Co - IP from siRNA - transfected HUVECs . graph shows relative of PlexinD1 - VEGFR2 association , in which values at 0 min are set to 1 . n = 3 per group .

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

Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.


Super - resolution of receptor colocalization in siRNA - transfected HUVECs 30 min after Sema3E stimulation . Left panels show immunolabeling of VEGFR2 ( green ) , PlexinD1 ( red ) , Nrp1 ( ) , with - blue asterisks indicating nuclei . Middle panels show magnified views of boxes in left panels . Right panels represent profiles along 5 μm arbitrary lines in middle panels . Scale bar , 10 µm . graph shows proportion of receptor complexes in PlexinD1 - positive vesicles . n = 10 per group .

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

Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light-blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in PlexinD1-positive vesicles. n = 10 cells per group.


Trajectory plots ( left ) rose plots ( right ) of HUVECs pre - treated with p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h . Red trajectories indicate with displacement to negative y - axis at end of analyses . P values for Rayleigh represent non - random distributions of endpoints . graphs show FMI along y - x - axes during migration . n = 90 per group .

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

Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.


Super - resolution of receptor colocalization in siRNA - transfected HUVECs 30 min after VEGF stimulation . VEGFR2 ( green ) , PlexinD1 ( red ) , Nrp1 ( ) are shown as in Fig 4E . Scale bar , 10 µm . graph shows proportion of receptor complexes in PlexinD1 - positive vesicles . Data of " no stimuli " are same from Fig 4E . n = 10 per group .

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

Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after VEGF stimulation. VEGFR2 (green), PlexinD1 (red), and Nrp1 (white) are shown as in Fig 4E. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in the PlexinD1-positive vesicles. Data of "no stimuli" are the same from Fig 4E. n = 10 cells per group.


Immunoblots in siRNA - transfected HUVECs . Blots for β - actin shown in ( C ) were used to confirm equal loading in ( D ) , as same lysates were analyzed in these experiments . graph shows changes of total VEGFR2 as a of values at 0 min . n = 3 per group .

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

Immunoblots in siRNA-transfected HUVECs. Blots for β-actin shown in (C) were used to confirm equal loading in (D), as the same cell lysates were analyzed in these experiments. The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min. n = 3 per group.


Immunoblots in siRNA - transfected HUVECs . Note early transient activation of PLCγ , Erk1 / 2 , Akt , but not p38 MAPK , in RhoJ - knockdown ECs .

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

Immunoblots in siRNA-transfected HUVECs. Note the early and transient activation of PLCγ, Erk1/2, and Akt, but not p38 MAPK, in RhoJ-knockdown ECs.


Immunoblots for ectopic RhoJ endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ - expressing vectors . graph shows of VEGFR2 relative to those of control set as 100 % . n = 3 per group .

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

Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ-expressing vectors. The graph shows protein levels of VEGFR2 relative to those of the control set as 100%. n = 3 per group.


Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model . Upper panels are snapshots of time - lapse imaging in which EC nuclei are labeled by SYTO dye ( green ) . In analyzed regions framed by dotted lines , centroids of first five EC nuclei from tip at 0 min are pseudocolored in magenta those of remaining EC nuclei are shown in cyan . Initial discrete labeling becomes intermixed over time ( see also Movie EV1 ) . Lower panels show trajectory analyses representing normalized positional changes of ECs during vessel elongation . Scale bar , 100

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

Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µ


of kinetics of EC migration . Values of forward reverse migration for RhojWT / WT are normalized to 1 . n = 33 vessel branches per group .

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

Quantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group.


of vessel elongation ( left graph ) . n = 33 vessel branches per group . Scatter plot shows relationship between " vessel elongation " " reverse migration " in RhojGFP / GFP aortic rings as evaluated by a Pearson 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 O O B-CONTROLLED_VAR O O O O O O O O O O O O

Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient.


