A HumanRPE ‐ 1 stably expressing low of mannosidase II ‐ mApple ( ManII ‐ mApple ) were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed stained using TFG antibodies imaged using confocal microscopy . Alternatively , were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . shown are projections of 3D data sets ( 4 μm in z ) . Merged with TFG in green ManII in red are shown . Scale bar , 5 μm . shown are representative of at least 10 analyzed for each condition . B Human RPE ‐ 1 were mock ‐ transfected ( control ) or transfected with a TFG siRNA for 60 h , then fixed stained using TFG GM130 antibodies imaged using confocal microscopy . Alternatively , were either treated with DMSO ( top row ) or brefeldin A ( BFA ) for 1 h ( middle row ) , followed by fixation , or washed into fresh media following DMSO or BFA treatment for 30 min ( bottom row ) , prior to fixation . shown are projections of 3D data sets ( 4 μm in z ) . Merged with TFG in green GM130 in red are shown . Scale bar , 5 μm . shown are representative of at least 10 analyzed for each condition .

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A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple (ManII‐mApple) were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and ManII in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.B Human RPE‐1 cells were mock‐transfected (control) or transfected with a TFG siRNA for 60 h, then fixed and stained using TFG and GM130 antibodies and imaged using confocal microscopy. Alternatively, cells were either treated with DMSO (top row) or brefeldin A (BFA) for 1 h (middle row), followed by fixation, or washed into fresh media following DMSO or BFA treatment for 30 min (bottom row), prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with TFG in green and GM130 in red are shown. Scale bar, 5 μm. Images shown are representative of at least 10 individual cells analyzed for each condition.


C Higher magnification views of indicated regions in ( B , boxed ) are shown ( GM130 distribution only ) . Scale bar , 2 μm .

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C Higher magnification views of the indicated regions in (B, boxed) are shown (GM130 distribution only). Scale bar, 2 μm.


A HumanRPE ‐ 1 stably expressing low of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . were subsequently treated with brefeldin A ( BFA ) for 1 h , followed by a wash into fresh media further incubation for 2 h in absence of BFA , prior to fixation . shown are projections of 3D data sets ( 4 μm in z ) . Merged with Sec31A in green ManII in red are shown ( representative of at least 15 analyzed ) . Scale bar , 5 μm .

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A HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with brefeldin A (BFA) for 1 h, followed by a wash into fresh media and further incubation for 2 h in the absence of BFA, prior to fixation. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A in green and ManII in red are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.


B HumanRPE ‐ 1 stably expressing low of mannosidase II ‐ mApple were transfected with a TFG siRNA for 60 h . were subsequently treated with BFA for 1 h , followed by a wash into fresh media further incubation for 1 h in absence of BFA , prior to fixation using α ‐ Sec16A α ‐ Sec31A antibodies . shown are projections of 3D data sets ( 4 μm in z ) . Merged with Sec31A ( green ) , ManII ( red ) , Sec16A ( blue ) are shown ( representative of at least 15 analyzed ) . Scale bar , 5 μm . C Higher magnification views of indicated regions in ( B , boxed ) are shown . Arrows highlight COPII ‐ positive transport carriers that contain cargo ManII , which are not juxtaposed to Sec16A ‐ labeled sites on ER . Additionally , arrowheads point out distinct foci in which COPII continues to associate with Sec16A ‐ labeled sites , indicating that COPII vesicle formation continues in absence of TFG . Scale bar , 1 μm .

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B HumanRPE‐1 cells stably expressing low levels of mannosidase II‐mApple were transfected with a TFG siRNA for 60 h. Cells were subsequently treated with BFA for 1 h, followed by a wash into fresh media and further incubation for 1 h in the absence of BFA, prior to fixation and staining using α‐Sec16A and α‐Sec31A antibodies. Images shown are projections of 3D data sets (4 μm in z). Merged images with Sec31A (green), ManII (red), and Sec16A (blue) are shown (representative of at least 15 cells analyzed). Scale bar, 5 μm.C Higher magnification views of the indicated regions in (B, boxed) are shown. Arrows highlight COPII‐positive transport carriers that contain the cargo ManII, which are not juxtaposed to Sec16A‐labeled sites on the ER. Additionally, arrowheads point out distinct foci in which COPII continues to associate with Sec16A‐labeled sites, indicating that COPII vesicle formation continues in the absence of TFG. Scale bar, 1 μm.


Formalin - fixed paraffin - embedded ( FFPE ) sections from control or Tgif1KO mice were immunostained with anti - pSmad2 antibody revealed by immunofluorescence ( IF ) DAPI . Representative pictures at 40x are shown ( n = 30 ) . Scale bars , 100 μM .

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Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM.


FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin eosin ( H & E ) or immunostained with antibodies to CK19 or Amylase revealed by . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Kaplan - Meyer survival of control , Tgif1KO , KC KTC mice . A regular mosaic two - colors was used to discriminate between control Tgif1KO mice .

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Kaplan-Meyer survival analysis of control, Tgif1KO, KC and KTC mice. A regular mosaic two-colors line was used to discriminate between control and Tgif1KO mice.


Pictures of whole pancreas spleen tissues from control , Tgif1KO , KC or KTC mice ( left ) . of pancreas was measured presented as dot plot ( n = 30 ) .

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Pictures of whole pancreas and spleen tissues from control, Tgif1KO, KC or KTC mice (left). Weight of pancreas was measured and presented as dot plot (n=30).


FFPE pancreatic sections from control , Tgif1KO , KC or KTC were stained with H & E or subjected to using antibodies to CK19 or α - SMA . Yellow arrows indicate PanINs in KC mice . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control, Tgif1KO, KC or KTC were stained with H&E or subjected to IHC analysis using antibodies to CK19 or α-SMA. Yellow arrows indicate PanINs in KC mice. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Representative pictures of dissected abdominal region of KC KTC mice . Yellow arrows indicate metastatic nodules in liver . Yellow dotted lines show pancreas .

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Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas.


FFPE sections of pancreas , liver , lungs or lymph - nodes from control , KC or KTC mice were stained with H & E or immunostained with anti - Vimentin antibody revealed by IF . Representative pictures at 4x ( Lymph node , H & E ) , 10x ( Liver , H & E ) 20x ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) are shown ( n = 18 to 30 ) . Scale bars , 1 , 000 μM ( Lymph node , H & E ) , 400 μM ( Liver , H & E ) 200 μM ( Pancreas , H & E ; Lung , H & E ; Lymph node , IF - Vimentin ) .

