LSM images of cross - sections through the heart of an adult control and ILK K . O . mouse four weeks after myocardial ischemia and reperfusion ( MI / R ) , showing the outer lateral region of the LV . Scale bars : 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1 - positive and CD31 - positive area ( normalised to total analysed myocardial area ) , respectively . ( n = 5 control and n = 7 ILK K . O . mice ) , P = 0 . 051 ( cardiac lymph vessel density in adult control versus adult ILK K . O . ) , * P = 0 . 025 ( cardiac blood vessel density in adult control versus adult ILK K . O . )

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

LSM images of cross-sections through the heart of an adult control and ILK K.O. mouse four weeks after myocardial ischemia and reperfusion (MI/R), showing the outer lateral region of the LV. Scale bars: 100 µm Overall cardiac lymph vessel and blood vessel density in heart sections as determined by Lyve1-positive and CD31-positive area (normalised to total analysed myocardial area), respectively. (n = 5 control and n = 7 ILK K.O. mice), P = 0.051 (cardiac lymph vessel density in adult control versus adult ILK K.O.), *P = 0.025 (cardiac blood vessel density in adult control versus adult ILK K.O.)


Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA . Scale bars : 50

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

Images of adult human LECs after 1 hour of BrdU incorporation and previous transfections with control or ILK siRNA. Scale bars: 50 µm


LEC proliferation as determined by the number of BrdU - positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF - C Cys156Ser ( n = 3 independent transfections per siRNA ) , * P = 0 . 032 ( control versus ILK - 1 ) , * P = 0 . 005 ( control versus ILK - 2 ) , * P = 0 . 0003 ( control versus ILK - 3 )

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

LEC proliferation as determined by the number of BrdU-positive cells normalised to the total number of LECs previously transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 3 independent transfections per siRNA), *P = 0.032 (control versus ILK-1), *P = 0.005 (control versus ILK-2), *P = 0.0003 (control versus ILK-3)


VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF - C Cys156Ser ( n = 4 ( control siRNA , ILK - 1 siRNA , and ILK - 3 siRNA ) , or n = 8 ( ILK - 2 siRNA ) independent transfections per siRNA ) , * P = 0 . 0001 ( control versus each siRNA )

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

VEGFR3 tyrosine phosphorylation as determined by ELISA of lysates from adult human LECs transfected with control siRNA or ILK siRNAs in the presence of VEGF-C Cys156Ser (n = 4 (control siRNA, ILK-1 siRNA, and ILK-3 siRNA), or n = 8 (ILK-2 siRNA) independent transfections per siRNA), *P = 0.0001 (control versus each siRNA)


LSM images of VEGFR3 / β1 integrin PLA dots in human LECs transfected with control or ILK siRNA . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs ( n = 5 independent transfections per siRNA ) , P = 0 . 234 ( control versus ILK - 1 ) , * P = 0 . 024 ( control versus ILK - 2 ) , * P = 0 . 001 ( control versus ILK - 3 )

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

LSM images of VEGFR3/β1 integrin PLA dots in human LECs transfected with control or ILK siRNA. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC after transfection with control siRNA or ILK siRNAs (n = 5 independent transfections per siRNA), P = 0.234 (control versus ILK-1), *P = 0.024 (control versus ILK-2), *P = 0.001 (control versus ILK-3)


LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes . Red dots are PLA dots composed of VEGFR3 and β1 integrin . Scale bars : 10 µm Quantification of VEGFR3 / β1 integrin PLA dots per human LEC with ( + ) or without ( - ) mechanical stretch ( n = 6 independent stretch chambers ) ,

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

LSM images of PLA dots in human LECs that were kept unstretched or mechanically stretched for 30 minutes. Red dots are PLA dots composed of VEGFR3 and β1 integrin. Scale bars: 10 µm Quantification of VEGFR3/β1 integrin PLA dots per human LEC with (+) or without (-) mechanical stretch (n = 6 independent stretch chambers),


Western blot ( WB ) image of human LECs that were either kept unstretched or stretched for 30 minutes , and used for immunoprecipitation ( IP ) of HA - tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with ( + ) or without ( - ) mechanical stretch ; normalised to the respective amount of HA - tagged β1 integrin ( n = 3 ( unstretched ) or n = 5 ( stretched ) independent stretch

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

Western blot (WB) image of human LECs that were either kept unstretched or stretched for 30 minutes, and used for immunoprecipitation (IP) of HA-tagged β1 integrin from whole cell lysates with subsequent detection of interacting ILK in IP lysates Quantification of the ILK protein amount in IP lysates from LECs with (+) or without (-) mechanical stretch; normalised to the respective amount of HA-tagged β1 integrin (n = 3 (unstretched) or n = 5 (stretched) independent stretch chambers


Confocal images of HeLa cells stained with NF45 / NF90 antibodies and antibodies against nucleolin and fibrillarin . Scale bar , 10 μm .

Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm.


HEK293 cells were infected with lentiviruses containing negative control shRNA ( shNC ) or NF45 / NF90 specific shRNA ( shNF45 # 1 , shNF45 # 2 , shNF90 # 1 and shNF90 # 2 ) for 24 h , and then transfected with rDNA - Luc . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show the protein levels of NF45 and NF90 . Empty vector ( EV ) , FLAG - NF45 plasmid ( NF45 ) or FLAG - NF90 plasmid ( NF90 ) were transfected into HEK293 cells for 24 h , and then rDNA - Luc was co - transfected into the cells . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show the protein levels of FLAG - NF45 and FLAG - NF90 .

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

HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.   Empty vector (EV), FLAG-NF45 plasmid (NF45) or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then rDNA-Luc was co-transfected into the cells. Luciferase activity was measured after a further 24 h (n=3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90.


HEK293 cells were infected with lentiviruses containing negative control shRNA ( shNC ) or NF45 / NF90 specific shRNA ( shNF45 # 1 , shNF45 # 2 , shNF90 # 1 and shNF90 # 2 ) for 24 h , and then transfected with rDNA - Luc . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show the protein levels of NF45 and NF90 .

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

HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.


E - F The qPCR analysis of 45S pre - rRNA from HEK293 cells treated with indicated shRNAs ( E ) and plasmids ( F ) for 24 h . Western blots show the expression levels of NF45 , NF90 , FLAG - NF45 and FLAG - NF90 . ( n = 3 )

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

E-F The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45 and FLAG-NF90. (n = 3)


In Jurkat cells , a ChIP assay was performed with control IgG , NF45 and NF90 antibodies , and then the precipitated DNA was analyzed using qPCR with the aforementioned primers . The relative rDNA fold - enrichment was normalized to control IgG treatment . ( n = 3 )

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

In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3)


EMSA assay . Lane 1 ) Biotin - labled rDNA probe . Lane 2 ) Biotin - labled rDNA probe + nuclear protein . Lane 3 ) Biotin - labled rDNA probe + nuclear protein + 100 - fold molar excess of biotin - unlabeled specific competitor . Lane 4 ) Biotin - labled rDNA probe + nuclear protein + 100 - fold molar excess of biotin - unlabeled mutant competitor . Lane 5 ) Biotin - labled rDNA probe + nuclear protein + NF90 antibody . Lane 6 ) Biotin - labled rDNA probe + nuclear protein + NF45 antibody . The details were described in Materials and Methods section .

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

EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section.


The relative luciferase activity of HEK293T cells transfected with empty vector ( EV ) or FLAG - NF90 ( NF90 ) in combination with rDNA - Luc wild type ( WT ) or mutated rDNA - Luc reporter plasmid ( Mutant ) for 24 h . ( n = 3 ) Western blot shows FLAG - NF90 protein level .

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

The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA -Luc wild type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n=3) Western blot shows FLAG-NF90 protein level.


The relative luciferase activity of HEK293T cells transfected with empty vector ( EV ) or FLAG - NF90 ( NF90 ) in combination with

The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA


Co - IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV , and the following plasmids : GFP - S5 , GFP - L9 , GFP - B23 , GFP - UBF , GFP - C23 . * represents the target protein .

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

Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein.


Co - IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells .

Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells.


HEK293 cells were infected by lentivirus containing NF45 - specfic shRNA or NF90 - specfic shRNA , and then stimulated in serum for 0 , 3 and 6 h . Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot .

HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot.


ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0 , 3 and 6 h . ( n = 3 )

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

ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3)


ChIP analysis of NF45 and NF90 binding to the H42 . 9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h . ( n = 3 )

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

ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3)


Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin . Scale bar , 10 μm .

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

Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.


