Suppression of IFNγ - inducible PD - L1 expression by YAP5SA is compromised in miR - 130a KO A549 . PD - L1 expression were analyzed by qRT - PCR in IFNγ - treated control or miR - 130a KO A549 pre - transduced with lenti - YAP5SA

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

Suppression of IFNγ-inducible PD-L1 expression by YAP5SA is compromised in miR-130a KO A549 cells. PD-L1 expression levels were analyzed by qRT-PCR in IFNγ-treated control or miR-130a KO A549 cells pre-transduced with lenti-YAP5SA virus


Representative immunohistochemical for VGLL4 IRF2BP2 in human non - small lung cancer tissues . score is indicated . Scale bar : 50

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

Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μm


of VGLL4 IRF2BP2 on human non - small lung cancer tissue arrays containing 71 samples was quantified as described in Materials Methods section . Correlation of VGLL4 IRF2BP2 expressions was significant ( Pearson correlation ; R = 0 . 6341 , P < 0 . 001 ) Correlation of VGLL4 PD - L1 expression on human non - small lung cancer tissue arrays containing 71

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

Staining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samples


Kaplan - Meier survival of with high ( n = 74 ) or low ( n = 130 ) VGLL4 mRNA from GSE31210 , a lung cancer data set . Survival curves are calculated according to Kaplan - Meier

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

Kaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method


B , C . Detection of CST at stalled active forks with SIRF in U2OS ( B ) HCT116 ( C ) . To detect CST at stalled forks , were - labeled with EdU for 8 min , followed by 4 mM HU treatment for 3 h . To detect CST at active forks , SIRF was performed without HU treatment . Representative SIRF of CTC1 , STN1 PCNA on normal or stalled forks are shown . Scale bars : 10 µm . Relative SIRF was calculated by normalizing to untreated samples then to their respective EdU - SIRF signals . Mean value in each sample is labeled in graph . N : number of analyzed in each sample .

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

B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample.


Western blot showing STN1 knockdown . shLUC ( siRNA targeting luciferase ) was used as control .

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

Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control.


CST deficiency causes ssDNA accumulation upon fork stalling . HeLa with STN1 knockdown were incubated with 10 µM BrdU for 48 h , followed by 2 mM HU treatment for 3 h , subsequently stained with BrdU antibody under a non - denaturing condition to detect ssDNA . N , number of analyzed in each condition . Relative BrdU was quantitated by Image J .

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

CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Relative BrdU fluorescence intensity was quantitated by Image J.


Scheme of DNA fiber analysis and representative fiber images.


fiber of U2OS with STN1 knockdown its RNAi - resistant Flag - STN1 WT re - expression with without mirin treatment .

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

DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment.


fiber of BJ with STN1 knockdown by two siRNA with without mirin treatment .

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

DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment.


fiber of HCT116 with STN1 knockdown with without mirin treatment .

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

DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment.


fiber of U2OS showing that SMARCAL1 - mediated fork reversal is needed for nascent - stand degradation observed in CST - deficient .

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

DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells.


fiber of U2OS showing that ZRANB3 - mediated fork reversal is needed for nascent - strand degradation observed in CST - deficient .

DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells.


SIRF detection of MRE11 at stalled forks in STN1 - deficient U2OS . Representative SIRF of MRE11 at normal or stalled replication forks are shown . Scale bars : 10 µm .

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

SIRF detection of MRE11 at stalled forks in STN1-deficient U2OS cells. Representative SIRF images of MRE11 at normal or stalled replication forks are shown. Scale bars: 10 µm.


ssDNA accumulation in STN1 - deficient U2OS with without mirin treatment . Representative of native BrdU are shown .

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

ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.


Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human MRE11 .

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

Coomassie blue stained SDS-PAGE gel (15%) of purified human MRE11.


( i ) Scheme shows nuclease activity of MRE11 in degrading 5 ' Cy3 - labeled substrates ( 25 nt + 60 nt with phosphorothioate bonds on both ends ) . ( ii ) 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with indicated of MRE11 with or without Mn2 + cofactor at 37 ° C for 20 min . Reactions were stopped by SDS proteinase K resolved on 27 % denatured polyacrylamide gel . ( iii ) results are graphed , error bars represent standard deviation ( ± SD ) calculated from at least three independent experiments .

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 B-CONTROLLED_VAR O O O B-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

(i) Scheme shows the nuclease activity of MRE11 in degrading 5' Cy3-labeled substrates (25 nt + 60 nt with phosphorothioate bonds on both ends). (ii) The 5' Cy3-labeled substrates (80 nM) were incubated with the indicated concentration of MRE11 with or without the Mn2+ cofactor at 37°C for 20 min. Reactions were stopped by SDS and proteinase K and resolved on 27% denatured polyacrylamide gel. (iii) The results are graphed, and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.


Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human CST ( CTC1 wild - type ∆ 700N ) .

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

Coomassie blue stained SDS-PAGE gel (15%) of purified human CST complex (CTC1 wild- type and ∆700N).


binding activity of wild - type CST Δ700N mutant . 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with indicated concentrations of CST . Samples were analyzed by 0 . 8 % agarose gel .

DNA binding activity of wild-type CST complex and Δ700N mutant complex. The 5' Cy3- labeled substrates (80 nM) were incubated with the indicated concentrations of CST. Samples were analyzed by 0.8 % agarose gel.


MRE11 degradation . 5 ' Cy3 - labeled substrates ( 80 nM ) were pre - incubated with indicated concentrations of CST wild - type ( i ) or ∆ 700N mutant variant ( ii ) at 37 ° C for 5 min . Reactions were completed by adding MRE11 ( 200 nM ) for an additional 20 min incubation then stopped by SDS proteinase K . Samples were resolved in 27 % denatured polyacrylamide gel . results are graphed error bars represent standard deviation ( ± SD ) calculated from at least three independent experiments .

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 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

MRE11 degradation analysis. 5' Cy3-labeled substrates (80 nM) were pre-incubated with indicated concentrations of CST wild-type (i) or ∆700N mutant variant (ii) at 37°C for 5 min. Reactions were completed by adding MRE11 (200 nM) for an additional 20 min incubation and then stopped by SDS and proteinase K. Samples were resolved in 27% denatured polyacrylamide gel. The results are graphed and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.


Deletion of N - terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres . U2OS stably expressing vector ( V ) , WT Myc - CTC1 Myc - ∆ 700N were treated with or without HU co - stained with Myc ( red ) TRF2 ( green ) antibodies . Boxed areas are amplified in inserts to indicate CTC1 / TRF2 colocalization ( yellow ) . Scale bars : 10 μm . of of with > 3 CTC1 / TRF2 colocalization foci were from three independent experiments . In each experiment , > 150 from each sample were analyzed .

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

Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed.


∆ 700N does not affect CST formation . HEK293T were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 HA - TEN1 . Co - IP was performed with Myc antibody to pulldown His6 - STN1 HA - TEN1 . Full - Myc - CTC1 was prone to degradation during immunoprecipitation .

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

∆700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc- CTC1 or Myc-∆700N, His6-STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His6- STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation.


∆ 700N retains RAD51 interaction . HEK293T were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 , HA - TEN1 Flag - RAD51 or vector control ( V ) , treated with HU ( 2 mM , 16 h ) . Co - IP was performed with Flag antibody to pulldown Myc - CTC1 .

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

∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.


fiber of CTC1 depleted U2OS with RNAi - resistant WT or ∆ 700N re - expression . Mean values in each sample are listed at top of graph . N , number of analyzed in each condition , * * *

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

DNA fiber analysis of CTC1 depleted U2OS cells with RNAi-resistant WT or ∆700N re-expression. Mean values in each sample are listed at the top of the graph. N, the number of cells analyzed in each condition, ***


Micronuclei ( MN ) formation in BRCA2 - STN1 - deficient . Co - depletion was achieved by transfecting siBRCA2 in U2OS expressing shSTN1 . Representative DAPI are shown . Arrows point to MN . Scale bars : 20 µm . Average percentages of nuclei containing MN from three independent experiments are presented . In each experiment , > 300 nuclei were analyzed per sample .