Labeling for YFP / GFP ( green ) , CD31 ( red ) , ETS transcription factor ERG ( blue ) in retinal vascular fronts of P5 CAG - MerCreMer : R26R - EYFPflox / WT CAG - MerCreMer : Rhojfloxflox mice after intraperitoneal ( i . p . ) injections of 10 µg of 4 - hydroxytamoxifen ( 4OHT ) at P1 . Scale bar , 50 µm . Relative contribution of YFP / GFP - positive ECs to tip positions . n = 16 per group . Values for CAG - MerCreMer : R26R - EYPflox / WT are normalized to 1 .

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

Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.   Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.


Labeling for GFP ( green ) CD31 ( red ) in retina of P4 RhojGFP / GFP mouse . Scale bar , 200 µm ( left ) ; 20 µm ( right ) .

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

Labeling for GFP (green) and CD31 (red) in retina of P4 RhojGFP/GFP mouse. Scale bar, 200 µm (left); 20 µm (right).


Labeling for EdU ( green ) , ERG ( red ) , CD31 ( gray ) in retinas of P4 RhojWT / WT RhojGFP / GFP mice 2 h after i . p . EdU injections . Scale bar , 20 µm . graph shows EC proliferation index in retinal capillaries behind angiogenic fronts . n = 12 per group .

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

Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.


Labeling for CD31 in retinas of P4 P7 RhojWT / WT RhojGFP / GFP mice . Scale bars , 500 µm .

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

Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.


Morphometric analyses of retinal vessels in RhojWT / WT ( P4 , n = 18 ; P7 , n = 8 ) RhojGFP / GFP ( P4 , n = 16 ; P7 , n = 11 ) mice .

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

Morphometric analyses of retinal vessels in RhojWT/WT (P4, n = 18; P7, n = 8) and RhojGFP/GFP (P4, n = 16; P7, n = 11) mice.


Labeling for CD31 in retinas of P4 Rhojflox / flox Pdgfb - iCreERT2 : Rhojflox / flox ( RhojiΔEC ) mice after single i . p . injection of 100 µg of 4OHT at P1 . Scale bar , 500 µm .

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

Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.


Morphometric analyses of retinal vessels in P4 Rhojflox / flox ( n = 7 ) Rhoji ∆ EC ( n = 6 ) mice .

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

Morphometric analyses of retinal vessels in P4 Rhojflox/flox (n = 7) and Rhoji∆EC (n = 6) mice.


Relative expression of Vegfa Sema3e mRNA from WT mouse retinas . n = 3 per group .

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

Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas. n = 3 per group.


Labeling for GFP ( green ) CD31 ( red ) in retina of P18 OIR - RhojGFP / WT mouse . Scale bar , 200

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

Labeling for GFP (green) and CD31 (red) in retina of P18 OIR-RhojGFP/WT mouse. Scale bar, 200 µ


Labeling for CD31 in retinas of P18 OIR - Rhojflox / flox OIR - Rhoji ∆ EC mice after daily i . p . injection of 200 µg of 4OHT from P12 . Scale bar , 500 µm . of areas of neovascular tufts in P18 OIR - Rhojflox / flox OIR - Rhoji ∆ EC mice . n = 6 per group .

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

Labeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm. Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.


( A ) Western blot of Beclin ‐ 1 FACS profiles following siRNA treatment in HeLa stably expressing a HA ‐ Beclin ‐ 1 construct resistant to siRNA targeting endogenous Beclin ‐ 1 .

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

(A) Western blot of Beclin‐1 and FACS profiles following siRNA treatment in HeLa cells and cells stably expressing a HA‐Beclin‐1 construct resistant to the siRNA targeting endogenous Beclin‐1.


( B ) FACS profiles of treated with indicated siRNA following nocodazole blockage release .

(B) FACS profiles of cells treated with indicated siRNA following nocodazole blockage and release.


( C , D ) of in mitosis ( C ) of each step of mitosis ( D ) by H3Ser10P for Control Beclin ‐ 1 siRNA ‐ treated . Error bars represent standard deviations ( s . d . ) ( n = number of counted ) calculated on three independent experiments . Two three asterisks indicate significant results ( P = 0 . 0028 P = 0 . 0006 , respectively ) .

