108 mRNA ( B ) . 150 beads from 15 for each condition . Arrowheads indicate beads that showed no movement on surface through recording . Data information : data were collected from three independent experiments . Unpaired two - t - was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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A, B. Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos. Bead tracks every 500 ms over 2 s are shown (A). Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants, which can be fully rescued by co-injecting with Xenopus ccdc108 mRNA (B). 150 beads from 15 embryos for each condition. Arrowheads indicate beads that showed no movement on the embryo surface through the recording. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


C , D . Defective cilia formation in ccdc108 morphants . at stage 27 were fixed stained with acetylated tubulin ( Ac - tub ) antibody / or Cep164 antibody . mRNA of a - bound form of GFP ( mGFP ) was co - injected with each morpholino to indicate targeted . Representative confocal ( C ) 3D - SIM ( D ) of epidermis for each condition plots show significantly reduced cilia number in Ccdc108 depleted . ( C ) 20 of 20 ( D ) > 80 MCCs from 6 for each condition . Data information : data were collected from three independent experiments . Unpaired two - t - was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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

C, D. Defective cilia formation in ccdc108 morphants. Embryos at stage 27 were fixed and stained with the acetylated tubulin (Ac-tub) antibody and/or Cep164 antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino to indicate targeted cells. Representative confocal images (C) and 3D-SIM images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


F . Defective cilia formation occurs in ccdc108 CRISPR mutants . at one stage were injected with Cas9 with or without sgRNA against ccdc108 ( 108sg ) fixed at stage 27 . Representative 3D - SIM plot show significantly reduced cilia number in ccdc108 CRISPR mutants . > 70 MCCs from 6 for each condition . membranes ( mGFP , purple ) , cilia ( Ac - tub , green ) basal bodies ( Cep164 , yellow ) were labeled with indicated antibodies . Data information : data were collected from three independent experiments . Unpaired two - t - was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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F. Defective cilia formation occurs in ccdc108 CRISPR mutants. Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 (108sg) and fixed at stage 27. Representative 3D-SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants. > 70 MCCs from 6 embryos for each condition. Cell membranes (mGFP, purple), cilia (Ac-tub, green) and basal bodies (Cep164, yellow) were labeled with indicated antibodies. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


G . In situ hybridization chain reaction ( HCR ) reveals that ccdc108 mRNA is specially detected in MCCs . at stage 35 were fixed subjected to in situ HCR ( red ) . were then incubated with Ac - tub antibody ( cyan ) DAPI ( gray ) .

G. In situ hybridization chain reaction (HCR) reveals that ccdc108 mRNA is specially detected in MCCs. Embryos at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the Ac-tub antibody (cyan) and DAPI (gray).


C , D . Representative confocal plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts ( C ) olfactory placodes ( D ) . Zebrafish were fixed stained with Ac - tub antibody ( green ) , phalloidin ( purple ) Hoechst ( blue ) . Data information : Quantitative data were from three independent repeats . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . Mean ± s . e . m values are presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts (C) and olfactory placodes (D). Zebrafish embryos were fixed and stained with the Ac-tub antibody (green), phalloidin (purple) and Hoechst (blue). Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). Mean ± s.e.m values are presented.


E . Scanning electron microscope of zebrafish display a failure in cilia formation in Ccdc108 depleted olfactory placodes .

E. Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes.


H . Representative 3D - SIM plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs . mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days subjected to immunostaining . GFP - CETN1 ( blue ) marked mEPCs infected with lentivirus . Basal bodies ( CEP164 , purple ) cilia ( Ac - tub , green ) were labeled with indicated antibodies . > 60 MCCs from three independent repeats for each condition were counted . Data information : Quantitative data were from three independent repeats . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . mean ± s . d . values are presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative 3D-SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs. mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining. GFP-CETN1 (blue) marked mEPCs infected with lentivirus. Basal bodies (CEP164, purple) and cilia (Ac-tub, green) were labeled with indicated antibodies. > 60 MCCs from three independent repeats for each condition were counted. Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). mean ± s.d. values are presented.


A . Depletion of Ccdc108 causes disorganized basal distribution in MCCs . Representative 3D - SIM ( x - y ) 3D - reconstructions ( x - z ) of MCCs of show a failure of apical trafficking of basal bodies in Ccdc108 depleted . membranes ( mGFP , blue ) , basal bodies ( Cetn1 , green ) distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Select of Cep164 channel was zoomed to show centriolar Cep164 accumulations . Orange arrowheads numbers mark centriole . > 70 MCCs from 6 for each condition were counted . Data information : Quantitative data were from three independent experiments . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

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A. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


B . Transmission electron microscopy of Xenopus displaying aberrant apical trafficking docking of centrioles in Ccdc108 depleted MCCs . Orange arrowheads mark centrioles . of centrioles docked to plasma in each field were scored . At least 7 for each sample were counted .

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B. Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs. Orange arrowheads mark centrioles. Percentage of centrioles docked to plasma membrane in each field were scored. At least 7 cells for each sample were counted.