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

Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. Co-depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentages of nuclei containing MN from three independent experiments are presented. In each experiment, >300 nuclei were analyzed per sample.


Anaphase bridges ( arrows ) in BRCA2 - STN1 - deficient U2OS . Scale bar : 10 µm . Average percentages of anaphase bridges from three independent experiments are presented .

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

Anaphase bridges (arrows) in BRCA2- and STN1-deficient U2OS cells. Scale bar: 10 µm. Average percentages of anaphase bridges from three independent experiments are presented.


γH2AX induced by BRCA2 knockdown STN1 knockdown in U2OS . Nuclei containing ≥ 5 foci were considered as positive γ - H2AX . Results were from three independent knockdown experiments . In each experiment , > 80 were analyzed per sample .

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

γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells. Nuclei containing ≥ 5 foci were considered as positive γ-H2AX staining. Results were from three independent knockdown experiments. In each experiment, >80 cells were analyzed per sample.


Co - depletion of STN1 BRCA2 significantly impairs replication . Scale bar : 50 µm . Results were from three independent knockdown experiments . In each experiment , > 180 nuclei were analyzed per sample .

O O O O B-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

Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Scale bar: 50 µm. Results were from three independent knockdown experiments. In each experiment, >180 nuclei were analyzed per sample.


Co - depletion of STN1 BRCA2 increases chromosome instabilities . U2OS with siBRCA2 / or shSTN1 knockdown were treated with HU ( 2 mM , 3 h ) . Representative metaphase show aberrant chromosomes ( red arrows ) . Boxed areas are amplified shown at bottom of . Scale bars : 20 µm . Two independent knockdown chromosome spread experiments were performed . N , number of metaphase spreads analyzed in each sample .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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

Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. N, the number of metaphase spreads analyzed in each sample.


SIRF of RAD51 binding to forks . Representative RAD51 SIRF at normal or stalled forks are shown . Scale bars : 10 µm . Two independent treatments were performed scatter plot of RAD51 - bound replication sites from one experiment is shown . Mean values in each sample are listed at top of graph . N , number of analyzed in each sample in one experiment .

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 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

SIRF analysis of RAD51 binding to forks. Representative RAD51 SIRF images at normal or stalled forks are shown. Scale bars: 10 µm. Two independent treatments were performed and scatter plot of RAD51-bound replication sites from one experiment is shown. Mean values in each sample are listed at the top of the graph. N, number of cells analyzed in each sample in one experiment.


RAD51 foci formation in U2OS CTC1 knockdown was rescued by RNAi - resistant WT but not by ∆ 700N . were treated with HU ( 2 mM , 16 h ) stained with anti - RAD51 . Representative are shown . Scale bars : 10 µm . Scatter plot of RAD51 foci number with without HU treatment is shown . Mean values in each condition are listed at top of graph .

B-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 B-CONTROLLED_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 B-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

RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph.


fiber of nascent - strand degradation in U2OS with STN1 RAD51 co - depletion . Three independent treatments were performed results from one biological replicate are shown . N , number of analyzed in each sample in one experiment . Western blots showing knockdown in fiber assays are included .

O O O O O O O O O B-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

DNA fiber analysis of nascent-strand DNA degradation in U2OS cells with STN1 and RAD51 co-depletion. Three independent treatments were performed and results from one biological replicate are shown. N, the number of DNA fibers analyzed in each sample in one experiment. Western blots showing protein knockdown in DNA fiber assays are included.


F Whole - mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury . G Immunostaining of injured hypoglossal nerves for GFP cytochrome c ( Cytc ) at 5 days after injury . H Number of mitochondria in injured axons of Atf3 : BAC2 Tg Rpt3 CKO mice at 5 days after injury ( n = 5 mice per group ) . I Total number of GFP - positive injured motor neurons in Atf3 : BAC2 Tg Rpt3 CKO mice at 5 days after injury ( n = 4 mice per group ) . J Number of GFP - positive mitochondria in soma at 5 days after injury . Data are shown as mean ± s . e . m . ( n = 3 mice ) . Data information : Data are shown as mean ± s . e . m . , * * P = 0 . 0005 in ( H ) , no significant difference ( P = 0 . 8915 ) in ( I ) * P = 0 . 0068 in ( J ) , determined by unpaired t - . Dashed lines reveal hypoglossal nucleus in axon in ( F G ) . Scale bars , 5 μm in ( F ) 2 . 5 μm in ( G ) .

O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole-mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury. G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c (Cytc) at 5 days after injury. H Number of mitochondria in injured axons of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 5 mice per group). I Total number of GFP-positive injured motor neurons in Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 4 mice per group). J Number of GFP-positive mitochondria in soma at 5 days after injury. Data are shown as the mean ± s.e.m. (n= 3 mice). Data information: Data are shown as the mean ± s.e.m., ** P = 0.0005 in (H), no significant difference (P = 0.8915) in (I) and * P = 0.0068 in (J), determined by unpaired t-test. Dashed lines reveal hypoglossal nucleus in axon in (F and G). Scale bars, 5 μm in (F) and 2.5 μm in (G).


C Thionine - stained motor neurons of Atf3 : BAC2 Tg Rpt3 CKO mice at 7 , 14 17 days after hypoglossal nerve axotomy . Arrows indicate loss of motor neurons . D graph showing of surviving motor neurons on injured side compared with that on control side . Data are shown as mean ± s . e . m . ( * * P = 0 . 0002 , * * * P < 0 . 0001 , determined by unpaired t - , n = 5 mice per group at each time point ) . Data information : Dashed lines outline hypoglossal nucleus in XII , hypoglossal nucleus ; cc , central canal . Scale bars , 150 μm in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Thionine-stained motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice at 7, 14 and 17 days after hypoglossal nerve axotomy. Arrows indicate the loss of motor neurons. D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side. Data are shown as the mean ± s.e.m. (** P = 0.0002, *** P < 0.0001, determined by unpaired t-test, n = 5 mice per group at each time point). Data information: Dashed lines outline hypoglossal nucleus in XII, hypoglossal nucleus; cc, central canal. Scale bars, 150 μm in C)


C Hypoglossal motor neurons of Atf3 : BAC2 Tg Rpt3 CKO mice before at 5 days ( 5d ) 28 days ( 28d ) after injury , immunostained by AnkG GFP . Arrows indicate AIS stained by AnkG . Scale bars , 20 μm . D of number of AIS in control ( Cont ) injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 043 , * * P = 0 . 0162 , * * * P = 0 . 008 , * * * * P < 0 . 0001 , determined by one - way ANOVA followed by Tukey post hoc , n . d . , not detected , n = 5 mice for each group . E of AIS in control ( Cont ) injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 0087 , * * P = 0 . 0024 , * * * P = 0 . 0007 , determined by one - way ANOVA followed by Tukey post hoc , n . d . , not detected , n = 5 mice for each 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice before and at 5 days (5d) and 28 days (28d) after injury, immunostained by AnkG and GFP. Arrows indicate the AIS stained by AnkG. Scale bars, 20 μm. D Quantification of the number of the AIS in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.043, ** P = 0.0162, *** P = 0.008, **** P < 0.0001, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group. E Quantification of the AIS length in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.0087, ** P = 0.0024, *** P = 0.0007, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.


B Ubiquitination of AnkG . lysates of COS - 7 expressing indicated truncated AnkG tagged with GST HA - Ubiquitin were pulled down with glutathione Sepharose beads , followed by western blotting with anti - HA anti - GST antibodies .

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

B Ubiquitination assay of AnkG protein. The lysates of COS-7 cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin were pulled down with glutathione Sepharose beads, followed by western blotting with anti-HA and anti-GST antibodies.


C Western blot of hypoglossal nuclei ( left ) nerves ( right ) at 5 days after injury with anti - AnkG , RPT3 GAPDH antibodies . AnkG products of 190 / 270 / 480 kDa are indicated by arrowheads . positions of degraded products by calpain are shown by asterisks at ~ 95 kDa 72 kDa .