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FFPE sections of pancreas, liver, lungs or lymph-nodes from control, KC or KTC mice were stained with H&E or immunostained with anti-Vimentin antibody and revealed by IF. Representative pictures at 4x (Lymph node, H&E), 10x (Liver, H&E) and 20x (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin) are shown (n=18 to 30). Scale bars, 1,000 μM (Lymph node, H&E), 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin).


volume metastasis in live KCLuc KTCLuc mice were analyzed by IVIS bioluminescence . Following imaging live , pancreas , brain , liver lung were dissected analyzed again by IVIS bioluminescence ( n = 6 , 5 out of 6 mice developed metastasis to liver lung ) .

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Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence. Following imaging live animals, pancreas, brain, liver and lung were dissected and analyzed again by IVIS bioluminescence (n= 6, 5 out of 6 mice developed metastasis to liver and lung).


Total lysates from pancreas of control , KC or KTC mice were pooled analyzed by immunoblotting using antibodies to pSmad2 , Smad2 or JunB ( n = 6 ) .

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Total cell lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2, Smad2 or JunB (n=6).


Expression of JunB in pancreas from control , KC or KTC mice ( same as in D ) was analyzed by qRT - PCR ( n = 6 ) . Data are expressed as mean ± . * * p < 0 . 01 ; on a two - Student ' s .

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Expression of JunB in pancreas from control, KC or KTC mice (same as in D) was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; based on a two-tailed Student's test.


extracts from MIAPaCa - 2 ( left ) control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody analyzed by immunoblotting using antibodies to Twist1 or TGIF1 ( A - C ) .

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Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C).


extracts from control or Tgif1KO mice ( right ) were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading , total lysates were analyzed by immunoblotting using antibodies to Twist1 , TGIF1 or β - Actin .

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Protein extracts from control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.


extracts from MIAPaCa - 2 ( left ) control were immunoprecipitated using normal IgG , anti - TGIF1 or anti - Twist1 antibody analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C , lysates were treated with DNase I before coimmunopreciptation .

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Protein extracts from MIAPaCa-2 cells (left) and control were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 In C, cell lysates were treated with DNase I before coimmunopreciptation.


Panc - 1 were treated with TGF - β for 30 min , extracts were immunoprecipitated with anti - TGIF1 antibody analyzed by immunoblotting using anti - Twist1 antibody . To control for activation of TGF - β signaling , total lysates were analyzed by immunoblotting using anti - pSmad2 antibody .

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Panc-1 cells were treated with TGF-β for 30 min, and protein extracts were immunoprecipitated with anti-TGIF1 antibody and analyzed by immunoblotting using anti-Twist1 antibody. To control for activation of TGF-β signaling, total cell lysates were analyzed by immunoblotting using anti-pSmad2 antibody.


PL45 stably expressing Dox - inducible control or sh - targeting TGIF were treated with vehicle or Dox for 48 hr analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) . In left panel , of bands was assessed by densitometry , result was presented as a of Twist1 / TGIF1 .

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PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1.


PL45 stably expressing Dox - inducible TGIF1 were treated with vehicle or Dox for 48 hr analyzed for binding of TGIF1 to Twist1 promoter by ChIP agarose gel .

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PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


Pancreatic from control or Tgif1KO mice was analyzed for binding of TGIF1 to Twist1 promoter by ChIP agarose gel .

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Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.


PL45 stably expressing both Dox - inducible TGIF1 Tam - inducible Twist1 ( Twist1ER ) were treated with Tam / or Dox for 48 hr analyzed for Twist1 expression by immunoblotting or qRT - PCR ( n = 6 ) .

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PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).


MIAPaCa - 2 were transfected with wild - type or mutated ( Twist1 binding site ) Twist1 luciferase reporters ( TGTLuc ) together with increasing amounts of Twist1 expression vector in presence of empty vector ( EV ) or TGIF1 expression vector . Luciferase activity was measured 48 hr following transfection normalized on basis of co - transfected Renilla luciferase ( n = 6 ) .

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MIAPaCa-2 cells were transfected with wild-type or mutated (Twist1 binding site) Twist1 luciferase reporters (TGTLuc) together with increasing amounts of Twist1 expression vector in the presence of empty vector (EV) or TGIF1 expression vector. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).


Total lysates from pancreas of control , KC or KTC mice were pooled analyzed by immunoblotting using antibodies to E - Cadherin , Vimentin , p16Ink4A β - Actin ( n = 6 ) .

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Total lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E-Cadherin, Vimentin, p16Ink4A and β-Actin (n=6).


Expression of Cdh1 , Vimentin , p16Ink4A in pancreas from control , KC or KTC mice ( same as in C ) was analyzed by qRT - PCR ( n = 6 ) .

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Expression of Cdh1, Vimentin, and p16Ink4A in pancreas from control, KC or KTC mice (same as in C) was analyzed by qRT-PCR (n=6).


FFPE pancreatic sections from control , KC or KTC mice were stained with H & E or immunostained with antibodies to E - Cadherin , Vimentin or p16Ink4a revealed by IF or . Representative pictures at 20x are shown ( n = 30 ) . Scale bars , 200 μM .

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FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.


Kaplan - Meyer survival of control , KTC KTWC mice .

Kaplan-Meyer survival analysis of control, KTC and KTWC mice.


FFPE sections of pancreas or liver from control , KTC or KTWC mice were stained with H & E or immunostained with anti - CK19 antibody revealed by . Representative pictures at 10x ( Liver , H & E ) or 20x ( Pancreas , H & E ; Pancreas , - CK19 ) are shown ( n = 30 ) . Scale bars , 400 μM ( Liver , H & E ) 200 μM ( Pancreas , H & E ; Pancreas , - CK19 ) .

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FFPE sections of pancreas or liver from control, KTC or KTWC mice were stained with H&E or immunostained with anti-CK19 antibody and revealed by IHC. Representative pictures at 10x (Liver, H&E) or 20x (Pancreas, H&E; Pancreas, IHC-CK19) are shown (n=30). Scale bars, 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Pancreas, IHC-CK19).


Expression of Cdh1 , Vimentin p16Ink4A in pancreas from control , KTC or KTWC mice was analyzed by qRT - PCR ( n = 6 ) .

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Expression of Cdh1, Vimentin and p16Ink4A in pancreas from control, KTC or KTWC mice was analyzed by qRT-PCR (n=6).


Specificity of anti - phospho - TGIF1 ( pTGIF1 ) antibody . extracts from Tgif1 - / - MEFs reconstituted with empty vector ( EV ) , wild - type ( TGIF1 ) or non - phosphorylated form ( TGIF1 . 2TA ) of Flag - TGIF1 were immunoblotted with anti - phospho - TGIF1 or β - Actin as a loading control .