C . Representative 3D - SIM reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs . were fixed stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Apical of MCCs in each condition were measured ; 80 MCCs from 6 for each condition . D . Representative confocal show a significant reduction in apical actin following depletion of Ccdc108 . boundaries apical actin network were marked with mGFP ( green ) phalloidin ( purple ) phalloidin measured plotted . > 80 MCCs from 6 for each condition . Data information : Quantitative data were from three independent experiments . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

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C. Representative 3D-SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Apical area of MCCs in each condition were measured ; 80 MCCs from 6 embryos for each condition. D. Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple) and phalloidin intensity levels measured and plotted. > 80 MCCs from 6 embryos for each condition. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


A . Live imaging of MCCs shows distribution of Ccdc108 in MCCs cilia basal bodies . expressing GFP - Ccdc108 ( green ) Cetn4 - RFP ( purple ) were imaged at stage 27 . To better visualize ciliary structures , maximum projections of stacks for regions containing cilia basal bodies are shown separately .

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A. Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies. Embryos expressing GFP-Ccdc108 (green) and Cetn4-RFP (purple) were imaged at stage 27. To better visualize ciliary structures, maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately.


C . Ccdc108 interacts with IFT . Interactions were further determined by coimmunoprecipitation of endogenous IFT from HEK293T with GFP - Trap agarose beads .

C. Ccdc108 interacts with IFT proteins. Interactions were further determined by coimmunoprecipitation of endogenous IFT proteins from HEK293T cells with GFP-Trap agarose beads.


D . Live imaging of MCCs shows both Ccdc108 Ift74 localize to ciliary axoneme basal bodies in MCCs . expressing mCherry - Ccdc108 ( green ) GFP - Ift74 ( purple ) were imaged at stage 27 .

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D. Live imaging of MCCs shows both Ccdc108 and Ift74 proteins localize to the ciliary axoneme and the basal bodies in MCCs. Embryos expressing mCherry-Ccdc108 (green) and GFP-Ift74 (purple) were imaged at stage 27.


A - C . IFT - interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus : ( A ) impaired multiciliogenesis , ( B ) defective apical migration / docking of centrioles , ( C ) reduced enrichment of apical F - actin . at stage 27 were fixed stained with acetylated tubulin ( A ; Ac - tub ; green ) , Cep164 ( B ; purple ) Cetn1 ( B ; green ) antibodies or phalloidin ( purple ) . mRNA of a - bound form of GFP was co - injected with each morpholino to indicate targeted ( A , B ) . Typical D - SIM ( A , B ) confocal ( C ) are present . were treated as described in Figure 1C expressed mGFP or GFP - Ccdc108 mutants . Plots show results from three independent experiments . ( A ) > 70 MCCs from 6 , ( C ) > 70 MCCs from 6 for each condition . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are presented .

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A-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal images (C) are present. Embryos were treated as described in Figure 1C and expressed mGFP or GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6 embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.


A , ift74 CRISPR mutants display defective cilia formation . at one stage were injected fixed at stage 27 with two independent sgRNA targeting ift74 . Representative confocal of Xenopus epidermis , plots display defective ciliation . ; 60 MCCs from 6 membranes ( mGFP ) , cilia ( Ac - tub , green ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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A, ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative confocal images of the Xenopus epidermis, and plots display defective ciliation. ;60 MCCs from 6 embryos Cell membranes (mGFP), cilia (Ac-tub, green) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


B . ift74 CRISPR mutants display defective cilia formation . at one stage were injected fixed at stage 27 with two independent sgRNA targeting ift74 . Representative 3D - SIM of Xenopus epidermis , plots display defective ciliation . ; 70 MCCs from 6 for each condition . membranes ( mGFP ) , cilia ( Ac - tub , green ) basal bodies ( Cep164 ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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B. ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative 3D-SIM images of the Xenopus epidermis, and plots display defective ciliation. ;70 MCCs from 6 embryos for each condition. Cell membranes (mGFP), cilia (Ac-tub, green) and basal bodies (Cep164) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C . Ift74 depletion affects centriole apical migration in MCCs . Representative 3D - SIM ( x - y ) 3D - reconstructions ( x - z ) of MCCs show failure of apical trafficking of basal bodies . Basal bodies ( Cetn1 , green ) distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Quantitative plot shows a slight significant reduction in basal bodies by depletion of Ift74 . > 70 MCCs from 6 for each condition . D . Representative 3D - SIM plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion . > 70 MCCs from 6 for each condition . were fixed stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ift74 depletion affects centriole apical migration in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs show failure of apical trafficking of basal bodies. Basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74. >70 MCCs from 6 embryos for each condition. D. Representative 3D-SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion. >70 MCCs from 6 embryos for each condition. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


E . Representative confocal plot show a significant reduction in apical F - actin enrichment upon Ift74 depletion . > 70 MCCs from 6 for each condition . boundaries apical actin network were marked with mGFP ( green ) phalloidin ( purple ) . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images and plot show a significant reduction in apical F-actin enrichment upon Ift74 depletion. >70 MCCs from 6 embryos for each condition. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple). Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C - E . Ccdc108 depletion affects centriolar distribution of IFT - B during centriole migration in MCCs . were co - injected with GFP - IFTs ( C : Ift74 ; D : Ift80 ; E : Ift88 ; green ) , Cetn4 - RFP ( purple ) each morpholino , imaged at stage 18 . HA tagged Ccdc108 were co - expressed to for rescue of phenotypic defects . Xenopus were subjected to immunoblotting to evaluate expression . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of IFT - B ( Ift74 , Ift80 Ift88 ) , Ccdc108 - IFT interaction deficient mutants are unable to rescue decreased centriolar accumulation of IFT - B . > 40 MCCs from 6 for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are also presented .

