TDU - deleted or HF4A VGLL4 mutations interacted with IRF2BP by co - immunoprecipitation analysis in HEK293T

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TDU-deleted or HF4A VGLL4 mutations interacted with IRF2BP by co-immunoprecipitation analysis in HEK293T cells


TDU - deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co - immunoprecipitation analysis in HEK293T

TDU-deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co-immunoprecipitation analysis in HEK293T cells


Expression of VGLL4 - WT or VGLL4 - HF4A enhances PD - L1 expression in A549 cells by immunoblot analysis . A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated

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Expression of VGLL4-WT or VGLL4-HF4A enhances PD-L1 expression in A549 cells by immunoblot analysis. A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodies


VGLL4 - HF4A rescues PD - L1 expression in VGLL4 - knockdown A549 cells . A549 cells were transfected with siRNA targeting to the 3 " UTR of VGLL4 mRNA and followed by transduction with lenti - VGLL4 - HF4A virus . Cell lysates were analyzed by western blot with indicated

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VGLL4-HF4A rescues PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with siRNA targeting to the 3"UTR of VGLL4 mRNA and followed by transduction with lenti-VGLL4-HF4A virus. Cell lysates were analyzed by western blot with indicated antib


Expression of VGLL4 - HF4A rescues tumor growth of VGLL4 - knockdown LLC cells in C57BL / 6 mice . Control , Vgll4 knockdown or Vgll4 knockdown together with VGLL4 - HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice . Tumor volumes were measured 15 days for nude mice and 21 days for C57BL / 6 mice after tumor cell

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Expression of VGLL4-HF4A rescues tumor growth of VGLL4-knockdown LLC cells in C57BL/6 mice. Control, Vgll4 knockdown or Vgll4 knockdown together with VGLL4-HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice. Tumor volumes were measured 15 days for nude mice and 21 days for C57BL/6 mice after tumor cell inoculation


Expression of VGLL4 - HF4A attenuates the T cell - meditated tumor cell killing . Activated T cells and A549 cells were co - cultured in 24 - well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal

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Expression of VGLL4-HF4A attenuates the T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal violet


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Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodies


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Correlation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodies


Immunoblot analysis of A549 cells transfected with VGLL4 - FLAG or VGLL4 - HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control

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Immunoblot analysis of A549 cells transfected with VGLL4-FLAG or VGLL4-HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cells


Deficiency of VGLL4 reduces IRF2BP2 protein levels . Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and

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Deficiency of VGLL4 reduces IRF2BP2 protein levels. Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4


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The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodies


qRT - PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549

qRT-PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cells


A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies . Relative IRF2BP2 expression levels were quantified in the right

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A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panel


Lys48 - linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells . HEK293T cells were transfected with indicated plasmids , treated with MG132 for 3 h before harvest , and subjected to immunoprecipitation and immunoblot analysis as

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Lys48-linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells. HEK293T cells were transfected with indicated plasmids, treated with MG132 for 3 h before harvest, and subjected to immunoprecipitation and immunoblot analysis as indicated


Lys48 - linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells . HEK293T cells were transfected with indicated siRNA and plasmids , and subjected to immunoprecipitation and immunoblot analysis as

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Lys48-linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells. HEK293T cells were transfected with indicated siRNA and plasmids, and subjected to immunoprecipitation and immunoblot analysis as indicated


IRF2 inhibits IRF1 transcriptional activity . A549 cells transfected with human PD - L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase

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IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD-L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assay


Knockdown IRF2 restores PD - L1 expression in VGLL4 - knockdown A549 cells . Gene expression levels were analyzed by qRT - PCR in A549 cells transfected or transduced as

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Knockdown IRF2 restores PD-L1 expression in VGLL4-knockdown A549 cells. Gene expression levels were analyzed by qRT-PCR in A549 cells transfected or transduced as indicated


Overexpression of IRF1 increases PD - L1 expression in VGLL4 - knockdown A549 cells . A549 cells were transfected with VGLL4 siRNA or together with HA - IRF1 plasmid and total cell lysates were analyzed by western

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Overexpression of IRF1 increases PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with VGLL4 siRNA or together with HA-IRF1 plasmid and total cell lysates were analyzed by western blot


IRF2 is released from PD - L1 promoter upon IFNγ stimulation . IRF2 - 3 × HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP - qPCR analysis in the PD - L1 promoter using control and HA antibodies . Normal mouse IgG was used as

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IRF2 is released from PD-L1 promoter upon IFNγ stimulation. IRF2-3×HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP-qPCR analysis in the PD-L1 promoter using control and HA antibodies. Normal mouse IgG was used as control


Increased association of IRF2 with PD - L1 promoter in IRF2BP2 knockout ( KO ) A549 cells revealed by ChIP - qPCR analysis . Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower

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Increased association of IRF2 with PD-L1 promoter in IRF2BP2 knockout (KO) A549 cells revealed by ChIP-qPCR analysis. Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panel


Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti - HA antibody in IRF2 - 3 × HA knockin A549

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Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti-HA antibody in IRF2-3×HA knockin A549 cells


Loss of IRF2BP2 attenuated IFNγ - inducible PD - L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD - L1 protein

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Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD-L1 protein level


Loss of IRF2BP2 attenuated IFNγ - inducible PD - L1 mRNA expression revealed by qRT - PCR analysis in A549 control and IRF2BP2 knockout

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Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 mRNA expression revealed by qRT-PCR analysis in A549 control and IRF2BP2 knockout cells


miR - 130a represses VGLL4 and PD - L1 protein levels . A549 cells were transfected with control and miR - 130a mimic and subjected to immunoblot

miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysis


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected to

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Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblot


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected t qRT - PCR

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Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected t qRT-PCR analysis


Decreased YAP target gene expression by inhibition of miR - 130a using its sponge in A549

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Decreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cells


YAP5SA expression attenuates IFNγ - inducible PD - L1 expression . A549 cells were transduced with control , YAP - 5SA or YAP - S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot

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YAP5SA expression attenuates IFNγ-inducible PD-L1 expression. A549 cells were transduced with control, YAP-5SA or YAP-S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysis


qRT - PCR analysis of cell lysates from control , YAP - 5SA or YAP - S94A expressing A549 cells treated with IFNγ for the indicated

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qRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated times


Control or YAP - overexpressing LLC cells were transplanted into nude mice ( upper panel ) or C57BL / 6 ( lower panel ) mice , and tumor - growth kinetics was measured at the indicated times . Tumor volumes at the end time points were analyzed and shown at the right

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Control or YAP-overexpressing LLC cells were transplanted into nude mice (upper panel) or C57BL/6 (lower panel) mice, and tumor-growth kinetics was measured at the indicated times. Tumor volumes at the end time points were analyzed and shown at the right panel


miR - 130a regulates IRF1 3 ′ UTR sensor activity . A549 cells were transfected with WT or mutant IRF1 3 ′ UTR sensor plasmids together with miR - 130a mimic . Sensor activities were determined by dual luciferase

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miR-130a regulates IRF1 3′UTR sensor activity. A549 cells were transfected with WT or mutant IRF1 3′UTR sensor plasmids together with miR-130a mimic. Sensor activities were determined by dual luciferase assay


YAP5SA induces miR - 130a expression and represses IRF1 expression . A549 cells were transduced with control or YAP - 5SA lentivirus and subjected to qRT - PCR

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YAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysis


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible IRF1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected to qRT - PCR

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Inhibition of miR-130a by its sponge enhances IFNγ-inducible IRF1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to qRT-PCR analysis


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

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

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


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

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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 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

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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 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 .

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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 the control .

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


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 .

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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 .

Scheme of DNA fiber analysis and representative fiber images.


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

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


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

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


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

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


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

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


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

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 cells . Representative SIRF images of MRE11 at normal or stalled replication forks are shown . Scale bars : 10 µm .

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

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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 .

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


( 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 .

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(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 complex ( CTC1 wild - type and ∆ 700N ) .

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


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 .

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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 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 .

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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 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 .

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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 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 .

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∆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 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 .

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∆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.


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 , * * *

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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 - 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 .

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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 - and STN1 - deficient U2OS cells . Scale bar : 10 µm . Average percentages of anaphase bridges from three independent experiments are presented .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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 .

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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 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 ) .

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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 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 )

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ,

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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 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

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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 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 )

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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 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 ) .

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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 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 .

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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 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 .

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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 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 )

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

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

O O 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 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 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 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

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 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 )

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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 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 ) .

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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 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 .

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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 ) , 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 .

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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 . 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 ) .

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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 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 .

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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 ) . 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 ) .

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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 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 ) .

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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 . 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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 . The full pictures are shown in Fig EV3 . Scale bar , 1 cm .

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


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

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


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

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


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 .

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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 ) 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 .

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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 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 .

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

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


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 .

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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 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 .

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

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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 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 .

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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 - and ABA - treated pDOG1shDOG1 : : LUC plants . Scale bar : 1cm .

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


E RT - qPCR analysis of DOG1 sense mRNA level .

E RT-qPCR analysis of DOG1 sense mRNA level.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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

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


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

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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 cells as in a . Arrows show reformation tubules . e , Three - dimensional reconstruction of a cell from d .

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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 images ( seconds ) of starved LAMP1 - YFP - expressing NRK cells .

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


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

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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 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 .

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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.