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Specificity of anti-phospho-TGIF1 (pTGIF1) antibody. Cell extracts from Tgif1-/- MEFs cells reconstituted with empty vector (EV), wild-type (TGIF1) or non-phosphorylated form (TGIF1.2TA) of Flag-TGIF1 were immunoblotted with anti-phospho-TGIF1 or β-Actin as a loading control.


Expression of pTGIF1 Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry . Representative pictures at different stages ( 40x ) are shown .

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Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry. Representative pictures at different stages (40x) are shown.


percentages of samples with high versus low expression of pTGIF1 Twist1 in normal tissues versus PDAC tissues are shown . Scale bars , 100 μM .

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The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown. Scale bars, 100 μM.


MIAPaCa - 2 were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 resistance colonies were pooled ( n = 6 ) . Expression of Twist1 was examined by qRT - PCR ( D ) .

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MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D).


MIAPaCa - 2 were transfected with TGIF1 or TGIF1 . 2TD expression vectors , selected with G418 resistance colonies were pooled ( n = 6 ) . Binding of TGIF1 to Twist1 promoter was examined by ChIP agarose gel .

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MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.


BxPC3 were transfected with TGTLuc together with TGIF1 or TGIF1 . 2TA in presence or absence of KrasG12D . Luciferase activity was measured 48 hr following transfection normalized on basis of co - transfected Renilla luciferase ( n = 6 ) .

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BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).


BxPC3 were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1 . 2TA in presence or absence of KrasG12D luciferase activity was measured 48 hr following transfection as described in ( E ) ( n = 6 ) .

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BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in (E) (n=6).


Representative microscope pictures ( 20x ) of MIAPaCa - 2 , Suit - 2 , Capan - 2 Panc - 1 . Scale bars , 200 μM .

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Representative microscope pictures (20x) of MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells. Scale bars, 200 μM.


Expression of TGIF1 Twist1 in MIAPaCa - 2 , Suit - 2 , Capan - 2 Panc - 1 was analyzed by immunoblotting .

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Expression of TGIF1 and Twist1 in MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells was analyzed by immunoblotting.


MIAPaCa - 2 , Suit - 2 , Capan - 2 Panc - 1 were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . extracts were analyzed 48 hr following transfection by immunoblotting using anti - Flag antibody ( C ) .

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MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti-Flag antibody (C).


MIAPaCa - 2 , Suit - 2 , Capan - 2 Panc - 1 were transfected with empty vector ( EV ) , wild - type ( TGIF1 ) or phosphorylation mimic mutant ( TGIF1 . 2TD ) of TGIF1 . were selected with neomycin for 2 - 3 , counted using an automatic counter ( D ) ( n = 6 ) .

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MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cells were selected with neomycin for 2-3 weeks, and counted using an automatic cell counter (D) (n=6).


Asynchronously growing human U - 2 OS were treated with 0 . 5 Gy of IR , allowed to recover for indicated time periods , stained for 53BP1 content for cycle resolved of 53BP1 foci in by QIBC .

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Asynchronously growing human U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, and stained for 53BP1 and DNA content for cell cycle resolved quantification of 53BP1 foci in individual cells by QIBC.


Representative from QIBC in ( A ) . Scale bars , 10 μm .

Representative images from the QIBC analysis in (A). Scale bars, 10 μm.


were treated with 0 . 5 Gy of IR in absence or presence of 0 . 4 M sorbitol , fixed 1h later , 53BP1 assembly at DSBs was analyzed by QIBC . Treatments as in ( C ) ; RIF1 accumulation at DSBs was analyzed by QIBC .

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Cells were treated with 0.5 Gy of IR in absence or presence of 0.4 M sorbitol, fixed 1h later, and 53BP1 assembly at DSBs was analyzed by QIBC. Treatments as in (C); RIF1 accumulation at DSBs was analyzed by QIBC.


U - 2 OS were treated with 0 . 5 Gy of IR , allowed to recover for indicated time periods , stained for 53BP1 MDC1 , analyzed by QIBC . 53BP1 foci intensities are plotted against MDC1 foci intensities , representative are shown .

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U-2 OS cells were treated with 0.5 Gy of IR, allowed to recover for the indicated time periods, stained for 53BP1 and MDC1, and analyzed by QIBC. 53BP1 foci intensities are plotted against MDC1 foci intensities, and representative images are shown.


U - 2 OS stably expressing GFP - MDC1 or GFP - 53BP1 were treated with 1 Gy of IR analyzed by live microscopy at 15 min intervals . Representative are provided kinetics of GFP - MDC1 GFP - 53BP1 foci formation dissolution are shown as single tracks . Bold lines represent averages from n = 29 for GFP - MDC1 n = 18 for GFP - 53BP1 .

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U-2 OS cells stably expressing GFP-MDC1 or GFP-53BP1 were treated with 1 Gy of IR and analyzed by live cell microscopy at 15 min intervals. Representative images are provided and kinetics of GFP-MDC1 and GFP-53BP1 foci formation and dissolution are shown as single cell tracks. Bold lines represent averages from n=29 for GFP-MDC1 and n=18 for GFP-53BP1 cells.


U - 2 OS were treated with 1 Gy of IR fixed at indicated time - points . Mean 53BP1 foci was analyzed from more than 1500 per time - point . Mean ( solid ) standard deviation from mean ( dashed lines ) are indicated . same as in ( A ) were analyzed for mean MDC1 foci . Mean ( solid ) standard deviation from mean ( dashed lines ) are indicated .

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U-2 OS cells were treated with 1 Gy of IR and fixed at the indicated time-points. Mean 53BP1 foci intensity was analyzed from more than 1500 cells per time-point. Mean (solid line) and standard deviation from the mean (dashed lines) are indicated. The same cells as in (A) were analyzed for mean MDC1 foci intensity. Mean (solid line) and standard deviation from the mean (dashed lines) are indicated.


GFP - 53BP1 were treated with 25 ng / ml NCS to induce breaks imaged at 30 minutes intervals . Examples of GFP - 53BP1 fusions ( green arrowheads magnified regions ) fissions ( blue arrowheads magnified regions ) are shown .

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GFP-53BP1 cells were treated with 25 ng/ml NCS to induce DNA breaks and imaged at 30 minutes intervals. Examples of GFP-53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown.


53BP1 - RFP , in which endogenous 53BP1 gene locus had been engineered by CRISPR / Cas9 to express 53BP1 - mScarlet from natural promoter , were treated with NCS ( 25 ng / ml ) imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads magnified regions ) fissions ( blue arrowheads magnified regions ) are shown on left in higher magnification on right .

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53BP1-RFP cells, in which the endogenous 53BP1 gene locus had been engineered by CRISPR/Cas9 to express 53BP1-mScarlet from the natural promoter, were treated with NCS (25 ng/ml) and imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) and fissions (blue arrowheads and magnified regions) are shown on the left and in higher magnification on the right.