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

C-E. Ccdc108 depletion affects centriolar distribution of IFT-B proteins during centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D: Ift80; E: Ift88; green), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.


A , B . Centriolar distribution of ciliary adhesion related Fak Cp110 are not reduced following Ccdc108 depletion . were co - injected with GFP - Cp110 ( A ) GFP - Fak ( B ) , Cetn4 - RFP ( purple ) each morpholino , imaged at stage 18 . > 30 MCCs from 6 for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

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

A, B. Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion. Embryos were co-injected with GFP-Cp110 (A) GFP-Fak (B), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C , D . Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs . were co - injected with GFP - Drg1 ( C ) GFP - RBD ( D ) , Cetn4 - RFP ( purple ) each morpholino , imaged at stage 18 . HA tagged Ccdc108 were co - expressed to for rescue of phenotypic defects . Xenopus were subjected to immunoblotting to evaluate expression . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of Drg1 RBD , neither of Ccdc108 - IFT interaction deficient mutants can rescue centriolar phenotypic defects . > 30 MCCs from 6 for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - t - was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs. Embryos were co-injected with GFP-Drg1 (C) GFP-RBD (D), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD, and neither of Ccdc108-IFT interaction deficient mutants can rescue the centriolar phenotypic defects. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


Total number of SNVs / InDels ( allele frequency ≥ 20 % ) detected in samples ( x - axis ) plotted against number of SNVs / InDels detected in corresponding PDX samples ( y - axis ) ; R² coefficient of determination Number of preserved SNVs / InDels ( black bars ) those that were not detected in PDX models ( grey bars ) in relation to their allele frequencies ( AF ) ; % - fraction of preserved

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

Total number of SNVs/InDels (allele frequency≥20%) detected in primary samples (x-axis) plotted against the number of SNVs/InDels detected in corresponding PDX samples (y-axis); R² coefficient of determination Number of preserved SNVs/InDels (black bars) and those that were not detected in PDX models (grey bars) in relation to their allele frequencies (AF); % - fraction of preserved mutations


Large ( > 1Mb ) CNA ( amplifications - shades of violet ; deletions - shades of red ) identified by low coverage whole genome sequencing in each of corresponding

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

Large (>1Mb) CNA (amplifications- shades of violet; deletions - shades of red) identified by low coverage whole genome sequencing in each of the patients and corresponding PDXs


Allele frequencies ( AF ) of mutations detected in ( PRI ) ' samples ( x - axis ) at initial diagnosis ( INI ) relapse ( REL ) plotted against AF of corresponding mutations detected in matched PDX models ( y - axis ) for relapse type 1 ( left ) relapse type 2 ( right ) ; black - mutations preserved between PDX , blue - mutations , which were selected for / against in PDX models , green - relapse specific mutations selected for in INIPDX , absent or detected with a very low AF in ' diagnosis sample , orange - subclonal mutations lost in PDX , red - clonal mutations lost in PDX , grey - mutations , which AF was affected by low blast content ( BC ) at relapse Allele frequencies of mutations preserved between ' samples ( x - axis ) PDX samples ( y - axis ) ; PCC - Pearson ' s correlation

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

Allele frequencies (AF) of mutations detected in primary (PRI) patients' samples (x-axis) at initial diagnosis (INI) and relapse (REL) plotted against AF of corresponding mutations detected in matched PDX models (y-axis) for relapse type 1 (left) and relapse type 2 patients (right); black - mutations preserved between primary and PDX, blue - mutations, which were selected for/against in PDX models, green - relapse specific mutations selected for in INIPDX, absent or detected with a very low AF in primary patients' diagnosis sample, orange - subclonal mutations lost in PDX, red - clonal mutations lost in PDX, grey - mutations, which AF was affected by low blast content (BC) at relapse Allele frequencies of all mutations preserved between primary patients' samples (x-axis) and PDX samples (y-axis); PCC- Pearson's correlation coefficient


Unsupervised hierarchical clustering on average degree of methylation of 500 most variable promoters ( red - low / violet - high methylation ) ; * - relapse ( REL ) sample of P4 with blast content of 8 %

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

Unsupervised hierarchical clustering based on the average degree of methylation of the 500 most variable promoters (red - low/violet -high methylation levels); * - relapse (REL) sample of patient P4 with blast content of 8%


degree of mean promoter methylation in ' samples plotted against degree of mean promoter methylation in PDX samples ; ρ - Spearman ' s rank correlation coefficient ; median combined ranks across hexagonal bins are shown as a gradient according to color