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

C Western blot of hypoglossal nuclei (left) and nerves (right) at 5 days after injury with anti-AnkG, RPT3 and GAPDH antibodies. AnkG products of 190/ 270/ 480 kDa are indicated by arrowheads. The positions of the degraded products by calpain are shown by asterisks at ~95 kDa and 72 kDa.


C localization of AnkG GFP - labeled mitochondria in AIS of uninjured MN of Thy1 - Mito Tg mice injured motor neurons of Atf3 : BAC2 Tg Rpt3 CKO mice . D Graphs show GFP AnkG scans corresponding to ( C ) over a 20 μm running from soma to axon . E Graph represents ( % ) of GFP in AIS , compared with that in AIS of Thy1 - Mito mouse ( n = 5 mice per group ) . Data information : For ( E , data are shown as mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in ( E determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . In closed arrowheads denote presence of AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in C ,

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 localization of AnkG and GFP-labeled mitochondria in the AIS of uninjured MN of Thy1-Mito Tg mice and injured motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice. D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to (C) over a 20 μm line running from the soma to the axon. E Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Thy1-Mito mouse (n = 5 mice per group). Data information: For (E, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in (E determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in C,


F Whole - mount observation of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in proximal region of peripheral hypoglossal ( XII ) nerve as shown in ( A ) . Dashed lines outline axon . G Graph shows mitochondrial number in hypoglossal ( XII ) axon ( n = 5 mice ) . Data information : For G , data are shown as mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in G ) determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 5 μm

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole-mount observation of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in the proximal region of peripheral hypoglossal (XII) nerve as shown in (A). Dashed lines outline the axon. G Graph shows mitochondrial number in hypoglossal (XII) axon (n = 5 mice). Data information: For G, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in


I Altered localization of GFP - labeled mitochondria in AIS of injured motor neurons of AnkG shRNA AAV - infected Rpt3 CKO mice at 5 days after injury . J scans of GFP AnkG corresponding to ( I ) . K Graph represents ( % ) of GFP in AIS , compared with that in AIS of Atf3 : BAC2 Tg mouse treated with AnkG shRNA ( n = 5 mice per group ) . Data information : For data are shown as mean ± s . e . m . , * * P = 0 . 0064 in ( K ) determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . In I ) , closed arrowheads denote presence of AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in I )

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

I Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury. J Fluorescent intensity scans of GFP and AnkG corresponding to (I). K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0064 in (K) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in I)


L Thionine - stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury . Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs . Dashed lines outline hypoglossal nucleus . XII , hypoglossal nucleus ; cc , central canal . M of surviving motor neurons on injured side compared with that on control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs ( n = 4 mice per group ) . Data information : For M ) , data are shown as mean ± s . e . m . , * P = 0 . 0405 , * * P = 0 . 003 in ( M ) , determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 100 μm in ( L ) .

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

L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal. M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).


B Whole - mount visualization of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in sciatic nerve . Dashed lines indicate boundary of axon . Scale bar , 5 μm . C Representative kymographs showing behavior of mtCFP in uninjured sciatic nerves of Thy1 - Mito Tg mtGFP in injured sciatic nerves of Atf3 ; BAC2 Rpt3 CKO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole-mount visualization of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in sciatic nerve. Dashed lines indicate the boundary of axon. Scale bar, 5 μm. C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1-Mito Tg and mtGFP in injured sciatic nerves of Atf3;BAC2 and Rpt3 CKO mice.


D - F number of mitochondria ( D ) , of motile mitochondria ( E ) stationary mitochondrial site ( F ) in axons . Data are shown as mean ± s . e . m . * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( D ) , * * P = 0 . 0006 , * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( E ) , determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected , n = 5 mice for each 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F The number of mitochondria (D), percentage of motile mitochondria (E) and stationary mitochondrial site (F) in axons. Data are shown as the mean ± s.e.m. *** P = 0.0001, **** P < 0.0001 in (D), ** P = 0.0006, *** P = 0.0001, **** P < 0.0001 in (E), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected, n = 5 mice for each group.


C Immunostaining of spinal cord in Atf3 : BAC Tg Atf3 ; SOD1 mice at P70 . boxed regions are magnified in lower panels . Arrows indicate GFP - ATF3 - positive motor neurons ( MNs ) . Dashed lines outline edge of gray matter in spinal cord . D ( % ) of GFP - positive MNs ( black bar ) in ChAT - positive MNs of Atf3 : BAC Atf3 ; SOD1 mice at P70 ( n = 5 mice per each group ) . E ( % ) of ATF3 , MMP - 9 or osteopontin ( OPN ) - positive MNs ( black bar ) in GFP - positive MNs of Atf3 ; SOD1 mice at P65 - 70 ( n = 5 - 7 mice per group ) . Data information : For ( D , E , data are shown as mean ± s . e . m . , Scale bars , 50 μm in ( C ; upper panel ) , 20 μm in ( C ; lower )

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 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 B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O 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 Immunostaining of spinal cord in Atf3:BAC Tg and Atf3;SOD1 mice at P70. The boxed regions are magnified in lower panels. Arrows indicate GFP- and ATF3- positive motor neurons (MNs). Dashed lines outline the edge of the gray matter in spinal cord. D The percentage (%) of GFP-positive MNs (black bar) in ChAT-positive MNs of Atf3:BAC and Atf3;SOD1 mice at P70 (n = 5 mice per each group). E The percentage (%) of ATF3, MMP-9 or osteopontin (OPN)-positive MNs (black bar) in GFP-positive MNs of Atf3;SOD1 mice at P65-70 (n = 5-7 mice per group). Data information: For (D, E, data are shown as the mean ± s.e.m., Scale bars , 50 μm in (C; upper panel), 20 μm in (C; lower)


F Immunostaining of AnkG in ChAT - positive spinal MN of Atf3 : BAC SOD1 in GFP - positive spinal MN of Atf3 ; SOD1 mouse at P70 . G of AIS ( n = 5 mice per group ) . Data information : For G , data are shown as mean ± s . e . m . , no significant difference in ( G ) determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 10 μm in (

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunostaining of AnkG in ChAT-positive spinal MN of Atf3:BAC and SOD1 and in GFP-positive spinal MN of Atf3;SOD1 mouse at P70. G Quantification of the AIS length (n = 5 mice per group). Data information: For G, data are shown as the mean ± s.e.m., no significant difference in (G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in (F


H localization of AnkG GFP - labeled mitochondria in uninjured spinal MN of Thy1 - Mito ( without ATF3 induction ) , traumatically sciatic nerve - injured MN of Atf3 : BAC ( with ATF3 induction ) pathologically damaged MN of Atf3 ; SOD1 mice ( with ATF3 induction ) . Closed arrowheads denote presence of AIS , while open arrowheads show disappeared AIS . I scan of GFP AnkG in corresponding to ( H ) . J ( % ) of GFP in AIS , compared with that in AIS of uninjured MNs in Thy1 - Mito mice ( n = 5 mice per group ) . Data information : For data are shown as mean ± s . e . m . , * * P = 0 . 0008 , * * * P = 0 . 0007 in ( J ) , * * determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 5 μm in ( H ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O 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 O O O O O O O O O O O O O O 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 The localization of AnkG and GFP-labeled mitochondria in uninjured spinal MN of Thy1-Mito (without ATF3 induction), traumatically sciatic nerve-injured MN of Atf3:BAC (with ATF3 induction) and pathologically damaged MN of Atf3;SOD1 mice (with ATF3 induction). Closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. I Fluorescent intensity scan of GFP and AnkG in corresponding to (H). J The percentage (%) of GFP intensity in the AIS, compared with that in the AIS of uninjured MNs in Thy1-Mito mice (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0008, *** P = 0.0007 in (J), ** determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in (H).