GFP - 53BP1 were left untreated , or treated with APH ( 0 . 5 μM ) or ATRi ( 1 μM ) to induce replication stress - associated heritable lesions , were imaged at 30 minutes intervals . Examples of 53BP1 fusions ( green arrowheads magnified regions ) are shown .

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GFP-53BP1 cells were left untreated, or treated with APH (0.5 μM) or ATRi (1 μM) to induce replication stress-associated heritable DNA lesions, and cells were imaged at 30 minutes intervals. Examples of 53BP1 fusions (green arrowheads and magnified regions) are shown.


- induced optoDroplet formation of Cry2 - mCherry - 53BP1 , Cry2 - mCherry - 53BP1 W1495A , Cry2 - mCherry - MDC1 . were imaged at 15 seconds intervals . Representative of optoDroplet formation before 6 minutes after induction are shown . Quantifications from single - QIBC of 2 - 3 independent experiments are shown with mean ( solid ) standard deviation from mean ( dashed lines ) indicated .

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Light-induced optoDroplet formation of Cry2-mCherry-53BP1, Cry2-mCherry-53BP1 W1495A, and Cry2-mCherry-MDC1. Cells were imaged at 15 seconds intervals. Representative images of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated.


Cry2 - mCherry - 53BP1 W1495A optoDroplet fusion at 15 seconds time resolution . Fusing optodroplets are highlighted by green arrowheads in magnified insets .

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Cry2-mCherry-53BP1 W1495A optoDroplet fusion at 15 seconds time resolution. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.


- induced optoDroplet formation of indicated Cry2 - mCherry - 53BP1 constructs . were imaged at 15 seconds intervals . Representative quantifications of optoDroplet formation before 6 minutes after induction are shown . Quantifications from single - QIBC of 2 - 3 independent experiments are shown with mean ( solid ) standard deviation from mean ( dashed lines ) indicated . Red bars indicate part of 53BP1 that was expressed as Cry2 - mCherry fusion .

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Light-induced optoDroplet formation of the indicated Cry2-mCherry-53BP1 constructs. Cells were imaged at 15 seconds intervals. Representative images and quantifications of optoDroplet formation before and 6 minutes after light induction are shown. Quantifications from single-cell QIBC analysis of 2-3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion.


Cry2 - mCherry - 53BP1 1203 - 1972 W1495A optoDroplets undergo fusion events upon induction . Fusing optodroplets are highlighted by green arrowheads in magnified insets .

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Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets undergo fusion events upon light induction. Fusing optodroplets are highlighted by green arrowheads and in the magnified insets.


- dependent optoDroplet formation exemplified by two neighboring that differ in their Cry2 - mCherry - 53BP1 1203 - 1972 W1495A expression level . were imaged at 15 seconds intervals . Quantifications of optoDroplet formation in lowly expressing ( mClow ) highly expressing ( mChigh ) from single - QIBC of 3 independent experiments are shown with mean ( solid ) standard deviation from mean ( dashed lines ) indicated .

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Concentration-dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2-mCherry-53BP1 1203-1972 W1495A expression level. Cells were imaged at 15 seconds intervals. Quantifications of optoDroplet formation in lowly expressing cells (mClow) and highly expressing cells (mChigh) from single-cell QIBC analysis of 3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated.


Cry2 - mEGFP - 53BP1 1203 - 1972 W1495A assembles into FokI - induced repair compartments within seconds after induction . FokI was induced for 1h where indicated before were - activated imaged at 15 seconds intervals . Red arrowheads mark FokI - induced lesion with mCherry accumulation .

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Cry2-mEGFP-53BP1 1203-1972 W1495A assembles into FokI-induced DNA repair compartments within seconds after light induction. FokI was induced for 1h where indicated before cells were light-activated and imaged at 15 seconds intervals. Red arrowheads mark the FokI-induced lesion with mCherry accumulation.


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Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later. Endogenous BRCA1 and p53 were co-stained.


As in ( A ) , comparing Cry2 - mCherry - 53BP1 constructs with without BRCT domains . Quantifications of p53 assembly into 53BP1 optoDroplets from single - QIBC of 500 - 600 per condition are shown below with mean ( solid ) standard deviation from mean ( dashed lines ) indicated . Red bars indicate part of 53BP1 that was expressed as Cry2 - mCherry fusion .

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As in (A), comparing Cry2-mCherry-53BP1 constructs with and without the BRCT domains. Quantifications of p53 assembly into 53BP1 optoDroplets from single-cell QIBC analysis of 500-600 cells per condition are shown below with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion.


Western blot of p53 induction upon damage ( 10 Gy , 2h ) with without 0 . 4 M Sorbitol .

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Western blot of p53 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M Sorbitol.


qPCR of p21 induction upon damage ( 10 Gy , 2h ) with without 0 . 4 M sorbitol . Mean ± standard deviation is indicated .

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qPCR of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. Mean ± standard deviation is indicated.


Western blot of p21 induction upon damage ( 10 Gy , 2h ) with without 0 . 4 M sorbitol .

Western blot of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol.


QIBC cycle on content Cyclin A upon damage with without 0 . 4 M sorbitol . were irradiated with 2 Gy in absence or presence of 0 . 4 M sorbitol . After 1h of recovery , sorbitol - containing medium was exchanged with fresh medium , were allowed to recover for an additional 7h . Percentages in G1 S / G2 are provided .

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QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0.4 M sorbitol. Cells were irradiated with 2 Gy in absence or presence of 0.4 M sorbitol. After 1h of recovery, sorbitol-containing medium was exchanged with fresh medium, and cells were allowed to recover for an additional 7h. Percentages in G1 and S/G2 are provided.


( A ) - comparison of proliferation of T - after treatment with dexamethasone ( Dexa ) . mean values of three biological replicates ± SD analyzed by two - way ANOVA are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(A) Cell count-based comparison of cellular proliferation of T-ALL cells after treatment with dexamethasone (Dexa). Individual and mean values of three biological replicates ± SD analyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( B ) Flow cytometric of by AnnexinV in T - lines upon treatment with indicated Dexa concentrations . is represented by number of AnnexinV positive ( AnnexinV + ) , apoptotic as a ( % ) of total . Mean values of n ≥ 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated Dexa concentrations. Cell death is represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells. Mean values of n≥4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( C ) Expression of human GRα LRH - 1 mRNA in human T - lines were determined by probe - real - time quantitative PCR , calculated as relative expression compared to beta - actin used to determine GR : LRH - 1 ( right ) . mean values of four biological replicates ± SD are shown statistically significant differences between CEM - C7 GC - resistant lines were determined by one - way ANOVA .