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

The degree of the mean promoter methylation in patients' samples plotted against the degree of mean promoter methylation in PDX samples; ρ - Spearman's rank correlation coefficient; median combined ranks across hexagonal bins are shown as a gradient according to the color legend


TSS enrichment score of primary samples (PRI) and PDXs


Reads number per peak in samples ( x - axis ) plotted against reads number in corresponding PDX model ; PCC = Pearson ' s correlation coefficient )

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

Reads number per peak in primary samples (x-axis) plotted against the reads number in corresponding PDX model; PCC=Pearson's correlation coefficient)


Fraction of peaks shared between PDX corresponding leukemia for ATAC peaks with a certain height ( x - axis ; average number of reads that map into an accessible region called by Macs2 )

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

Fraction of peaks shared between PDX and corresponding primary leukemia for ATAC peaks with a certain height (x-axis; average number of reads that map into an accessible region called by Macs2)


MA plot of log2 fold changes between PDX samples plotted against mean of normalized read per peak , red - differentially accessible regions ( p < 0 . 05 ) as analyzed by

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

MA plot of log2 fold changes between primary and PDX samples plotted against the mean of normalized read counts per peak, red - differentially accessible regions (p<0.05) as analyzed by DESeq


Relative expression of HIF1A - AS2 transcript in indicated lines under normoxia condition were monitored by qRT - PCR represented as 39 minus Ct values .

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

Relative expression levels of the HIF1A-AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT-PCR analysis and represented as 39 minus Ct values.


Sanger sequencing validation of editing events on HIF1A - AS2 . editing frequencies of indicated sites were quantitated on three independent experiments shown in Table EV 1 . Asterisks mark sites undergoing statistically significant increases in editing .

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Sanger sequencing validation of RNA editing events on HIF1A-AS2. The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1. Asterisks mark the sites undergoing statistically significant increases in RNA editing.


- IP of ADAR1 in HUVECs , followed by real - time PCR of HIF - 1A HIF1A - AS2 RNAs . relative enrichment of transcript association was normalized to IgG . GAPDH was used as a non - binding control .

RNA-IP of ADAR1 in HUVECs, followed by real-time PCR analysis of the HIF-1A and HIF1A-AS2 RNAs. The relative enrichment of transcript association was normalized to the IgG. GAPDH was used as a non-binding control.


were cultured under normal or hypoxic condition . Relative of HIF1A - AS transcripts in total vs . poly ( A ) + fractions ( F ) were determined by qRT - PCR .

Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F) were determined by qRT-PCR.


were cultured under normal or hypoxic condition . Relative of HIF1A - AS transcripts in subcellular fractions ( G ) , were determined by qRT - PCR .

Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in subcellular fractions (G), were determined by qRT-PCR.


FISH of spatial distribution of HIF - 1A mRNA ( red ) HIF1A - AS2 transcripts ( ) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs . Nuclei were counterstained by DAPI . Scale bars , 10 μm .

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

FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm.


Expression of HIF1A - AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides ( ODN ) - knockdown . were then treated with CoCl2 for 5 hrs to mimic hypoxic condition , subjected to gene expression analyses : abundance of HIF - 1A HIF1A - AS2 was analyzed by real - time PCR normalized by 18s rRNA ( A ) .

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

Expression of HIF1A-AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides (ODN)-based knockdown. Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition, and subjected to gene expression analyses: The RNA abundance of HIF-1A and HIF1A-AS2 was analyzed by real-time PCR and normalized by 18s rRNA (A).


HIF - 1α level was monitored by western blot , with β - actin as internal control ( B ) .

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

HIF-1α protein level was monitored by western blot, with β-actin as the internal control (B).


run - on assays were performed to assess extent of nascent transcription of target genes under mock vs . CoCl2 treatment ( C )

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

Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes under mock vs. CoCl2 treatment (C)


run - on assays were performed to assess extent of nascent transcription of target genes in HIF1A - AS2 knockdown ( D ) .

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

Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A-AS2 knockdown cells (D).


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize polymerase II binding on isolated from MCF7 under CoCl2 treatment ( E ) , precipitated fragments were quantitatively analyzed by real - time PCR using primers for HIF1A - AS2 HIF - 1A promoters , normalized to values of IgG . HIF1A - AS2 corresponds to transcribed region .

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

HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment (E), The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize polymerase II binding on isolated from MCF7 in HIF1A - AS2 - knockdown vs . control ( F ) . precipitated fragments were quantitatively analyzed by real - time PCR using primers for HIF1A - AS2 HIF - 1A promoters , normalized to values of IgG . HIF1A - AS2 corresponds to transcribed region .

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

HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A-AS2-knockdown vs. control cells (F). The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HUVECs were transfected with ADAR1 - targeting siRNAs subjected to hypoxia treatment for 5 hrs . expression of ADAR1 , HIF - 1A HIF1A - AS2 transcripts were analyzed by qRT - PCR .

