( H ) The frequency of the different Beclin ‐ 1 phenotypes depended on the efficiency of Beclin ‐ 1 knockdown . Mitotic progression in cells treated with increasing concentrations of Beclin ‐ 1 siRNA was followed by video ‐ microscopy . Left panel : western blot of Beclin ‐ 1 upon RNAi treatment ; right panel : quantification of the appearance of the three different mitotic progression patterns , that is , prolonged mitosis with normal anaphase ( as C ) , with lagging chromatids ( as D ) and mitotic exit without clear anaphase ( as E ) upon increased Beclin ‐ 1 knockdown experiments . NEB , nuclear envelop breakdown ; RNAi , RNA interference ; siRNA , small interfering RNA ; YFP , yellow fluorescent protein .

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(H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.


( A ) Inter ‐ kinetochore distance is reduced in unaligned chromosomes of Beclin ‐ 1 ‐ depleted cells . The distance between each kinetochore pair was quantified in siRNA ‐ treated and control cells treated with nocodazole for 3 h before fixation . Cells were stained with anti ‐ CREST antibody and DAPI . Quantification of the distance between kinetochore pairs ( indicated by coloured bars ) is reported at the bottom and done on sister kinetochores present in the same focal plane . One asterisk indicates significant result ( P = 0 . 0365 ) . Graphs represent three independent experiments ( error bars = s . d . ) .

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(A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.).


( 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 .

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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 and CREST . Two asterisks indicate significant results , P = 0 . 0053 .

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


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

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


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

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


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

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


( 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 .

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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 ‐ cell lysates from control and Beclin ‐ 1 siRNA ‐ treated cells . siRNA , small interfering RNA .

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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 cell extracts expressing HA ‐ Beclin ‐ 1 or HA ‐ GFP . Input is 3 . 5 % of the 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 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 .

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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 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 . ) .

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(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 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 ) .

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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 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 .

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(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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 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 ) .

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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 fluorescence , statistical significance was analyzed by Anova test ( 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 ) and MHC ( red ) revealing reconstructed muscle innervation ( asterisks ) . B ) Superimposed image in ( a ) over LacZ stained image .

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


C ) Syn and 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 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 ) .

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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 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 .

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(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 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 .

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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 ) cells were the 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 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 .

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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 fluorescence intensity at the transcription site for 5 ' PP7 and 3 ' MS2 at GAL3 .

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


( 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 .

(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 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 .

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(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 ) 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 ) .

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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 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 .

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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 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 .

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(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 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 .

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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 and 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 DNA 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 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 .

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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 ) 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 .

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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 DNA containing UASwt and 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 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 .

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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 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 ) .

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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 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 .

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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 cell , showing the GAL10 TS ( arrow , left panel ) , single molecules of Gal4 ( arrows , middle panel ) and 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 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 .

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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 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 .

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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 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 .

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(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 .

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


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

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


( 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 .

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(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 . The fit of 2 linear lines reveals a burst duration of 152 . 4 ± 2 . 3s . n = 19 traces . Shaded area and errors indicate SEM .

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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 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 .

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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 the different signal durations of Gal4 binding and GAL10 transcription . Gal4 binding overlaps with the 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 .

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


( 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 ) .

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(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 ) 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 .

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(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 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 .

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(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 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 .

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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 . 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 .

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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 . 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 .

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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 . 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 ) .

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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 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 .

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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 images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes .

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 . 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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

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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 the patients and corresponding

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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 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

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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 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 %

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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%


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

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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

TSS enrichment score of primary samples (PRI) and PDXs


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

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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 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 )

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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 primary and PDX samples plotted against the mean of normalized read counts per peak , red - differentially accessible regions ( p < 0 . 05 ) as analyzed by

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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 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 .

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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 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 .

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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.


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 .

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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.


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 .

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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.


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

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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 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 .

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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 ) - 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 ) .

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

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


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

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


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

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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 ) 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 .

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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 ) 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 .

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

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α protein ( F ) by western

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 images and quantification of 2D capillary network formation assays on HUVECs subjected to the 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 RNA editing of the LIMD1 ( A ) and VHL ( B ) transcripts .

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


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 .

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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 analysis was done on the fractionated MCF7 cells with the 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 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 .

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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.