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

(C,D) Quantification of cells in mitosis (C) and quantification of each step of mitosis (D) by H3Ser10P staining for Control and Beclin‐1 siRNA‐treated cells. Error bars represent standard deviations (s.d.) (n=number of counted cells) calculated on three independent experiments. Two and three asterisks indicate significant results (P=0.0028 and P=0.0006, respectively).


( E ) Knockdown of Beclin ‐ 1 increases number of mitotic with misaligned chromosomes . siRNA ‐ treated HeLa were fixed in PFA , immunolabelled for α ‐ tubulin ( green ) stained with DAPI ( blue ) . arrows indicate misaligned chromosomes . of with misaligned chromosomes is shown at right ( n = number of counted ) . Three asterisks indicate significant results ( P = 0 . 0001 ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; HA , haemagglutinin ; PFA , paraformaldehyde ; siRNA , small interfering .

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

(E) Knockdown of Beclin‐1 increases the number of mitotic cells with misaligned chromosomes. siRNA‐treated HeLa cells were fixed in PFA, immunolabelled for α‐tubulin (green) and stained with DAPI (blue). White arrows indicate misaligned chromosomes. Quantification of cells with misaligned chromosomes is shown at the right (n=number of counted cells). Three asterisks indicate significant results (P=0.0001). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; HA, haemagglutinin; PFA, paraformaldehyde; siRNA, small interfering RNA.


Beclin ‐ 1 depletion prolongs prometaphase by delaying chromosome congression . ( A - H ) Time ‐ lapse films of HeLa stably expressing YFP ‐ α ‐ tubulin H2B ‐ Cherry treated with indicated siRNA . pictures are acquired every 10 min . Time lapse starts at envelope breakdown . ( A ) Mitosis in control . ( B ) Western blot of whole ‐ lysates from Control , Beclin ‐ 1 Beclin ‐ 1 + Mad2 siRNA ‐ treated . ( C - E ) Three different classes of mitotic progression were observed upon Beclin ‐ 1 depletion . Higher magnification of lagging chromatids in anaphase of Beclin ‐ 1 ‐ depleted is shown on right of ( D ) .

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

Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A-H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C-E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D).


( F ) prometaphase delay is dependent on MAD2 spindle checkpoint . treated simultaneously with siRNA targeting MAD2 Beclin ‐ 1 rapidly exit mitosis . ( G ) Average duration of mitosis in different classes of Beclin ‐ 1 ‐ depleted , compared to control MAD2 depletions . n = number of counted in three independent experiments . Error bars on graphs represent s . d .

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

(F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d.


( H ) frequency of different Beclin ‐ 1 depended on efficiency of Beclin ‐ 1 knockdown . Mitotic progression in 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 : of appearance of three different mitotic progression patterns , that is , prolonged mitosis with normal anaphase ( as C ) , with lagging chromatids ( as D ) mitotic exit without clear anaphase ( as E ) upon increased Beclin ‐ 1 knockdown experiments . NEB , envelop breakdown ; RNAi , interference ; siRNA , small interfering ; YFP , yellow .

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

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

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

(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 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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR 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 ) .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

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 ) .

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

D, 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 .

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

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 .

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

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 ) .

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

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 .

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

(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 .

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

(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 .

O O O O O O O O O O O O O O O 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). 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 .

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

(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 .

O O O O O O 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) 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 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 B-CONTROLLED_VAR 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 ) .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 ) .

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

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 .

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

(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 .

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

(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 .

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

(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 .

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

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

(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 .

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

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


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

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O 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) 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 .

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 B-CONTROLLED_VAR 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

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

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 .

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

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 ) .

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

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 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-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

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 .

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

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

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

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 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

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

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

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

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 .

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

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 .

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

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 .

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

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 .

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

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

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 ) .

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

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 O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 .

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