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The expression levels of ADAR1, HIF-1A and HIF1A-AS2 transcripts were analyzed by qRT-PCR.


HUVECs were transfected with ADAR1 - targeting siRNAs subjected to hypoxia treatment for 5 hrs . run - on was done to measure newly synthesized HIF - 1A HIF1A - AS2 RNAs in ADAR1 - knockdown , followed by real - time PCR of indicated transcripts .

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

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Nuclear run-on assay was done to measure the newly synthesized HIF-1A and HIF1A-AS2 RNAs in ADAR1-knockdown cells, followed by real-time PCR analysis of the indicated transcripts.


HUVECs were transfected with ADAR1 - targeting siRNAs subjected to hypoxia treatment for 5 hrs . ChIP of polII binding on region of HIF1A - AS2 HIF - 1A promoters . extent of occupancy was compared between control ADAR1 - knockdown . precipitated fragments were quantitatively analyzed by real - time PCR normalized by values of IgG .

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

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. ChIP analysis of RNA polII binding on chromatin region of the HIF1A-AS2 and HIF-1A promoters. The extent of occupancy was compared between control and ADAR1-knockdown cells. The precipitated DNA fragments were quantitatively analyzed by real-time PCR and normalized by the values of IgG.


binding of ADAR1 to HIF1A - AS2 HIF - 1A promoters under nomoxia ( 20 % O2 ) vs . hypoxia ( 1 % O2 , 5 hrs ) conditions , as assessed by ChIP .

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

Chromatin binding of ADAR1 to HIF1A-AS2 and HIF-1A promoters under nomoxia (20% O2) vs. hypoxia (1% O2, 5 hrs) conditions, as assessed by ChIP.


HUVECs were transfected with ADAR1 - targeting siRNAs subjected to hypoxia treatment for 5 hrs . 3 ' - end extension of HIF1A - AS2 transcription in ADAR1 knockdown ( GFP , control ) was examined by PCR with primers corresponding to depicted regions of HIF1A - AS2 locus . GAPDH serves as an internal control . locations are shown in kbp relative to transcription start site .

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

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The 3'-end extension of HIF1A-AS2 RNA transcription in ADAR1 knockdown cells (GFP, control) was examined by PCR with primers corresponding to the depicted regions of HIF1A-AS2 locus. GAPDH serves as an internal control. Sequence locations are shown in kbp and relative to the transcription start site.


HUVECs were transfected with ADAR1 - targeting siRNAs subjected to hypoxia treatment for 5 hrs . Expression of HIF - 1α ( F ) by western

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α protein (F) by western blot


HUVECs were transfected with ADAR1 - targeting siRNAs subjected to hypoxia treatment for 5 hrs . Expression of HIF - 1α downstream genes ( G ) by real - time PCR , as indicated . qRT - PCR results were normalized by expression of 18s rRNA .

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α downstream genes (G) by real-time PCR, as indicated. qRT-PCR results were normalized by the expression of 18s rRNA.


Transcriptome - wide changes in subjected to CoCl2 treatment , HIF1A - AS2 or ADAR1 knockdown were profiled by - Sequencing . For genes that undergo up - regulation upon CoCl2 treatment ( n = 505 ) , overall expression profiles of different groups are represented by . expression values ( represented by normalized read ) were displayed in shades of red or blue ( linear scale ) relative to means of corresponding values within experimental groups . Clusters of genes ( on unsupervised hierarchical clustering ) are indicated denoted by their experimental types / conditions .

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 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

Transcriptome-wide changes in the cells subjected to CoCl2 treatment, HIF1A-AS2 or ADAR1 knockdown were profiled by RNA-Sequencing. For genes that undergo up-regulation upon CoCl2 treatment (n = 505), overall expression profiles of the different cell groups are represented by the heatmap. The expression values (represented by normalized read counts) were displayed in shades of red or blue (linear scale) relative to the means of all corresponding values within individual experimental groups. Clusters of genes (based on unsupervised hierarchical clustering) are indicated and denoted by their experimental types/conditions.


GSEA comparing gene expression profile by gene up - regulated upon CoCl2 treatment those down - regulated in ADAR1 knockdown . curves correspond to observed enrichment score profiles in CoCl2 gene set to ADAR1 knockdown gene sets . bar ‐ code plot below enrichment score plot denote rank of each member in CoCl2 up - regulated gene list .

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

GSEA comparing the gene expression profile by gene up-regulated upon CoCl2 treatment and those down-regulated in ADAR1 knockdown. The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets. The bar‐code plot below enrichment score plot denote the rank of each member in the CoCl2 up-regulated gene list.


HUVECs were transfected with indicated combinations of siRNA ( GFP as control versus ADAR1 ) ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . expression of ADAR1 HIF - 1α ( E ) were analyzed respectively by immunoblotting .

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

HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. The expression levels of ADAR1 and HIF-1α protein (E) were analyzed respectively by immunoblotting.