ChIP analysis of NF45 and NF90 binding to the H42 . 9 loci in CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

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

ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)


qPCR analysis of pre - rRNA in CD3 + T cells treated with P / I for 6 h . ( n = 3 )

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

qPCR analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3)


CD3 + T cells were first silenced with shNC , shNF45 or shNF90 , and then treated with P / I to extract total RNA for qPCR analysis of pre - rRNA . ( n = 3 )

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

CD3+ T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3)


The analysis of rDNA - luc activity in Jurkat cells with or without NF90 silence upon P / I stimulation or not . ( n = 3 )

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

The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3)


Confocal images of Jurkat cells to show NF45 ( left panel ) or NF90 ( right panel ) ( green fluorescence ) colocalized with NFATc2 ( red fluorescence ) in nucleoli . Scale bar , 10 μm .

Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm.


Co - IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P / I .

Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I.


ChIP analysis of NFATc2 binding to the rDNA loci in CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)


Jurkat cells co - transfected with rDNA - luc ( wild type or mutant plasmid ) with NF90 and / or NFATc2 for 24 h , then rDNA - luc activity was detected after P / I sitmulation or not ( DMSO ) . ( n = 3 )

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

Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3)


ChIP analysis of NFATc2 binding to the H42 . 9 loci in NF90 - slienced CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

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

ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)


CD69 expression , an early marker of lymphocyte activation , in CD3 + T cells infected with shNC , shNF45 or shNF90 lentivirus upon stimulation with P / I for 12 h .

CD69 expression, an early marker of lymphocyte activation, in CD3+ T cells infected with shNC, shNF45 or shNF90 lentivirus upon stimulation with P/I for 12 h.


B - C PBMC cells with shNC , shNF45 or shNF90 lentivirus infection were stimulated with P / I for 12h , then the freshly replaced medium without P / I after another 24 h culture was harvested for IL - 2 and IFN - γ detections by ELISA . ( n = 3 )

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

B-C PBMC cells with shNC, shNF45 or shNF90 lentivirus infection were stimulated with P/I for 12h, then the freshly replaced medium without P/I after another 24 h culture was harvested for IL-2 and IFN-γ detections by ELISA.(n=3)


The proliferation of shNC , shNF45 or shNF90 - infected PBMCs was monitored using a CCK - 8 assay . ( n = 3 )

The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3)


Confocal images to show the subcellular localization of NFATc2 in CD3 + T cells treated with P / I and FK506 or CX5461 . Scale bar , 10 μm .

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

Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm.


Flow cytometry analysis of CD69 expression in P / I - stimulated CD3 + T cells treated with or without FK506 or CX5461 .

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

Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461.


G - H ELISA assays of IL - 2 and IFN - γ in CD3 + T cells with the indicated treatments . ( n = 3 )

G-H ELISA assays of IL-2 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3)


Flow cytometry analysis of CFSE - labeled CD3 + T cells in the presence of DMSO , FK506 and CX5461 for 0 , 3 and 7 d .

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

Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.


qPCR analysis of pre - rRNA expression from skin - and heart - transplanted mouse spleens . ( n = 5 )

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

qPCR analysis of pre-rRNA expression from skin- and heart-transplanted mouse spleens. (n = 5)


pre - rRNA expression levels of PBMC from renal transplantation patients . ( Stable group , n = 7 . ABMR group , n = 8 . TCMR group , n = 8 ) .

pre-rRNA expression levels of PBMC from renal transplantation patients. (Stable group, n = 7. ABMR group, n = 8. TCMR group, n = 8).


Skin graft survival curve in mice treated with DMSO , 2 . 0 mg / kg FK506 or 0 . 5 - 2 . 0 mg / kg CX5461 . ( n = 5 )

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

Skin graft survival curve in mice treated with DMSO, 2.0 mg/kg FK506 or 0.5 -2.0 mg/kg CX5461. (n = 5)


Representative images of HE staining of skin graft at seven days post transplantation . Scale bar , 50 μm .

Representative images of HE staining of skin graft at seven days post transplantation. Scale bar, 50 μm.


CD3 + T cells ( upper ) or CD4 + and CD8 + T cells ( lower ) in the spleens of skin - graft recipient mice were analyzed by FACS .

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of skin-graft recipient mice were analyzed by FACS.


G - H ELISA analysis of IL - 2 and IFN - γ levels in skin - graft recipient mouse serum at 4th day post transplantation . ( n = 5 )

G-H ELISA analysis of IL-2 and IFN-γ levels in skin-graft recipient mouse serum at 4th day post transplantation. (n = 5)


The survival time ( days ) of cardiac graft in mice treated with DMSO , 2 . 0 mg / kg FK506 or 2 . 0 mg / kg CX5461 . ( n = 5 )

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

The survival time (days) of cardiac graft in mice treated with DMSO, 2.0 mg/kg FK506 or 2.0 mg/kg CX5461. (n=5)


Representative images of HE and CD3 - IHC staining of cardiac graft at seven days post transplantation . NC , Scale bar , 100 μm .