K Whole - mount observation of mtGFP or mtCFP in sciatic nerve . Dashed lines outline axon . L number of mtGFP or mtCFP in sciatic nerve ( n = 5 mice ) Data information : For L ) , data are shown as mean ± s . e . m . , , * * P = 0 . 0036 , * * * * P < 0 . 0001 in ( L ) , determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 10 μm in K )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole-mount observation of mtGFP or mtCFP in sciatic nerve. Dashed lines outline the axon. L The number of mtGFP or mtCFP in sciatic nerve (n = 5 mice) Data information: For L), data are shown as the mean ± s.e.m., , ** P = 0.0036, **** P <0.0001 in (L), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in K)


B . Whole exome sequencing was carried out revealed a missense p . L165F variant ( c . 493 C > T ) in ALX1 homeodomain , heterozygous in parents ( ALX1165L / 165F ) , wildtype in unaffected sibling ( ALX1165L / 165L ) , homozygous in both affected subjects ( ALX1165F / 165F ) .

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

B. Whole exome sequencing was carried out and analysis revealed a missense p.L165F variant (c.493 C>T) in the ALX1 homeodomain, heterozygous in the parents (ALX1165L/165F), wildtype in the unaffected sibling (ALX1165L/165L), and homozygous in both affected subjects (ALX1165F/165F).


E . Immunofluorescence for pluripotent markers SSEA4 , OCT4 , SOX2 , TRA - 1 - 60 alkaline phosphatase of iPSC clones . One representative iPSC clone is shown for each genotype . Scale bar : 400 µm .

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

E. Immunofluorescence staining for pluripotent markers SSEA4, OCT4, SOX2, and TRA-1-60 and alkaline phosphatase staining of iPSC clones. One representative iPSC clone is shown for each genotype. Scale bar: 400 µm.


F . Expression of pluripotent ( OCT4 , NANOG ) , endoderm ( Endo . , AFP , GATA4 , FOXA2 ) , ectoderm ( Ecto . , NESTIN , GFAP , SOX1 ) , mesoderm ( Meso . , BRACH . ( BRACHYURY ) , RUNX1 , CD34 ) gene markers for ALX1165L / 165L ( green ) , ALX1165L / 165F ( red ) , ALX1165F / 165F ( blue ) iPSC relative to undifferentiated ( UND ) . Data are represented as pooled mean ± of three experiments on three clones from each genotype . Significance : p = 0 . 0167 for OCT4 , p = 0 . 0005 for NANOG , p = 0 . 000004 for AFP , p = 0 . 0082 for GATA4 , p = 0 . 0137 for FOXA2 , p = 0 . 00002 for NESTIN , p = 0 . 0167 for GFAP , p = 0 . 0014 for SOX1 , p = 0 . 0117 for BRACHYURY , p = 0 . 0008 for RUNX1 p = 0 . 0068 for CD34 when comparing undifferentiated differentiated ALX1165L / 165L iPSC . p = 0 . 0013 for OCT4 , p = 0 . 0011 for NANOG , p = 0 . 0000003 for AFP , p = 0 . 0003 for GATA4 , p = 0 . 0063 for FOXA2 , p = 0 . 0001 for NESTIN , p = 0 . 027 for GFAP , p = 0 . 000002 for SOX1 , p = 0 . 000009 for BRACHYURY , p = 3e - 9 for RUNX1 p = 0 . 000006 for CD34 when comparing undifferentiated differentiated ALX1165F / 165L iPSC . p = 0 . 0201 for OCT4 , p = 0 . 006 for NANOG , p = 1x10 - 12 for AFP , p = 5x10 - 13 for GATA4 , p = 0 . 0031 for FOXA2 , p = 0 . 0292 for NESTIN , p = 0 . 00001 for GFAP , p = 6x10 - 7 for SOX1 , p = 0 . 0204 for BRACHYURY , p = 0 . 0009 for RUNX1 p = 0 . 000003 for CD34 when comparing undifferentiated differentiated ALX1165F / 165F iPSC . Data from each clone were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-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 O O 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. Expression of pluripotent (OCT4, NANOG), endoderm (Endo., AFP, GATA4, FOXA2), ectoderm (Ecto., NESTIN, GFAP, SOX1), and mesoderm (Meso., BRACH. (BRACHYURY), RUNX1, CD34) gene markers for ALX1165L/165L (green), ALX1165L/165F(red), and ALX1165F/165F (blue) iPSC relative to undifferentiated cells (UND). Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype. Significance: p=0.0167 for OCT4, p=0.0005 for NANOG, p= 0.000004 for AFP, p=0.0082 for GATA4, p=0.0137 for FOXA2, p=0.00002 for NESTIN, p=0.0167 for GFAP, p=0.0014 for SOX1, p=0.0117 for BRACHYURY, p=0.0008 for RUNX1 and p=0.0068 for CD34 when comparing undifferentiated and differentiated ALX1165L/165L iPSC. p=0.0013 for OCT4, p=0.0011 for NANOG, p=0.0000003 for AFP, p=0.0003 for GATA4, p=0.0063 for FOXA2, p=0.0001 for NESTIN, p=0.027 for GFAP, p =0.000002 for SOX1, p=0.000009 for BRACHYURY, p=3e-9 for RUNX1 and p=0.000006 for CD34 when comparing undifferentiated and differentiated ALX1165F/165L iPSC. p=0.0201 for OCT4, p=0.006 for NANOG, p=1x10-12 for AFP, p=5x10-13 for GATA4, p=0.0031 for FOXA2, p=0.0292 for NESTIN, p=0.00001 for GFAP, p=6x10-7 for SOX1, p=0.0204 for BRACHYURY, p=0.0009 for RUNX1 and p=0.000003 for CD34 when comparing undifferentiated and differentiated ALX1165F/165F iPSC. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.


B . of iPSC iPSC - derived NCC at Days 0 , 14 , passage 4 following differentiation . Scale bars : 400 µm ( Day 0 ) , 200µm ( Day 14 , passage 4 ) .

O O O O O O O O O O O O O O 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. Images of iPSC and iPSC-derived NCC at Days 0, 14, and passage 4 following differentiation. Scale bars: 400 µm (Day 0), 200µm (Day 14, passage 4).


Gene expression across NCC differentiation of unaffected control ALX1165L / 165L ( green ) , heterozygous ALX1165L / 165F ( magenta ) homozygous ALX1165F / 165F iPSC : ALX1 , neural plate border specifier genes ZIC1 , PAX7 , PAX3 , MSX1 , MSX2 , DLX5 ; neural crest specifier genes FOXD3 , P75 , TFAP2A , SNAI2 , TWIST1 ; lineage specifier gene HAND2 . RT - qPCR relative expression values were normalized to RPLP0 GAPDH expression . Data is represented as pooled mean ± of three experiments on three clones from each genotype . Exact p - values are provided in Table EV1 . Data from each clone were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant .

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

Gene expression analysis across NCC differentiation of unaffected control ALX1165L/165L (green), heterozygous ALX1165L/165F (magenta) and homozygous ALX1165F/165F iPSC: ALX1, neural plate border specifier genes ZIC1, PAX7, PAX3, MSX1, MSX2, DLX5; neural crest specifier genes FOXD3, P75, TFAP2A, SNAI2, TWIST1; and lineage specifier gene HAND2. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. Exact p-values are provided in Table EV1. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.


A . Homozygous ALX1165F / 165F NCC ( blue ) showed an increase in sensitivity to apoptosis when compared to control ALX1165L / 165L NCC ( black ) . data on left represents mean of Annexin V positive , indicative of apoptosis , as determined by FACS , with data on right being an example of one such experiment . Apoptosis was induced by immersion in a 55 ° C water bath for ten minutes . Representative experiment for each condition is shown . Data is represented as pooled mean ± of three independent experiments . Data obtained of each clone from three independent experiments were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant . . * : Significantly different from basal apoptosis rate : p = 3x10 - 12 between control NCC basal apoptosis induced apoptosis , between ALX1165F / 165F NCC basal apoptosis induced apoptosis . * * : Significantly different from control NCC ( p = 0 . 0004 ) .