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(C) Expression levels of human GRα and LRH-1 mRNA in human T-ALL cell lines were determined by probe-based real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM-C7 and GC-resistant cell lines were determined by one-way ANOVA.


( D ) Immunoblots of GR , BimEL , BimL BimS Tubulin from T - treated for 24 h with 20 µM 3d2 or 30 µM Dexa . Representative results from n = 2 biological replicates are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(D) Immunoblots of GR, BimEL, BimL and BimS and Tubulin from T-ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa. Representative results from n=2 biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( E ) Immunoblots of GR , PARP ( loading control ) Tubulin ( cytoplasmatic loading control ) from ( CP ) nucleoplasmic ( NP ) lysates of T - treated with Dexa . Note : extracts were more highly concentrated ( approx . 6 - fold ) than fractions . Representative results from two biological replicates are shown .

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(E) Immunoblots of GR, PARP (nuclear loading control) and Tubulin (cytoplasmatic loading control) from cytoplasmic (CP) and nucleoplasmic (NP) lysates of T-ALL cells treated with Dexa. Note: Nuclear extracts were more highly concentrated (approx. 6-fold) than cytosolic fractions. Representative results from two biological replicates are shown.


( F ) GR activity in human T - transfected with a control luciferase reporter plasmid ( pGL3 ) ( F ) GR responsive ( GRE ) luciferase reporter treated with 3d2 / or Dexa . β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity calculated as relative to DMSO - treated pGL3 control . mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(F) GR activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) (F) GR responsive (GRE) luciferase reporter and treated with 3d2 and/or Dexa. β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( G ) LRH - 1 activity in human T - transfected with a control luciferase reporter plasmid ( pGL3 ) ( G ) LRH - 1 responsive ( 5xRE ) luciferase reporter treated with 3d2 β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity calculated as relative to DMSO - treated pGL3 control . mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(G) LRH-1 activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) and (G) LRH-1 responsive (5xRE) luciferase reporter and treated with 3d2 β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( H ) Flow cytometry - of BiFC in Jurkat transiently co - transfected with YFP1 - LRH - 1 GR - YFP2 treated for indicated time periods with Dexa . YFP + are shown as a ( % ) of viable . mean values ± SD of three biological replicates analyzed by one - way ANOVA are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0h ) was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(H) Flow cytometry-based quantification of BiFC in Jurkat cells transiently co-transfected with YFP1-LRH-1 and GR-YFP2 and treated for indicated time periods with Dexa. YFP+ cells are shown as a percentage (%) of viable cells. Individual and mean values ± SD of three biological replicates analyzed by one-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0h) was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


( A ) Daily counting - comparison of proliferation of T - after ( A ) treatment with 3d2 mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(A) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (A) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


( B ) Daily counting - comparison of proliferation of T - after ( B ) small hairpin - mediated LRH - 1 knockdown ( shLRH - 1 ) . mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(B) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (B) small hairpin RNA-mediated LRH-1 knockdown (shLRH-1). Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( C ) cycle distribution of T - treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry . Representative results from n = 3 biological replicates are shown . Stacked bars represent mean of technical triplicates ± SD . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(C) Cell cycle distribution of T-ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry. Representative results from n=3 biological replicates are shown. Stacked bars represent the mean of technical triplicates ± SD. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( D ) Simultaneous flow cytometric of cycle distribution by EdU / DAPI by AnnexinV in MOLT - 4 upon treatment with 3d2 . Dead are represented by number of AnnexinV positive ( AnnexinV + ) , apoptotic as a ( % ) of total cycle phase distribution as % of viable , AnnexinV negative ( AnnexinV - ) . Representative results from three biological replicates are shown . Bars represent mean of technical triplicates ± SD differences in AnnexinV + were analyzed by one - way ANOVA . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(D) Simultaneous flow cytometric analysis of cell cycle distribution by EdU/DAPI staining and cell death by AnnexinV staining in MOLT-4 cells upon treatment with 3d2. Dead cells are represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells and cell cycle phase distribution as % of viable, AnnexinV negative (AnnexinV-) cells. Representative results from three biological replicates are shown. Bars represent the mean of technical triplicates ± SD and differences in AnnexinV+ cells were analyzed by one-way ANOVA. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( E ) Flow cytometric of by AnnexinV in T - lines upon treatment with indicated 3d2 concentrations . Mean values of n = 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(E) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated 3d2 concentrations. Mean values of n=4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( F ) Flow cytometric of 3d2 - induced by AnnexinV in wild - type ( WT ) , Bcl - 2 overexpressing ( Bcl - 2 ) Z - VAD - FMK ( zVAD ) - treated CEM - C1 . Mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD analyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(F) Flow cytometric analysis of 3d2-induced cell death by AnnexinV staining in wild-type (WT), Bcl-2 overexpressing (Bcl-2) and Z-VAD-FMK (zVAD)-treated CEM-C1. Mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD analyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.


( G ) Immunoblots of full - ( cl . ) PARP , P - p38 , c - Myc , Caspase 3 ( Casp . 3 ) , cl . Casp . 3 , BimEL , BimL BimS Tubulin from MOLT - 4 treated with 40 µM 3d2 for indicated time periods . Representative results from two biological replicates are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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(G) Immunoblots of full-length and cleaved (cl.) PARP, P-p38, c-Myc, Caspase 3 (Casp. 3), cl. Casp. 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 for indicated time periods. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


( A ) Immunoblots of full - ( cl . ) PARP , BimEL , BimL BimS Tubulin from MOLT - 4 treated for 24 h with 10 µM dexamethasone ( Dexa ) 3d2 . Representative results from three biological replicates are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control .

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(A) Immunoblots of full-length and cleaved (cl.) PARP, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated for 24 h with 10 µM dexamethasone (Dexa) and 3d2. Representative results from three biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.


( B ) Flow cytometric of by AnnexinV in MOLT - 4 treated with 3d2 , 30 µM Dexa / or 50 µM pan caspase inhibitor Z - VAD - FMK ( zVAD ) . mean values of technical triplicates of one representative out of n = 3 biological replicates ± SD are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control . Two - way ANOVA ( B ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(B) Flow cytometric analysis of cell death by AnnexinV staining in MOLT-4 cells treated with 3d2, 30 µM Dexa and/or 50 µM pan caspase inhibitor Z-VAD-FMK (zVAD). Individual and mean values of technical triplicates of one representative out of n=3 biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control. Two-way ANOVA (B ns = not significant, ** p < 0.01, *** p < 0.001.