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

Representative images of HE and CD3-IHC staining of cardiac graft at seven days post transplantation. NC, Scale bar, 100 μm.


CD3 + T cells ( upper ) or CD4 + and CD8 + T cells ( lower ) in the spleens of heart - graft recipient mice were analyzed by FACS .

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of heart-graft recipient mice were analyzed by FACS.


L - M ELISA analysis of IL - 2 and IFN - γ levels in heart - graft recipient mouse serum at 7th day post transplantation . ( n = 5 )

L-M ELISA analysis of IL-2 and IFN-γ levels in heart-graft recipient mouse serum at 7th day post transplantation. (n = 5)


qPCR analysis of pre - rRNA expression from transplanted mouse spleen . ( n = 5 )

qPCR analysis of pre-rRNA expression from transplanted mouse spleen. (n = 5)


FACS analysis of CD4 + , CD25 + and Foxp3 + Treg cells isolated from superficial cervical lymph nodes of heart - transplanted mice .

FACS analysis of CD4+, CD25+ and Foxp3+ Treg cells isolated from superficial cervical lymph nodes of heart-transplanted mice.


B : Growth charts of 2 probands each showing typical growth patterns of self - limited DP , without compromised linear growth before puberty . Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age .

O O O O O O O O O O O O O O O O O O O O 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: Growth charts of 2 probands each showing typical growth patterns of self-limited DP, without compromised linear growth before puberty. Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age.


C : Biological consequences of the 2 identified N - terminal mutations . Both WT and mutant N - terminal protein fragments ( p . Arg156Leu and p . Glu161Lys ) were expressed in HEK293 cells as demonstrated by western blotting . The GFP - tagged protein products of both were not detected in the conditioned media of mammalian cells , as compared to wild type ( WT ) , and appear to be retained in the intracellular compartment ( mean ± s . e . m . ; n = 3 ) . Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media . UT - untransfected negative control ; nd - not detected ; * : p = 0 . 01094 ( WT vs p . Arg156Leu ) and p = 0 . 04408 ( WT vs p . Glu161Lys ) by 2 - tailed t - test . It has to date not been possible to test cytoplasmic retention for the two C - terminal mutations due to difficulty expressing the full length or C - terminal protein fragment in mammalian cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Biological consequences of the 2 identified N-terminal mutations. Both WT and mutant N-terminal protein fragments (p.Arg156Leu and p.Glu161Lys) were expressed in HEK293 cells as demonstrated by western blotting. The GFP-tagged protein products of both were not detected in the conditioned media of mammalian cells, as compared to wild type (WT), and appear to be retained in the intracellular compartment (mean ± s.e.m.; n = 3). Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media. UT - untransfected negative control; nd - not detected; *: p=0.01094 (WT vs p.Arg156Leu) and p=0.04408 (WT vs p.Glu161Lys) by 2-tailed t-test. It has to date not been possible to test cytoplasmic retention for the two C-terminal mutations due to difficulty expressing the full length or C-terminal protein fragment in mammalian cells.


A - J : Igsf10 expression was not observed at E10 . 5 ( A , sagittal section ) but readily detectable at E12 . 5 , in the NM ( B and C , frontal sections ) and E14 . 5 , along the migratory path of GnRH neurons ( D , frontal section ) . Igsf10 positive cells were negative for Isolectin b4 ( marker of vasculature ) at all developmental stages ( E , at E14 . 5 ) . Incubation with the sense probe resulted in no signal at all stages ( E14 . 5 shown in F ) . Peripherin - positive cells olfactory axons were extending within the Igsf10 - positive NM ( G and H ) . Cxcl12 mRNA expression is shown in I ( E14 . 5 , frontal section ) . GnRH neurons are located in the MPOA by E17 . 5 ( J , frontal section ) in an Igsf10 negative area .

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A-J: Igsf10 expression was not observed at E10.5 (A, sagittal section) but readily detectable at E12.5, in the NM (B and C, frontal sections) and E14.5, along the migratory path of GnRH neurons (D, frontal section). Igsf10 positive cells were negative for Isolectin b4 (marker of vasculature) at all developmental stages (E, at E14.5). Incubation with the sense probe resulted in no signal at all stages (E14.5 shown in F). Peripherin-positive cells olfactory axons were extending within the Igsf10-positive NM (G and H). Cxcl12 mRNA expression is shown in I (E14.5, frontal section). GnRH neurons are located in the MPOA by E17.5 (J, frontal section) in an Igsf10 negative area.