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

A. Homozygous ALX1165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1165L/165L NCC (black). The data on the left represents the mean percentage of Annexin V positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for ten minutes. Representative experiment for each condition is shown. Data is represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. . *: Significantly different from the basal apoptosis rate: p=3x10-12 between control NCC basal apoptosis and induced apoptosis, and between ALX1165F/165F NCC basal apoptosis and induced apoptosis. **: Significantly different from control NCC (p=0.0004).


B . Expression of cyclins CCNA2 ( blue ) CCND1 ( orange ) in NCC at passages 2 3 of ALX1165L / 165L ALX1165F / 165F NCC . RT - qPCR relative expression values were normalized to RPLP0 GAPDH expression . Data is represented as pooled mean ± of three experiments on three clones from each genotype . * : Significantly different from control NCC at passage 2 ( p = 0 . 001 between control ALX1165F / 165F NCC at passage 2 for CCNA2 , p = 0 . 0052 for CCND1 ) . * * : Significantly different from control NCC at passage 3 ( p = 0 . 0494 between control ALX1165F / 165F NCC for CCNA2 , p = 0 . 0008 for CCND1 ) .

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

B. Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1165L/165L and ALX1165F/165F NCC. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. *: Significantly different from control NCC at passage 2 (p=0.001 between control and ALX1165F/165F NCC at passage 2 for CCNA2, p=0.0052 for CCND1). **: Significantly different from control NCC at passage 3 (p=0.0494 between control and ALX1165F/165F NCC for CCNA2, p=0.0008 for CCND1).


C . Activated Sorting ( FACS ) experiments showed that control ALX1165L / 165L NCC ( green ) exhibited increased expression of mesenchymal markers CD90 , CD105 , CD73 with culture time ( passages 1 through 4 ) , whereas homozygous ALX1165F / 165F NCC ( blue ) showed a consistent expression of markers expressed at passage 1 throughout . Further , control ALX1165L / 165L NCC showed a downregulation of CD57 expression with culture time , while ALX1165F / 165F NCC maintained same level of CD57 across passages . Data are presented as mean of positive - stained across passage numbers . Data obtained of each clone from three independent experiments were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC . For CD90 , p = 0 . 0013 at passage 3 p = 0 . 0207 at passage 4 . For CD105 , p = 0 . 0016 at passage 2 , p = 0 . 00004 at passage 3 p = 0 . 0021 at passage 4 . For CD73 , p = 0 . 0060 at passage 2 , p = 0 . 00004 at passage 3 p = 0 . 0114 at passage 4 . For CD57 , p = 0 . 0026 at passage 2 , p = 0 . 000003 at passage 3 p = 0 . 000007 at passage 4 .

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

C. Fluorescence Activated Cell Sorting (FACS) experiments showed that control ALX1165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive-stained cells across passage numbers. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. *: Significantly different from control NCC. For CD90, p=0.0013 at passage 3 and p=0.0207 at passage 4. For CD105, p=0.0016 at passage 2, p=0.00004 at passage 3 and p=0.0021 at passage 4. For CD73, p=0.0060 at passage 2, p=0.00004 at passage 3 and p=0.0114 at passage 4. For CD57, p=0.0026 at passage 2, p=0.000003 at passage 3 and p=0.000007 at passage 4.


A . Mutant ALX1165F / 165F NCC ( blue ) exhibited a migration defect in timed coverage of central clearing of wound when compared with control ALX1165L / 165L NCC ( black ) . Data is presented as recovery of central circular clear of wound by migrating NCC at end of a 24 - hour period . For pictures , were stained in serum free media containing 3 . 6µM CellTracker Green CMFDA ( Life Technologies ) for 30 min at 37 ° C allowed to recover for 30 min before starting experiment . were acquired every 6 hours using a Keyence BZ - X800 microscope . Surface analyses percentages of coverage were measured using ImageJ software ( NIH ) . NCC were monitored over 24 hours . data are represented as average of of closure ± . Scale bar = 200µm . Data obtained of each clone from three independent experiments were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC ( p < 0 . 0001 ) .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mutant ALX1165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L/165L NCC (black). Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24-hour period. For fluorescent pictures, cells were stained in serum free media containing 3.6µM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 hours using a Keyence BZ-X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200µm. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.*: Significantly different from control NCC (p<0.0001).


B . Multiplex of BMP2 BMP9 in supernatant of cultured NCC showed that ALX1165F / 165F NCC ( blue ) secrete less BMP2 more BMP9 compared to control ALX1165L / 165L NCC ( green ) . Data are represented as pooled mean ± of three clones from each genotype . Statistical significance was determined with an ANOVA . A p - value < 0 . 05 was considered significant . * : Significantly different from control NCC ( p = 0 . 0424 for BMP2 p = 0 . 0192 for BMP9 ) .

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

B. Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F/165F NCC (blue) secrete less BMP2 and more BMP9 compared to control ALX1165L/165L NCC (green). Data are represented as pooled mean ± SEM of three clones from each genotype. Statistical significance was determined with an ANOVA test. A p-value <0.05 was considered significant. *: Significantly different from control NCC (p =0.0424 for BMP2 and p=0.0192 for BMP9).


C . Addition of soluble BMP2 or CV2 , a BMP9 antagonist , to culture medium could partially rescue migration defect of ALX1165F / 165F NCC . At beginning of , 10 , 50 , or 100 ng / ml of soluble BMP2 , CV2 , or a combination of two at 100 ng / ml each were added to culture medium , were monitored over next 24 hours . data are represented as average of of closure ± . Scale bar : 400µm .

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

C. Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400µm.


A . Dissected flatmount wild type alx1 - / - zebrafish larvae craniofacial cartilages after Alcian blue , anterior points to left of page in . ventral cartilages appear normal , but alx1 - / - anterior neurocranium ( ANC ) appears narrow , with midline element that is derived from frontonasal NCC being absent . Meckel ' s cartilage ( arrow , MC ) is also diminutive . Scale bar : 200 µm .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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. Dissected flatmount wild type and alx1-/- zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1-/- anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. The Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 µm.


B . Zebrafish alx1 mutants ( blue ) show reduced detectable expression of alx1 but increased expression of alx3 , alx4a compared to wild type controls ( green ) . alx4b expression are similar between wild type alx1 - / - lines . Data is represented as mean of pooled from three different clutches . RT - qPCR relative expression values were normalized to elfa 18S expression using ΔΔCT method . Data from each clutch were pooled mathematical mean was calculated . was used to determine standard error . To statistical significance , an ANOVA was performed . A p - value < 0 . 05 was considered to be statistically significant . Statistical significance denoted by * ; p < 0 . 0001 between WT zebrafish alx1 - / - at measured time points ; at 10 ss , 24 hpf 36 hpf for alx3 ; at 24 hpf 36 hpf for alx4a . Refer to Table EV2 for p - values .

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

B. Zebrafish alx1 mutants (blue) show reduced detectable expression of alx1 but increased expression of alx3, alx4a compared to wild type controls (green). alx4b expression levels are similar between wild type and alx1-/- lines. Data is represented as the mean of all pooled embryos from three different clutches. The RT-qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method. Data from each clutch were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. Statistical significance denoted by *; p<0.0001 between WT zebrafish and alx1-/- at all measured time points; at 10 ss, 24 hpf and 36 hpf for alx3; and at 24 hpf and 36 hpf for alx4a. Refer to Table EV2 for p-values.


C . Dissected flatmount of zebrafish injected with Alx1DN , after Alcian blue . developed an absence of frontonasal derived median portion of anterior neurocranium ( ANC ) a profound hypoplasia of Meckel ' s ventral cartilages . In most severely affected zebrafish , a nearly abrogated ANC was observed . Scale bar : 200 µm .

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

C. Dissected flatmount of zebrafish embryos injected with Alx1DN, after Alcian blue staining. The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium (ANC) and a profound hypoplasia of the Meckel's and ventral cartilages. In the most severely affected zebrafish, a nearly abrogated ANC was observed. Scale bar: 200 µm.