( C ) Flow cytometric of by AnnexinV in control or Bim knockout ( Bim KO ) CEM - C7 treated with 3d2 30 µM Dexa . Mean values of technical triplicates of one representative of n = 4 biological replicates ± SD are shown . Data information : Two - way ANOVA C , ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout (Bim KO) CEM-C7 cells treated with 3d2 and 30 µM Dexa. Mean values of technical triplicates of one representative of n=4 biological replicates ± SD are shown. Data information: Two-way ANOVA C, ns = not significant, ** p < 0.01, *** p < 0.001.


( D ) Immunoblots of BimEL , BimL BimS , Bcl - 2 Tubulin from MOLT - 4 expressing a non - ( shcontrol ) or LRH - 1 - ( shLRH - 1 ) targeting small hairpin construct .

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(D) Immunoblots of BimEL, BimL and BimS, Bcl-2 and Tubulin from MOLT-4 expressing a non- (shcontrol) or LRH-1- (shLRH-1) targeting small hairpin RNA construct.


( E ) Flow cytometric of by AnnexinV in control or glucocorticoid receptor knockout ( GR KO ) MOLT - 4 treated with 3d2 30 µM Dexa . Mean values of n = 3 biological replicates ± SD are shown . Data information : Two - way ANOVA E ) ; ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 .

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(E) Flow cytometric analysis of cell death by AnnexinV staining in control or glucocorticoid receptor knockout (GR KO) MOLT-4 cells treated with 3d2 and 30 µM Dexa. Mean values of n=3 biological replicates ± SD are shown. Data information: Two-way ANOVA E); ns = not significant, ** p < 0.01, *** p < 0.001.


( F / G ) mRNA expression of human GR ( F ) Bim ( G ) in shcontrol shLRH - 1 CEM - C1 were determined by real - time quantitative PCR after treatment with 1 µM Dexa for indicated times . Fold induction relative to untreated shcontrol sample ( 0h ) was calculated after normalization to beta - actin . values of technical triplicates of one representative of two biological replicates ± SD are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control .

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(F/G) mRNA expression levels of human GR (F) and Bim (G) in shcontrol and shLRH-1 CEM-C1 cells were determined by real-time quantitative PCR after treatment with 1 µM Dexa for indicated times. Fold induction relative to the untreated shcontrol sample (0h) was calculated after normalization to beta-actin. Individual values of technical triplicates of one representative of two biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.


( H ) Immunoblots of full - ( cl . ) PARP , GR phospho - AMPK ( P - AMPK ) , c - Myc , full - cl . Caspase 3 , BimEL , BimL BimS Tubulin from MOLT - 4 treated with 40 µM 3d2 / or 10 µM Dexa . Representative results from two biological replicates are shown . Data information : For displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø ) was used as a solvent control .

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(H) Immunoblots of full-length and cleaved (cl.) PARP, GR and phospho-AMPK (P-AMPK), c-Myc, full-length and cl. Caspase 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 and/or 10 µM Dexa. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø) was used as a solvent control.


( A ) Ex vivo response of human T acute lymphoblastic leukemia ( T - ) - derived xenografts ( PDX ) , co - cultured on hTERT - immortalized bone marrow MSCs , to treatment with indicated concentrations of 3d2 / or dexamethasone ( Sample 1 , 3 4 : 1 nM ; Sample 2 : 0 . 1 nM ) . viability was quantified by live using CyQuant combined with automated image calculated as ( % ) of dimethyl sulfoxide treated control . Data information : displayed experiments display one biological replicate .

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(A) Ex vivo response of human T cell acute lymphoblastic leukemia (T-ALL) patient-derived xenografts (PDX), co-cultured on hTERT-immortalized primary bone marrow MSCs, to treatment with indicated concentrations of 3d2 and/or dexamethasone (Sample 1, 3 and 4: 1 nM; Sample 2: 0.1 nM). Cell viability was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage (%) of the dimethyl sulfoxide treated control. Data information: All displayed experiments display one biological replicate.


( B ) Synergy scores ( Z - scores ) between 3d2 dexamethasone of human T - - derived xenografts ( PDX ) were calculated from viability curves of PDX samples treated with increasing concentrations of 3d2 / or dexamethasone using SynergyFinder tool . Z - scores ≥ 0 indicate additivity or synergism < 0 antagonism . Data information : displayed experiments display one biological replicate .

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(B) Synergy scores (Z-scores) between 3d2 and dexamethasone of human T-ALL patient-derived xenografts (PDX) were calculated from the viability curves of PDX samples treated with increasing concentrations of 3d2 and/or dexamethasone using SynergyFinder tool. Z-scores ≥ 0 indicate additivity or synergism and < 0 antagonism. Data information: All displayed experiments display one biological replicate.


( C ) Expression of human GRα , LRH - 1 Bim mRNA in human PDX were determined by real - time quantitative PCR , calculated as relative expression compared to beta - actin used to determine GR : LRH - 1 ( right ) . Data information : displayed experiments display one biological replicate .

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(C) Expression levels of human GRα, LRH-1 and Bim mRNA in human PDX were determined by real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Data information: All displayed experiments display one biological replicate.


( E ) In vivo leukemia progression of human T - PDX transplanted into NSG mice treated for 3 with vehicle ( PBS control ) , 40 mg / kg 3d2 or 10 mg / kg Dexamethasone starting 3 days post transplantation . Engraftment was calculated as % of human vs total ( human + mouse ) CD45 positive ( CD45 + ) as assessed by flow cytometric of peripheral blood . Data information : displayed experiments display one biological replicate .

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(E) In vivo leukemia progression of human T-ALL PDX transplanted into NSG mice treated for 3 weeks with vehicle (PBS control), 40 mg/kg 3d2 and or 10 mg/kg Dexamethasone starting 3 days post transplantation. Engraftment was calculated as % of human vs total (human+mouse) CD45 positive (CD45+) cells as assessed by flow cytometric analysis of peripheral blood. Data information: All displayed experiments display one biological replicate.


( a ) Immunofluorescence labeling of wildtype mouse dorsal skin samples ( Dnmt1f / f , skin from P3 ) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte - specific knockout ( Dnmt1Δ / Δep ) results in almost absence of a specific in epidermis in hair follicles . Dashed lines indicate dermal - epidermal border dotted lines indicate hair follicles .

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(a) Immunofluorescence labeling of wildtype mouse dorsal skin samples (Dnmt1f/f, skin from P3) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte-specific knockout (Dnmt1Δ/Δep) results in the almost absence of a specific staining in the epidermis and in hair follicles. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.


( d ) Knockout of Dnmt1 results in lower of methylation in epidermis in hair follicles as determined by anti 5 - mC labeling of P3 skin . Dashed lines indicate dermal - epidermal border dotted lines indicate hair follicles .