K - N : In human 11pcw brains , IGSF10 expression pattern was similar to that observed in mouse , with GnRH neurons interspersed in an IGSF10 - positive NM ( K - N , frontal sections , see also schematic in P ) . IGSF10 positive cells were also detected in the olfactory bulb ( OB , panel O ) . Sense probe resulted in no specific signal ( panel N ) .

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K-N: In human 11pcw brains, IGSF10 expression pattern was similar to that observed in mouse, with GnRH neurons interspersed in an IGSF10-positive NM (K-N, frontal sections, see also schematic in P). IGSF10 positive cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).


A : Levels of Igsf10 expression in native NIH3T3 cells , and cells stably expressing a scrambled ( sh - scr ) or Igsf10 ( sh - Igsf10 ) shRNA .

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A: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.


B and C : migration of GN11 cells from aggregates into NIH3T3 sh - scr ( B ) or sh - Igsf10 ( C ) . D : Analysis of the migration index ( see Methods ) .

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B and C: migration of GN11 cells from aggregates into NIH3T3 sh-scr (B) or sh-Igsf10 (C).D: Analysis of the migration index (see Methods).


E : RT - PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points . Expected band size : 905 bp .

E: RT-PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points. Expected band size: 905 bp.


F : Representative time - lapse analysis of mispair control ( Control Sp - MO and Control ATG - MO ) and Igsf10 morpholinos ( Igsf10 Sp - MO and Igsf10 ATG - MO ) injected Tg ( gnrh3 : EGFP ) embryos and larvae , at different time - points . Images show lateral view of live larvaehead ( 48 and 72 hpf ) and dorsal view ( 4 and 5 dpf ) of live larvaehead ; anterior is left . White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos . Brackets show the extension of the projections towards the hypothalamus . White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus , indicated with Δ ( hpf - hours post fertilisation ; dpf - days post fertilisation . ) Scale bar , 100 um ( panels at 48 and 72hpf in F ) , 150 um ( panels at 4 and 5dpf in F ) . Panel G : Effect of Igsf10 Splice - site morpholino injection observed at 48hrs . Quantification of the percentage of embryos showing an abnormal GnRH3 - neuron phenotype was significantly higher in Igsf10 Splice - site - MO ( Sp - MO ) injected embryos compared to relative controls . * : p = 0 . 02422 , * * : p = 0 . 00101 , * * * : p = 0 . 00020 , * * * * : p = 0 . 00001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Representative time-lapse analysis of mispair control (Control Sp-MO and Control ATG-MO) and Igsf10 morpholinos (Igsf10 Sp-MO and Igsf10 ATG-MO) injected Tg(gnrh3:EGFP) embryos and larvae, at different time-points. Images show lateral view of live larvaehead (48 and 72 hpf) and dorsal view (4 and 5 dpf) of live larvaehead; anterior is left. White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos. Brackets show the extension of the projections towards the hypothalamus. White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus, indicated with Δ (hpf - hours post fertilisation; dpf - days post fertilisation.) Scale bar, 100 um (panels at 48 and 72hpf in F), 150 um (panels at 4 and 5dpf in F). Panel G: Effect of Igsf10 Splice-site morpholino injection observed at 48hrs. Quantification of the percentage of embryos showing an abnormal GnRH3-neuron phenotype was significantly higher in Igsf10 Splice-site-MO (Sp-MO) injected embryos compared to relative controls. *: p=0.02422, **: p=0.00101, ***: p=0.00020, ****: p=0.00001.


( A ) 12 - lead ECG of patient 1 . ( B ) 12 - lead ECG of patient 2 . ( C ) ECG of patient 2 during epinephrine test ( A - represents baseline ECG ; B - ECG at 17 min and 47 s of the beginning of epinephrine infusion ( 0 . 2 μg / kg of epinephrine ) and C - ECG after the end of epinephrine infusion ) .

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(A) 12-lead ECG of patient 1. (B) 12-lead ECG of patient 2. (C) ECG of patient 2 during epinephrine test (A- represents baseline ECG; B- ECG at 17 min and 47 s of the beginning of epinephrine infusion (0.2 μg/kg of epinephrine) and C- ECG after the end of epinephrine infusion).