D . Lineage tracing experiments in control Alx1DN mutant revealed aberrant migration of anterior cranial NCC when alx1 is disrupted . In control , anterior cranial NCC always migrate to contribute to median portion of ANC . In contrast , anterior cranial NCC labeled in Alx1DN fail to migrate to median ANC , where ANC structure is narrower labeled cranial NCC are found in an anterior lateral ectopic location ( asterisks ) . Scale bar : 250µm .

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

D. Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted. In the control animal, the anterior cranial NCC always migrate to contribute to the median portion of the ANC. In contrast, the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC, where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location (white asterisks). Scale bar: 250µm.


A Representative picture of carrying pASDOG1 : : LUC sprayed with mock solution , ABA or GA imaged 24 h later . full pictures are shown in Fig EV2 Appendix Fig S1 . Scale bar , 1 cm .

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

A Representative picture of plants carrying pASDOG1::LUC sprayed with mock solution, ABA or GA and imaged 24 h later. The full pictures are shown in Fig EV2 and Appendix Fig S1. Scale bar, 1 cm.


B of emitted per after treatment with ABA GA , compared with mock treatment .

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

B Quantification of emitted light intensity per plant after treatment with ABA and GA, compared with mock treatment.


C Antisense expression is reduced in response to drought conditions . full pictures are shown in Fig EV3 . Scale bar , 1 cm .

O 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 Antisense expression is reduced in response to drought conditions. The full pictures are shown in Fig EV3. Scale bar, 1 cm.


D of emitted per for control drought - treated .

D Quantification of emitted light intensity per plant for control and drought-treated plants.


E Strand - specific RT - qPCR asDOG1 expression of 3 - day Col - 0 ( WT ) drought - treated vs . control .

O O O 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 Strand-specific RT-qPCR asDOG1 expression analysis of 3-day Col-0 (WT) drought-treated vs. control plants.


F asDOG1 expression level ( black ) is reduced while DOG1 sense expression ( grey ) is increased after application of ABA . Leaves of mature Col - 0 ( WT ) 40 - day - old were sprayed with ABA collected 0 , 6 10 h later for extraction RT - qPCR . Signals were normalized against level of UBC transcript . data points are averages for at least three biological replicates are normalized against mock - treated value . Error bars represent standard deviation . * * * * represent t - P - values of < 0 . 05 < 0 . 001 , respectively .

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

F The asDOG1 expression level (black line) is reduced while DOG1 sense expression (grey line) is increased after application of ABA. Leaves of mature Col-0 (WT) 40-day-old plants were sprayed with ABA and collected 0, 6 and 10 h later for RNA extraction and RT-qPCR. Signals were normalized against the level of the UBC transcript. The data points are the averages for at least three biological replicates and are normalized against the mock-treated value. Error bars represent standard deviation.* and *** represent t-test P-values of < 0.05 and < 0.001, respectively.


A Col - 0 ( WT ) dog1 - 3 mutant were either watered normally or subjected to water withdrawal for 5 days then watered again allowed to recover for 2 days . A picture of a representative experiment is shown . Scale bar , 2 cm .

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

A Col-0 (WT) and dog1-3 mutant plants were either watered normally or subjected to water withdrawal for 5 days and then watered again and allowed to recover for 2 days. A picture of a representative experiment is shown. Scale bar, 2 cm.


B of recovered dead Col - 0 ( WT ) , null - 3 dog1 - 4 ; were scored as dead when unable to grow produced offspring after an extended recovery period .

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 Quantification of recovered and dead Col-0 (WT), null-3 and dog1-4 plants; plants were scored as dead when unable to grow and produced offspring after an extended recovery period.


C of DOG1 sense expression in drought - treated vs . control Col - 0 ( WT ) dog1 - 3 mutant .

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

C Analysis of DOG1 sense expression in drought-treated vs. control Col-0 (WT) and dog1-3 mutant plants.


D dog1 - 3 mutant are defective in regulation of majority of tested drought marker genes . Col - 0 ( WT ) dog1 - 3 mutant were subjected to drought for 3 days expression of five marker genes was determined using RT - qPCR . Error bars represent standard deviation , * , * * * * * represent t - P - values of < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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

D dog1-3 mutant plants are defective in the regulation of the majority of the tested drought marker genes. Col-0 (WT) and dog1-3 mutant plants were subjected to drought for 3 days and the expression of five marker genes was determined using RT-qPCR. Error bars represent standard deviation, and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


A expressing LUC fused with full - DOG1 ( pDOG1LUC : : DOG1 ) were sprayed with mock solution or ABA 10 , 20 40 days after germination analysed 24 h after treatment . graphs show mean emitted per .

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

A Plants expressing LUC fused with full-length DOG1 (pDOG1LUC::DOG1) were sprayed with mock solution or ABA 10, 20 and 40 days after germination and analysed 24 h after treatment. The graphs show mean emitted light intensity per plant.


B Representative picture of mock - ABA - treated pDOG1LUC : : DOG1 . full picture is shown in Fig EV2C D . Scale bar , 1 cm .

O O O O O O B-CONTROLLED_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

B Representative picture of mock- and ABA-treated pDOG1LUC::DOG1 plants. The full picture is shown in Fig EV2C and D. Scale bar, 1 cm.


C A truncated construct that lacks DOG1 antisense promoter region ( pDOG1shDOG1 : : LUC ) is not induced by ABA is highly expressed throughout development . graphs show mean emitted per .

O O O O O O B-MEASURED_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

C A truncated construct that lacks the DOG1 antisense promoter region (pDOG1shDOG1::LUC) is not induced by ABA and is highly expressed throughout development. The graphs show mean emitted light intensity per plant.


D Representative picture of mock - ABA - treated pDOG1shDOG1 : : LUC . Scale bar : 1cm .

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

D Representative picture of mock- and ABA-treated pDOG1shDOG1::LUC plants. Scale bar: 1cm.


E RT-qPCR analysis of DOG1 sense mRNA level.


F Strand - specific RT - qPCR of antisense after 10 h of ABA treatment in 10 - , 20 - 40 - day - old Col - 0 .

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

F Strand-specific RT-qPCR analysis of antisense levels after 10 h of ABA treatment in 10-, 20- and 40-day-old Col-0 plants.


G pASDOG1 : : LUC reporter is silenced by application of ABA , while mutation of TATA elements in asDOG1 promoter ( pASDOG1 ∆ TATA : : LUC ) leads to attenuation of expression in presence absence of ABA .

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-CONTROLLED_VAR O

G The pASDOG1::LUC reporter is silenced by the application of ABA, while mutation of TATA elements in the asDOG1 promoter (pASDOG1∆TATA::LUC) leads to attenuation of expression in the presence and absence of ABA.


H Mutation of TATA elements in asDOG1 promoter leads to high - level DOG1 sense expression non - responsiveness to ABA . Error bars represent standard deviation * , * * * * * represent t - P - values of < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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 Mutation of TATA elements in the asDOG1 promoter leads to high-level DOG1 sense expression and non-responsiveness to ABA. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


A expressing truncated asDOG1 promoter - deficient version pDOG1shDOG1 : : LUC are more resistant to drought than expressing full - genomic version pDOG1 : : LUC - DOG1 . Three independent lines are shown for each construct , following 10 days of water deprivation . Scale bar , 2 cm .

O O O O 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

A Plants expressing the truncated asDOG1 promoter-deficient version pDOG1shDOG1::LUC are more resistant to drought than plants expressing the full-length genomic version pDOG1::LUC-DOG1. Three independent lines are shown for each construct, following 10 days of water deprivation. Scale bar, 2 cm.


B RT - qPCR of selected drought marker genes in mock - ABA - treated Col - 0 ( WT ) two independent pDOG1shDOG1 : : LUC transgenic lines . Error bars represent standard deviation * , * * * * * represent t - P - values of < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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

B RT-qPCR quantification of selected drought marker genes in mock- and ABA-treated Col-0 (WT) and two independent pDOG1shDOG1::LUC transgenic lines. Error bars represent standard deviation and *, ** and *** represent t-test P-values of < 0.05, < 0.01 and < 0.001, respectively.


a , LAMP1 + lysosome quantity in NRK ( dotted outline ) starved for hours ( h ) . Error bars show s . e . m . ( n = 3 ) .