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(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.


( h ) Hematoxylin Eosin ( H & E ) , immunolabeling for KERATIN 10 ( KRT10 ) , LORICRIN ( LOR ) KERATIN 6 ( KRT6 ) expression of dorsal skin sections ( P5 ) of control Dnmt1Δ / Δep mice .

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(h) Hematoxylin and Eosin (H&E) staining, immunolabeling for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.


( c ) Flow cytometric of respective immune populations from epidermis of control Dnmt1Δ / Δep mice of different postnatal . are shown as of viable . Data are mean ± , two - t - . ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 ( P3 ) , 4 ( P5 ) 4 ( P6 + P7 ) . Data information : Scale bars , 50 µm .

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(c) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SEM, two-tailed t-test. ns, not significant, * p≤0.05, ** p≤0.01, **** p≤0.0001, n=3 (P3), 4 (P5) and 4 (P6 + P7). Data information: Scale bars, 50 µm.


( d ) Relative mRNA expression of immune related genes in control Dnmt1Δ / Δep mice at P3 ( n = 3 ) , P5 ( n = 5 ) P7 ( n ≥ 3 ) . Data are mean ± . P values are calculated using one - way ANOVA with post hoc Tukey multiple comparison , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Data information : Scale bars , 50 µm .

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(d) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ/Δep mice at P3 (n=3), P5 (n=5) and P7 (n≥3). Data are mean ± SEM. P values are calculated using one-way ANOVA with post hoc Tukey multiple comparison test, * p≤0.05, ** p≤0.01, *** p≤0.001 , **** p≤0.0001. Data information: Scale bars, 50 µm.


( a ) Relative mRNA expression of epidermal Intracisternal A - type particle ( IAP ) during different postnatal time points ( P0 n ≥ 3 , P3 n = 3 , P5 n = 5 , P7 n ≥ 3 mice ) , which were compared using one way - ANOVA with post hoc Tukey multiple comparison . Data are mean ± . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

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(a) Relative mRNA expression levels of epidermal Intracisternal A-type particle (IAP) during different postnatal time points (P0 n≥3, P3 n=3, P5 n=5, P7 n≥3 mice), which were compared using one way-ANOVA with post hoc Tukey multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01.


( c ) Relative expression of immune related genes of epidermis from P7 of control ( n ≥ 7 ) , Dnmt1Δ / Δep , ( n ≥ 8 ) , MAVS knockout ( Mavs - / - , n = 7 ) Dnmt1Δ / Δep Mavs - / - ( n = 6 ) mice were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison . Data are mean ± . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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(c) Relative expression of immune related genes of the epidermis from P7 of control (n≥7), Dnmt1Δ/Δep, (n≥8), MAVS knockout (Mavs-/-, n=7) and Dnmt1Δ/Δep Mavs-/- (n=6) mice were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.


Immortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( c , d ) PicoGreen of PBS - DAC - treated keratinocytes for 48h detected micronuclei ( arrow ) , which were analyzed in a blinded manner , compared using two - t - . Data are mean ± , * p ≤ 0 . 05 ( n = 3 biological replicates ) ( d ) . Data information : Scale bars , 20

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Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). Data information: Scale bars, 20 µm


Immortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( e ) Co - labeling of by PicoGreen by cGAS in PBS DAC treated keratinocytes . Within enlarged sections on right arrows point at / cGAS double - positive blebs micronuclei . ( f ) Relative expression of CCL5 IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way - ANOVA with post hoc Dunnett multiple comparison . Data are mean ± . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ( n = 4 biological replicates ) . Data information : Scale bars , , 50

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Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (e) Co-labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes. Within enlarged sections on the right arrows point at DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates). Data information: Scale bars, , 50 µm


( c ) Representative of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f / f , Dnmt1Δ / Δep , Dnmt1Δ / Δep Cgas - / - mice . Dashed lines indicate dermal - epidermal border . ( d ) of CD45 - positve . Tissue sections approximately 1 cm in were scanned of CD45 positive per of nuclei was quantified using ImageJ . Sections obtained from five mice per genotype were analyzed calculated values were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison . Data are mean ± . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . Data information : Scale bars , 100

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(c) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f/f, Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice. Dashed lines indicate the dermal-epidermal border. (d) Quantification of CD45-positve cells. Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ. Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ** p≤0.01, *** p≤0.001. Data information: Scale bars, 100 µm


( e ) Epidermis obtained from P7 control , Dnmt1Δ / Δep , Cgas - / - Dnmt1Δ / Δep Cgas - / - mice was analyzed for relative mRNA expression of immune related genes using one way - ANOVA with post hoc Holm - Sidak multiple comparison . Data are mean ± . ns not significant , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 . At least 7 mice per genotype were used .

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(e) Epidermis obtained from P7 control, Dnmt1Δ/Δep, Cgas-/- and Dnmt1Δ/Δep Cgas-/- mice was analyzed for relative mRNA expression levels of immune related genes using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, **** p≤0.0001. At least 7 mice per genotype were used.


52 - day - old ( A ) of genotypes indicated grown in LDs . Scale bars : 5 cm . B - C number of rosette leaves time to flowering at time of flowering of genotypes shown in A numbers indicate independent transgenic lines . letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of indicated genotypes . Data information : In ( B - C data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD ) .

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52-day-old (A) plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B-C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B-C data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).


D - E qPCR results showing mRNA expression of CIB1 , FT in genotypes indicated . numbers indicate independent transgenic lines . transcript of CIB1 or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates for each of these two technical replicates were performed .

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D-E qPCR results showing mRNA expression of CIB1, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


F . 25 - day - old ( F ) of genotypes indicated grown in LDs . Scale bars : 5 cm . , G - H . number of rosette leaves time to flowering at time of flowering of genotypes shown in numbers indicate independent transgenic lines . letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of indicated genotypes . Data information : In G - H ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD ) .

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F. 25-day-old plants (F) of the genotypes indicated grown in LDs. Scale bars: 5 cm. , G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).


, I - J . qPCR results showing mRNA expression of CO , FT in genotypes indicated . numbers indicate independent transgenic lines . transcript of CO or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates for each of these two technical replicates were performed .

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, I-J. qPCR results showing mRNA expression of CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


Immunoblots showing CIB1 ( A ) in WT co - 1 backgrounds ( A ) over course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with anti - Myc antibody , stripped re - probed with anti - actin antibody ( ACT ) .

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Immunoblots showing CIB1 (A) protein levels in WT and co-1 backgrounds (A) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).


B . Immunoblots showing CO ( B ) in WT cibh backgrounds ( B ) over course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with anti - Myc antibody , stripped re - probed with anti - actin antibody ( ACT ) .