HUVECs were transfected with indicated combinations of siRNA ( GFP as control versus ADAR1 ) ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . For in ( E ) , expression of heat shock - encoding genes ( HSPA1A , HSPA1B , HSPA6 ) with reported roles in angiogenesis were profiled by qRT - PCR .

O O O O 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

HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. For cells in (E), RNA expression of heat shock protein-encoding genes (HSPA1A, HSPA1B, and HSPA6) with reported roles in angiogenesis were profiled by qRT-PCR.


Montage of representative of 2D capillary network formation assays on HUVECs subjected to above experimental manipulation . Scale bars , 2000 μm .

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Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation. Scale bars, 2000 μm.


Sanger sequencing validation of editing of LIMD1 ( A ) VHL ( B ) transcripts .

Sanger sequencing validation of RNA editing of the LIMD1 (A) and VHL (B) transcripts.


Downregulation of ADAR1 in MCF7 was done using specific siRNAs . were then subjected to hypoxia treatment for 5 hrs . extent of knockdown gene alterations was monitored by western blotting using antibodies targeting LIMD1 , VHL , PHD1 , PHD2 , ADAR1 HIF - 1α , as indicated β - actin was used as loading control .

Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs. Cells were then subjected to hypoxia treatment for 5 hrs. The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1, VHL, PHD1, PHD2, ADAR1 and HIF-1α, as indicated β-actin was used as loading control.


To characterize subcellular distribution of target transcripts , real - time PCR was done on fractionated MCF7 with indicated experimental treatments .

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To characterize subcellular distribution of target transcripts, real-time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments.


Translation efficiency was determined by quantifying transcript association with polyribosome fraction , using ADAR1 - depleted MCF7 with hypoxia treatment . abundance of LIMD1 in polysomes was analyzed by real - time PCR normalized by eEF1α .

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Translation efficiency was determined by quantifying transcript association with the polyribosome fraction, using ADAR1-depleted MCF7 cells with hypoxia treatment. The abundance of LIMD1 in polysomes was analyzed by real-time PCR and normalized by the eEF1α levels.


To inspect HIF - 1α abundance modification with ubiquitin , anti - HIF - 1α , anti - ubiquitylation ( Ub ) , anti - VHL immunoprecipitation experiments were performed , coupled with immunoblotting detection of ubiquitylation HIF - 1α , as indicated . MCF7 transfected with ADAR1 siRNA treated with hypoxia were used . signals were quantified using ImageJ software . fold changes of were determined on basis of immunoreactive signals normalized by input ( In ) , with control ( siGFP ) group being represented as 1 .

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To inspect HIF-1α protein abundance and modification with ubiquitin, anti- HIF-1α, anti-ubiquitylation (Ub), and anti-VHL immunoprecipitation experiments were performed, coupled with immunoblotting detection of ubiquitylation and HIF-1α, as indicated. MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used. Protein signals were quantified using the ImageJ software. The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input (In), with the control (siGFP) group being represented as 1.


B , Western blot showing no detectable Wbp2 in 4 week old mutant brain compared to wt littermate controls . 5μg of lysate were subjected to 10 % SDS - PAGE . β - tubulin was used as loading control . Wbp2 l Wbp2 s refer to long short isoforms respectively .

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B, Western blot showing no detectable Wbp2 protein in 4 week old mutant brain compared to wt littermate controls. 5μg of the protein lysate were subjected to 10% SDS-PAGE. β-tubulin was used as loading control. Wbp2 l and Wbp2 s refer to the long and short isoforms respectively.


C , Quantitative real - time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants ( n = 3 ) inner ears eyes , compared to wt littermate controls ( n = 3 ) . Heterozygotes show intermediate . Hprt was used as a control are normalised to wt .

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C, Quantitative real-time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants (n=3) inner ears and eyes, compared to wt littermate controls (n=3). Heterozygotes show intermediate levels. Hprt was used as a control and levels are normalised to wt levels.


D , F , X - gal of Wbp2 hets at P14 showing Wbp2 expression ( blue ) in main cochlear structures : stria vascularis ( black arrowhead in D ) , spiral prominence ( empty arrowhead in D ) , Reissner ' s ( arrow in D ) , strong expression in spiral ganglion ( red arrowheads in E ) , IHCs OHCs in organ of Corti ( arrowheads in F ) , pillar spiral limbus ( empty arrowhead in F ) . Scale bars : D : 50μm , E , F : 20μm . : inner hair ; ohc : outer hair . No X - gal is observed in wt controls ( not shown ) . X - gal reaction is always .

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D, F, X-gal staining of Wbp2 hets at P14 showing Wbp2 expression (blue) in all the main cochlear structures: the stria vascularis (black arrowhead in D), spiral prominence (empty arrowhead in D), Reissner's membrane (arrow in D), strong expression in the spiral ganglion cells (red arrowheads in E), IHCs and OHCs in the organ of Corti (arrowheads in F), pillar cells and spiral limbus (empty arrowhead in F). Scale bars: D: 50μm, E,F: 20μm. ihc: inner hair cells; ohc: outer hair cells. No X-gal staining is observed in wt controls (not shown). The X-gal reaction is always cytoplasmic.