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, LAMP1+ lysosome size and quantity in NRK cells (dotted outline) starved for hours (h). Error bars show s.e.m. (n = 3).


b , Starved NRK expressing LAMP1 - YFP show tubules ( arrows ) . Box is expanded on right .

b, Starved NRK cells expressing LAMP1-YFP show tubules (arrows). Box is expanded on the right.


c , Immuno - ( gold marks LAMP1 ) of starved NRK . Continuity ( arrow ) illustrated between autolysosome ( red star ) tubule .

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

c, Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule.


d , LAMP1 - YFP expressing NRK - LC3 as in a . Arrows show reformation tubules . e , Three - dimensional reconstruction of a from d .

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

d, LAMP1-YFP expressing NRK-LC3 cells as in a. Arrows show reformation tubules. e, Three-dimensional reconstruction of a cell from d.


f , Time - lapse ( seconds ) of starved LAMP1 - YFP - expressing NRK .

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

f, Time-lapse images (seconds) of starved LAMP1-YFP-expressing NRK cells.


g , Time - lapse ( minutes ) of starved LAMP1 - PAGFP or LAMP1 - RFP - expressing NRK after photo - activation . Scale bars , 5 µm .

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

g, Time-lapse images (minutes) of starved LAMP1-PAGFP or LAMP1-RFP-expressing NRK cells after photo-activation. Scale bars, 5 µm.


a - c , Starved LAMP1 - YFP NRK stained with Lysotracker or DQ - BSA . c , Right , of purified lysosomes . d , Magnification ( 4 × ) of shown in c . Arrows indicate reformation tubules arrowhead indicates autolysosome .

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

a-c, Starved LAMP1-YFP NRK cells stained with Lysotracker or DQ-BSA. c, Right, TEM of purified lysosomes. d, Magnification (4×) of images shown in c. Arrows indicate reformation tubules and arrowhead indicates autolysosome.


e , f , Immunofluorescence ( e ) immuno - EM ( f ) using LAMP1 antibody of starved LAMP1 - YFPNRKcells shown in c .

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

e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of starved LAMP1-YFPNRKcells shown in c.


g , Density gradient fractions of NRK starved for times shown blotted for LAMP2 or Cathepsin D . Middle panel shows 8 - h fractions 7 9 analysed by . Fraction 10 is gradient top .

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

g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10  is the  gradient top.


h , Starved LAMP1 - PAGFP NRK were photo - activated ( 4 h ) imaged at 12 h with Lysotracker ( upper ) or DQ - BSA ( lower ) . Boxes show enlargements . R ( r ) = Pearson ' s coefficient . Scale bars , 5 µm .

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

h, Starved LAMP1-PAGFP NRK cells were photo-activated (4 h) and imaged at 12 h with Lysotracker (upper) or DQ-BSA (lower). Boxes show enlargements. R(r) = Pearson's coefficient. Scale bars, 5 µm.


a , ( bottom ; scale bar , 5 µm ) ( top ; scale bar , 10 µm ) of NRK - LC3 starved for times shown with enlargements ( boxes ) .

O O O O O O O O O O O O O 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, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes).


b , d , Immunoblots of shown in a with indicated antibodies ( d , after transfection with non - specific ( NS ) or atg5 RNAi ) . c , Autophagy ( black lines show independent experiments ) phospho - S6K ( densitometry of data shown in b , red ) .

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

b, d, Immunoblots of cells shown in a with indicated antibodies (d, after transfection with non-specific (NS) or atg5 RNAi). c, Autophagy (black lines  show  independent experiments) and phospho-S6K percentage (densitometry of data shown in b, red line).


e - g , LAMP1 - YFP NRK - LC3 starved for 2 h then treated with 100 nM rapamycin for another 4 h ( total , 6 h ) or 6 h ( total , 8 h ) analysed by blotting ( e ) as in b or microscopy ( f ; scale bar , 5 µm ) with ( g ) . Error bars , s . e . m . ; n = 3 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-g, LAMP1-YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.


a , LAMP1 / LC3NRK were starved for 4 h treated with GTPγS for 6 h of further starvation .

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

a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation.


b , Rab7Q67L - GFP - transfected LC3 - CFP / Lamp1 - RFP NRK starved for 10 h .

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

b, Rab7Q67L-GFP-transfected LC3-CFP/Lamp1-RFP NRK cells starved for 10 h.


c , Rab7 - CFP NRK starved for 2 h treated with 100 nM rapamycin for 8 h of further starvation .

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

c, Rab7-CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation.


d , LAMP1 - YFP NRK - LC3 starved for times shown with 1 µg ml - 1 leupeptin blotted with indicated antibodies . e , in d imaged . .

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

d, LAMP1-YFP NRK-LC3 cells starved for times shown with 1 µg ml-1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged..


f , Fibroblast from with Scheie syndrome ( GM01256 ) or healthy controls starved blotted with indicated antibodies . g , in f imaged . Lysosome classified by Image - ProPlus ( IPP ) software .

O O O O O O O O O O O O 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, Fibroblast cells from patients with Scheie syndrome (GM01256) or healthy controls starved and blotted with indicated antibodies. g, Cells in f imaged. Lysosome size classified by Image-ProPlus (IPP) software.


A Representative show expression of YAP in normal cervical tissues , early - stage cervical cancer ( stage I II ) , advanced - stage cervical cancer ( stage III IV ) . Note significant increase in YAP immunosignals ( brown ) in advanced - stage tumors . Tissue : 1 . 5 mm . B Positivity ( of positively stained relative to total number of in tissue section ) of YAP immunosignals in normal cancerous tissues . Data were analyzed with unpaired t - in GraphPad Prism 5 with Welch ' s correction . Bars represent means ± ( n = 10 for normal tissues ; n = 69 for tissues ; * * * P 0 . 0001 ) .

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

A Representative images show expression of YAP in normal cervical tissues, early-stage cervical cancer (stage I II), and advanced-stage cervical cancer (stage III IV). Note the significant increase in YAP immunosignals (brown staining) in the advanced-stage tumors. Tissue core size: 1.5 mm.B Positivity (percentage of positively stained cells relative to the total number of cells in the tissue section) of YAP immunosignals in the normal and cancerous tissues. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Bars represent means ± SEM (n = 10 for normal tissues; n = 69 for tumor tissues; ***P 0.0001).


C A representative image showing expression of YAP ( brown ) in normal cervical tissue . Scale bar = 20 μm . D - F Representative showing expression of YAP in ( D ) early - stage cervical cancer tissue ( stage Ib , T1N0M0 ) ; ( E ) medium - stage cervical cancer tissue ( stage IIb , T2N0M0 ) ; ( F ) advanced - stage cervical cancer tissue ( stage IIIb , T3N1M0 ) . Scale bar = 20 μm . G , H High - resolution showing location of YAP in ( G ) early - stage cervical cancer tissue ( H ) advanced - stage cervical cancer tissue . Scale bar = 20 μm .

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-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-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

C A representative image showing the expression of YAP (brown staining) in normal cervical tissue. Scale bar = 20 μm.D-F Representative images showing the expression of YAP in (D) early-stage cervical cancer tissue (stage Ib, T1N0M0); (E) medium-stage cervical cancer tissue (stage IIb, T2N0M0); and (F) advanced-stage cervical cancer tissue (stage IIIb, T3N1M0). Scale bar = 20 μm.G, H High-resolution images showing the cellular location of YAP in (G) early-stage cervical cancer tissue and (H) advanced-stage cervical cancer tissue. Scale bar = 20 μm.


I Multidimensional cancer genomics data results showing cross - cancer YAP gene alteration . histogram showed frequencies of YAP gene mutation , deletion , amplification across cancers . Data were extracted from TCGA database analyzed using cBioPortal online analyzing tools . results indicate that highest alteration frequency of YAP gene occurs in cervical cancer .

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 B-MEASURED_VAR O O O O O O

I Multidimensional cancer genomics data analysis results showing cross-cancer YAP gene alteration. The histogram showed the frequencies of YAP gene mutation, deletion, and amplification across cancers. Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools. The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer.