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B. Immunoblots showing CO (B) protein levels in WT and cibh backgrounds (B) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).


relative of CIB1 in WT co - 1 backgrounds over course of 20 hours under LDs were calculated Data information : Error bars represent standard deviation of three biological replicates of Western blots .

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The relative levels of CIB1 in WT and co-1 backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.


D . relative of CO in WT cibh backgrounds over course of 20 hours under LDs were calculated Data information : Error bars represent standard deviation of three biological replicates of Western blots .

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D. The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.


A . microscopy showing that CIB1 co - localizes with CO in speckles . Scale bars : 2 μm .

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A. Fluorescent microscopy images showing that CIB1 co-localizes with CO in nuclear speckles. Scale bars: 2 μm.


B . BiFC assays to show in vivo interactions . Epidermal of N . benthamiana leaves were co - transformated with cCFP - CO nYFP - CIB1 , with cCFP - CO nYFP , or with cCFP nYFP - CIB1 . BF , bright field . Merge , overlay of YFP bright field . Scale bars : 50 μm . Data information : For each experiment , two or more biological replicates were performed .

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B. BiFC assays to show in vivo protein interactions. Epidermal cells of N. benthamiana leaves were co-transformated with cCFP-CO and nYFP-CIB1, with cCFP-CO and nYFP, or with cCFP and nYFP-CIB1. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bars: 50 μm. Data information: For each experiment, two or more biological replicates were performed.


C . Split - LUC showing that CIB1 interacts with CO . Leaf epidermal of N . benthamiana were co - transformed with CO - nLUC cLUC - CIB1 or cLUC or nLUC with cLUC - CIB or cLUC . Data information : For each experiment , two or more biological replicates were performed .

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C. Split-LUC assay showing that CIB1 interacts with CO. Leaf epidermal cells of N. benthamiana were co-transformed with CO-nLUC and cLUC-CIB1 or cLUC or nLUC with cLUC-CIB or cLUC. Data information: For each experiment, two or more biological replicates were performed.


D . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 , 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag seedlings grown in LDs . Data information : For each experiment , two or more biological replicates were performed .

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D. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1, 35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag seedlings grown in LDs. Data information: For each experiment, two or more biological replicates were performed.


F . In vitro pull - down assays showing interaction between CIBC CO , or CIBC COΔN lack of interaction between CIB1C COΔC . His - TF tagged CO , COΔN , COΔC or His - TF tagged UVR8 were mixed with GST - CIB1C purified from E . coli , His antibody was used for in vitro pull - down . products were analyzed by immunoblots probed with anti - CIB1C antibody or anti - His antibody ( UVR8 , CO , COΔC , COΔN ) . Data information : For each experiment , two or more biological replicates were performed .

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F. In vitro pull-down assays showing the interaction between CIBC and CO, or CIBC and COΔN and the lack of interaction between CIB1C and COΔC. His-TF tagged CO, COΔN, COΔC or His-TF tagged UVR8 were mixed with GST-CIB1C purified from E. coli, and His antibody was used for the in vitro pull-down assay. The products were analyzed by immunoblots probed with the anti-CIB1C antibody or the anti-His antibody (UVR8, CO, COΔC, COΔN). Data information: For each experiment, two or more biological replicates were performed.


A . 28 - day - old of genotypes indicated grown in LDs . Scale bars : 5 cm . B , C . number of rosette leaves time to flowering at time of flowering of genotypes shown in A . letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of indicated genotypes . Data information : In ( B , C ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD ) .

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A. 28-day-old plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B, C. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B, C), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).


D . Immunoblots showing Myc - CIB1 CO - Flag in genotypes indicated . Samples were separated on 10 % SDS - PAGE , blotted , probed with anti - Myc antibody , stripped re - probed with anti - Flag antibody .

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D. Immunoblots showing Myc-CIB1 and CO-Flag protein levels in the genotypes indicated. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-Flag antibody.


E . qPCR results showing mRNA expression of FT in genotypes indicated . transcript in Col are set to 1 . 0 . Samples were collected from 7 - day - old seedlings of genotypes indicated at ZT16 . Data information : error bars indicate standard error within three biological replicates for each of these two technical replicates were performed .

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E. qPCR results showing mRNA expression of FT in the genotypes indicated. The transcript levels in Col are set to 1.0. Samples were collected from 7-day-old seedlings of the genotypes indicated at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


G . Box plots ( minimum , first quartile , median , third quartile , maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in expressing different reportors effectors . relative LUC activities normalized to REN activity are shown . LUC / REN in expressing ProFT alone is set to 1 . 0 . Data information : data are presented as mean ± SD , n = 4 . letters " a " to " g " indicate statistically significant differences determined by Tukey ' s HSD ( P ≤ 0 . 05 ) . Two biological replicates were performed .

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G. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The relative LUC activities normalized to the REN activity are shown. The LUC/REN in plants expressing ProFT alone is set to 1.0. Data information: data are presented as mean ± SD, n=4. The letters "a" to "g" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


A . microscopy showing that CRY2 , CIB1 CO co - localized to nucleus . Scale bars : 2

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A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μ


B . Co - IP assays showing that CRY2 , CIB1 CO form a . Samples were prepared from 10 - day - old WT or 35S : Myc - CIB1 / 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) , samples were collected at ZT16 .

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B. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16.


C . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) . Samples were collected at times indicated over course of one day .

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C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day.


E . Representative results of ChIP - qPCR assays . fragments ( ~ 500 bp ) were prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag seedlings , immunoprecipitated by anti - CRY2 , anti - Flag ( CO - Flag ) , anti - Myc ( Myc - CIB1 ) antibody or anti - preimmune IgG , precipitated was analyzed by qPCR using primer pairs indicated . IP / input of anti - preimmune is set to 1 . 0 . Data information : relative IP / input ratios are shown with their standard deviation ( n = 3 ) . Two biological replicates were performed .

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E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed.


F . Box plots ( minimum , first quartile , median , third quartile , maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in expressing different reportors effectors . LUC / REN in expressing ProFT alone is set to 1 . 0 . Immunoblots were done to show GFP - CRY2W374A , Myc - CIB1 Myc - CO in transient Dual - LUC . Samples were separated on 10 % SDS - PAGE , blotted , probed with anti - Myc antibody , with anti - GFP antibody . Data information : relative LUC activities normalized to REN activity are shown ( LUC / REN ) with their standard deviation ( n = 6 ) . letters " a " to " f " indicate statistically significant differences determined by Tukey ' s HSD ( P ≤ 0 . 05 ) . Two biological replicates were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody. Data information: the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.