A , E Mean ABR thresholds ( ± SD ) for clicks tone pips are plotted for wt ( green ) , het ( blue ) hom ( red ) mice at ages A , P14 ( wt , n = 3 ; het , n = 8 ; hom , n = 6 ) ; B , 4 ( wt , n = 38 ; het , n = 26 ; hom , n = 37 ) ; C , 14 ( wt , n = 10 ; hom , n = 14 ) ; D , 28 ( wt , n = 15 ; het , n = 5 ; hom , n = 25 ) ; E , 44 ( wt , n = 9 ; het , n = 2 ; hom , n = 11 ) . Grey symbols lines indicate thresholds of mutants . In B , open symbols represent thresholds under urethane anaesthesia ( see methods ) , showing no difference compared with ketamine / xylazine used for other thresholds . F , Mean thresholds for mutants aged 2 ( yellow ) , 4 ( purple ) , 14 ( cyan ) , 28 ( grey ) 44 ( black ) .

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A,E Mean ABR thresholds (± SD) for clicks and tone pips are plotted for wt (green), het (blue) and hom (red) mice at ages A, P14 (wt, n=3; het, n=8; hom, n=6); B, 4 weeks (wt, n=38; het, n=26; hom, n=37); C, 14 weeks (wt, n=10; hom, n=14); D, 28 weeks (wt, n=15; het, n=5; hom, n=25); E, 44 weeks (wt, n=9; het, n=2; hom, n=11). Grey symbols and lines indicate thresholds of individual mutants. In B, open symbols represent thresholds under urethane anaesthesia (see methods), showing no difference compared with ketamine/xylazine used for all other thresholds.F, Mean thresholds for mutants aged 2 weeks (yellow), 4 weeks (purple), 14 weeks (cyan), 28 weeks (grey) and 44 weeks (black).


G , Averaged click - evoked ABR waveforms from 4 old wt ( n = 23 , green ) mutants ( n = 34 , red ) , at 50dB sensation level ( SL ) ( left panel ) . SP ABR Wave 1 ( W1 ) are indicated by grey areas . Expanded averaged SP ABR W1 waveform patterns for 10 - 60dB SL in 10dB increments are plotted in green ( wt ; middle panel ) red ( mutants ; right panel ) . , to illustrate growth of SP W1 with stimulus level .

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G, Averaged click-evoked ABR waveforms from 4 weeks old wt (n=23, green) and mutants (n=34, red), at 50dB sensation level (SL) (left panel). SP and ABR Wave 1 (W1) are indicated by grey areas. Expanded averaged SP and ABR W1 waveform patterns for 10-60dB SL in 10dB increments are plotted in green (wt; middle panel) and red (mutants; right panel)., to illustrate the growth of SP and W1 with stimulus level.


H , I . Mean 2f1 - f2 DPOAE thresholds ( ± SD ) are plotted for wt ( green ) , heterozygote ( blue ) homozygous ( red ) mice aged 4 ( H ) or 21 ( I ) , as a function of f2 frequency .

H, I. Mean 2f1-f2 DPOAE thresholds (± SD) are plotted for wt (green), heterozygote (blue) and homozygous (red) mice aged 4 weeks (H) or 21 weeks (I), as a function of f2 frequency.


A . Pure tone audiogram recorded from one of probands , showing severe to profound bilateral asymmetric hearing loss .

A. Pure tone audiogram recorded from one of the probands, showing severe to profound bilateral asymmetric hearing loss.


B . Agarose gel trace of cDNA obtained from mouse wt P28 inner ear ( IE ) , P28 brain , adult P4 organ of Corti ( OC ) . Results show expression of two Wbp2 isoforms in brain at P28 very faint band for short isoform together with a strong band for long isoform in inner ear at P28 . If we look at just organ of Corti ( adult P4 ) , we observe a strong band for long isoform in P4 OC an almost undetectable band for short isoform , which was not even picked up by sequencing ( see C ) . Wbp2 l : long isoform ( 550 bp ) ; Wbp2 s : short isoform ( 480 bp ) .

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B. Agarose gel trace of cDNA obtained from mouse wt P28 inner ear (IE), P28 brain, adult and P4 organ of Corti (OC). Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28. If we look at just the organ of Corti (adult and P4), we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform, which was not even picked up by sequencing (see C). Wbp2 l: long isoform (550 bp); Wbp2 s: short isoform (480 bp).


D . Western blot showing predominant presence of Wbp2 long isoform in cochlea at P28 , with a short trace of short isoform showing up only when a higher of lysate ( 20μg ) is loaded . Both isoform are absent in Wbp2 - deficient mouse . Gapdh was used as a loading control . Wbp2 l : long isoform ; Wbp2 s : short isoform .

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D. Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28, with a short trace of the short isoform showing up only when a higher concentration of protein lysate (20μg) is loaded. Both isoform are absent in the Wbp2-deficient mouse. Gapdh was used as a loading control. Wbp2 l: long isoform; Wbp2 s: short isoform.