A , C , E Western blot showing of YAP phosphorylated YAP in ME180 lines [ ME180 - MXIV ( control ) , ME180 - YAP , ME180 - YAPS127A ] ( A ) ; HT3 lines [ HT3 - MXIV ( control ) , HT3 - YAP , HT3 - YAPS127A ] ( C ) ; Ect1 lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , Ect1 - YAPS127A ] ( E ) . β - Actin was used as a loading control .

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

A, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.


B , D , F Growth curves of YAP - overexpressed ME180 lines [ ME180 - MXIV ( control ) , ME180 - YAP , ME180 - YAPS127A ] ( B ) ; HT3 lines [ HT3 - MXIV ( control ) , HT3 - YAP , HT3 - YAPS127A ] ( D ) ; Ect1 lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , Ect1 - YAPS127A ] ( F ) . Each point represents mean ± ( n = 4 ) . * * * P < 0 . 0001 , ME180 - MXIV vs . ME180 - YAP ME180 - MXIV vs . ME180 - YAPS127A on day 8 . * * * P < 0 . 0001 , HT3 - MXIV vs . HT3 - YAP HT3 - MXIV vs . HT3 - YAPS127A on day 8 . * * * P < 0 . 0001 , Ect1 - MXIV vs . Ect1 - Ect1 - YAPS127A on day 4 . * P < 0 . 0074 , Ect1 - MXIV vs . Ect1 - YAP on day 4 .

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

B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.


G Western blot showing YAP in non - targeting control siRNA ( siCtrl ) - YAP siRNA ( siYAP ) - transfected HT3 .

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

G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.


H Proliferation of HT3 treated with control ( siCtrl ) or YAP siRNA ( siYAP ) . Each point represents mean ± ( n = 5 ) . * * * P < 0 . 001 compared with siCtrl ( siCtrl vs . siYAP , P = 0 . 0002 ) .

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

H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).


A Representative showing colonies formed by Ect1 - MXIV , Ect1 - YAP , Ect1 - YAPS127A after growth in soft agar for 9 days . Scale bar : 500 μm . B Quantitative data showing colony formation in Ect1 - MXIV , Ect1 - YAP , Ect1 - YAPS127A . Each bar represents mean ± of five independent experiments . Bars with different letters are significantly different from each other ( Ect1 - MXIV vs . Ect1 - YAP , P = 0 . 0003 ; Ect1 - MXIV vs . Ect1 - YAPS127A , P = 0 . 0002 ) .

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

A Representative images showing colonies formed by Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells after growth in soft agar for 9 days. Scale bar: 500 μm.B Quantitative data showing colony formation in Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells. Each bar represents mean ± SEM of five independent experiments. Bars with different letters are significantly different from each other (Ect1-MXIV vs. Ect1-YAP, P = 0.0003; Ect1-MXIV vs. Ect1-YAPS127A, P = 0.0002).


C , E Representative showing anchorage - independent growth of YAP - expressing HT3 ( C ) ME180 ( E ) cervical cancer lines . Anchorage - independent growth was determined by soft agar colony formation . Scale bar : 500 μm . D , F - quantitative showing differences of anchorage - independent growth in HT3 - MXIV , HT3 - YAP , HT3 - YAPS127A ( D ) ME180 - MXIV , ME180 - YAP , ME180 - YAPS127A ( F ) . anchorage - independent growth was determined using a CytoSelect 96 - Well Transformation kit , data are presented as relative units ( RFU ) . Each bar represents mean ± of four independent experiments . Bars with different letters are significantly different from each other ( HT3 - MXIV vs . HT3 - YAP , P = 0 . 0002 ; HT3 - MXIV vs . HT3 - YAPS127A , P < 0 . 0001 ; ME - MXIV vs . ME - YAP , P = 0 . 0011 ; ME - MXIV vs . ME - YAPS127A , P = 0 . 0013 ) .

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

C, E Representative images showing the anchorage-independent growth of YAP-expressing HT3 (C) and ME180 (E) cervical cancer cell lines. Anchorage-independent cell growth was determined by the soft agar colony formation assay. Scale bar: 500 μm.D, F Fluorescence-based quantitative analysis showing the differences of anchorage-independent growth in HT3-MXIV, HT3-YAP, and HT3-YAPS127A (D) and ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells (F). The anchorage-independent cell growth was determined using a CytoSelect 96-Well Cell Transformation Assay kit, and data are presented as relative fluorescent units (RFU). Each bar represents mean ± SEM of four independent experiments. Bars with different letters are significantly different from each other (HT3-MXIV vs. HT3-YAP, P = 0.0002; HT3-MXIV vs. HT3-YAPS127A, P < 0.0001; ME-MXIV vs. ME-YAP, P = 0.0011; ME-MXIV vs. ME-YAPS127A, P = 0.0013).


A , B Representative of tumorxenografts of ME180 - MXIV ( MX , right flank ) , ME180 - YAP ( A ) ( YAP , left flank ) , ME180 - YAPS127A ( B ) ( YAPS127A , left flank ) implanted in athymic nude mice . Red lines indicate edge of tumors . C growth curve of humantumorxenografts derived from control ( ME - MX ) YAP - overexpressing ME180cervical cancer lines ( ME - YAP , ME - YAPS127A ) implanted in athymic nude mice . Each point represents mean ± of six tumors ( control n = 10 ) .

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

A, B Representative images of tumorxenografts of ME180-MXIV (MX, right flank), ME180-YAP (A) (YAP, left flank), and ME180-YAPS127A (B) (YAPS127A, left flank) cells implanted in athymic nude mice. Red lines indicate the edge of tumors.C The growth curve of humantumorxenografts derived from control (ME-MX) and YAP-overexpressing ME180cervical cancer cell lines (ME-YAP, ME-YAPS127A) implanted in the athymic nude mice. Each point represents mean ± SEM of six tumors (control n = 10).


D Representative showing relative of tumors from control YAP - overexpressing ME180 cervical cancer . E average of xenografts from control YAP - overexpressing ME180 lines . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc . Each bar represents mean ± ( n = 10 for control , n = 6 for YAP YAPS127A groups ) . Bars with different letters are significantly different from each other ( MXIV vs . YAP , P = 0 . 0008 ; MXIV vs . YAPS127A , P < 0 . 0001 ) .

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

D Representative images showing the relative size of tumors from control and YAP-overexpressing ME180 cervical cancer cells.E The average weight of tumor xenografts from the control and YAP-overexpressing ME180 cell lines. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents the mean ± SEM (n = 10 for control, n = 6 for YAP and YAPS127A groups). Bars with different letters are significantly different from each other (MXIV vs. YAP, P = 0.0008; MXIV vs. YAPS127A, P < 0.0001).


F Western blot of YAP in xenografts derived from ME180 - MXIV , ME180 - YAP , ME180 - YAPS127A .

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

F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells.


G Expression of Ki67 in xenografts of ME180 - MXIV , ME180 - YAP , ME180 - YAPS127A implanted in athymic nude mice . Ki67 was visualized using an Alexa - 488 ( green ) - conjugated secondary antibody . Actin was visualized using an Alexa - 594 ( red ) - conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 20 μm . Note significant increase in Ki67 - positive in ME180 - YAP ME180 - YAPS127A xenografts .

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

G Expression of Ki67 in the tumor xenografts of ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells implanted in athymic nude mice. Ki67 was visualized using an Alexa-488 (green)-conjugated secondary antibody. Actin was visualized using an Alexa-594 (red)-conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 20 μm. Note the significant increase in the Ki67-positive cells in the ME180-YAP and ME180-YAPS127A tumor xenografts.


H Western blot showing YAP in ME180 transfected with lentiviral empty vector ( shCtrl ) or lentivirus - YAP shRNAs ( shYAP # 1 or shYAP # 2 ) .

O O O O B-CONTROLLED_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

H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector (shCtrl) or lentivirus-based YAP shRNAs (shYAP#1 or shYAP#2).