A . At P14 , afferent terminals below IHCs are slightly swollen in mutants ( neurofilament labelling in green , arrowheads to compare ; CtBP2 labels ribbons IHCnuclei in red ) . Scale bars : 10 μm . At P28 , Neurofilament / CtBP2 labelling in organ of Corti of 4 week old Wbp2 - deficient mice littermate controls shows severe swelling of IHCafferent terminals in mutants , especially in 24kHz region ( yellow arrowheads ) . pre - synaptic ribbons do not look as well aligned to terminals in mutants ( arrows ) . At this stage , we also observe swelling of OHCafferent terminals in 24kHz region ( empty arrowheads ) . Scale bars : 5 μm . : IHCnucleus ; p : pillar side ; m : modiolar side . B . of pre - synaptic ribbons per in 8 , 18 24kHz regions , showing no difference between mutants controls at P28 .

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A. At P14, afferent terminals below IHCs are slightly swollen in the mutants (neurofilament labelling in green, arrowheads to compare; CtBP2 labels ribbons and IHCnuclei in red). Scale bars: 10 μm. At P28,Neurofilament/CtBP2 labelling in the organ of Corti of 4 week old Wbp2-deficient mice and littermate controls shows severe swelling of IHCafferent terminals in the mutants, especially in the 24kHz region (yellow arrowheads). The pre-synaptic ribbons do not look as well aligned to the terminals in the mutants (white arrows). At this stage, we also observe swelling of OHCafferent terminals in the 24kHz region (empty arrowheads). Scale bars: 5 μm. ihc: IHCnucleus; p: pillar side; m: modiolar side.B. Counts of pre-synaptic ribbons per IHC in the 8, 18 and 24kHz regions, showing no difference between mutants and controls at P28.


C . of organ of Corti performed at P28 showing swollen afferent terminals below inner outer hair ( arrowheads for comparisons between mutants controls ) . Scale bars : 5 μm .

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C. TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells (arrowheads for comparisons between mutants and controls). Scale bars: 5 μm.


D . Neurofilament / CtBP2 labelling shows swollen retracting terminals ( arrows ) , especially in 24kHz regions in mutants at 8 ( arrows ) . Scale bars : 10μm . E . of pre - synaptic ribbons in 8 , 18 24kHz regions , showing no difference in their number per in mutants compared to littermate controls at 8 .

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D. Neurofilament/CtBP2 labelling shows swollen and retracting terminals (white arrows), especially in the 24kHz regions in the mutants at 8 weeks (arrows). Scale bars: 10μm.E. Counts of pre-synaptic ribbons in the 8, 18 and 24kHz regions, showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks.


A . We arrayed synapse aligned by ascending of post - synaptic site after GluR2 / 3 CtBP2 labelling . This is a composite image made of several synapses taken from a single from a single wt a single hom , representing double labelling experiments performed on 3 mutants 3 controls . Synapses in mutants show abnormal smaller green patches , suggesting reduced expression of GluR2 / 3 AMPA receptor subunits . Scale bar at bottom right : 1μm .

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A. We arrayed synapse images aligned by ascending size of the post-synaptic site after GluR2/3 and CtBP2 labelling. This is a composite image made of several synapses taken from a single IHC from a single wt and a single hom, representing double labelling experiments performed on 3 mutants and 3 controls. Synapses in the mutants show abnormal morphology and smaller green patches, suggesting reduced expression of the GluR2/3 AMPA receptor subunits. Scale bar at bottom right: 1μm.


B - G . of ribbon synapses of wt ( B , arrowheads for synaptic vesicles ) homs ( C - G ) at 4 of , showing a representative array of synaptic in 24 kHz cochlear region of mutants . While in C D ribbons look slightly abnormal in with misplaced synaptic vesicles ( arrowheads in C ) , we also observe orphan post - synaptic densities surrounded by floating synaptic vesicles ( arrowheads in E ) , ribbons with synaptic membranes ( arrow in F ) that have detached from ( asterisk labels original position of synapse before detachment ) are floating in swollen nerve terminal ( F ) , ribbons ( arrow in G ) that are detached from their densities ( asterisk in G ) . Scale bars : B - E : 200 nm ; F , G : 500 nm . nt : nerve terminal ; psd : post - synaptic density ; snt : swollen nerve terminal .

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B-G. TEM images of IHC ribbon synapses of wt (B, arrowheads for synaptic vesicles) and homs (C-G) at 4 weeks of age, showing a representative array of synaptic phenotypes in the 24 kHz cochlear region of mutants. While in C and D the ribbons look slightly abnormal in size with misplaced synaptic vesicles (arrowheads in C), we also observe orphan post-synaptic densities surrounded by floating synaptic vesicles (arrowheads in E), ribbons with synaptic membranes (arrow in F) that have detached from the IHC membrane (the asterisk labels the original position of the synapse before detachment) and are floating in the swollen nerve terminal (F), ribbons (arrow in G) that are detached from their densities (asterisk in G). Scale bars: B-E: 200 nm; F,G: 500 nm. nt: nerve terminal; psd: post-synaptic density; snt: swollen nerve terminal.