( E ) ECG of patient 3 ( subject IV : 13 ) showing mild QTc prolongation .

(E) ECG of patient 3 (subject IV:13) showing mild QTc prolongation.


( F ) ICD interrogation of patient 3 ( subject IV : 13 ) reveals an episode of VT .

(F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.


( A - H ) Expression of Tecrl in mouse development . Expression is not observed in ( A ) the cardiac crescent at E7 . 5 but is prominent in ( B ) the inflow tract ( ift ) region at E8 . 5 , particularly in ( C ) the left sinus venosus ( lsv ) . ( D ) At E9 . 5 , Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract . ( E - G ) From E10 onwards , Tecrl is expressed in the somites . ( H ) At E14 . 5 , Tecrl expression is present in the entire myocardium ; [ atrium ( a ) , ventricle ( v ) , neural tube ( nt ) , lung ( lu ) , right atrium ( ra ) , left atrium ( la ) , right ventricle ( rv ) and left ventricle ( lv ) ] .

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(A-H) Expression of Tecrl in mouse development. Expression is not observed in (A) the cardiac crescent at E7.5 but is prominent in (B) the inflow tract (ift) region at E8.5, particularly in (C) the left sinus venosus (lsv). (D) At E9.5, Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract. (E-G) From E10 onwards, Tecrl is expressed in the somites. (H) At E14.5, Tecrl expression is present in the entire myocardium; [atrium (a), ventricle (v), neural tube (nt), lung (lu), right atrium (ra), left atrium (la), right ventricle (rv) and left ventricle (lv)].


( I ) mRNA expression analyses of TECR and TECRL by RT - qPCR in human tissues demonstrates preferential expression of Tecrl in the heart ; [ Bone marrow ( Bone M . ) , heart , skeletal muscle ( Sk . Muscle ) , uterus , liver , spleen , thymus , thyroid , prostate , brain , lung , small intestine ( Small I . ) and colon ]

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(I) mRNA expression analyses of TECR and TECRL by RT-qPCR in human tissues demonstrates preferential expression of Tecrl in the heart; [Bone marrow (Bone M.), heart, skeletal muscle (Sk.Muscle), uterus, liver, spleen, thymus, thyroid, prostate, brain, lung, small intestine (Small I.) and colon]


( J ) Localization of MYC - Tecrl in COS - 1 cells . The accumulation of MYC - Tecrl is perinuclear consistent with it being localized to the ER .

(J) Localization of MYC-Tecrl in COS-1 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER.


( K - M ) Co - localization of MYC - Tecrl and the ER marker Calnexin in H10 cells . Nuclei stained with DAPI . Scale bar : 10 μm .

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(K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.


( A ) Skin fibroblasts ( left ) from IV : 13 , homozygous for the TECRLc . 331 + 1G > A mutation were reprogrammed to hiPSCs ( center ) , which expressed the pluripotency markers NANOG and SSEA4 ( right ) . Scale bars : 100 μm .

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(A) Skin fibroblasts(left) from IV:13, homozygous for the TECRLc.331+1G>A mutation were reprogrammed to hiPSCs(center), which expressed the pluripotency markers NANOG and SSEA4(right). Scale bars: 100 μm.


( B ) TECRLHom - hiPSCs have a normal karyotype ( left ) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines ( right ) .

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(B) TECRLHom-hiPSCs have a normal karyotype(left) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines (right).


( C ) TECRLHom - hiPSCs generate derivatives of mesoderm ( left ) , endoderm ( center ) and ectoderm ( right ) . Scale bars : 25

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(C) TECRLHom-hiPSCs generate derivatives of mesoderm(left), endoderm(center) and ectoderm(right). Scale bars: 25 μm


( F ) ACTN2 immunostaining of CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs . Scale bar : 25 μm .

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(F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm.


( G ) Analysis of RT - PCR products by gel electrophoresis . Amplification products of TECRL exons 2 - 4 from CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs ( left ) and coding sequence of TECRL from CTRL - and TECRLHom - hiPSC - CMs ( right ) .

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(G) Analysis of RT-PCR products by gel electrophoresis. Amplification products of TECRL exons 2-4 from CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs(left) and coding sequence of TECRL from CTRL- and TECRLHom-hiPSC-CMs(right).


( A ) Representative traces of [ Ca2 + ] i transients in Indo - 1 AM - loaded hiPSC - CMs paced at 1 Hz . ( B ) Time to reach 50 % of [ Ca2 + ] i transient amplitude , t1 / 2 ; [ Ca2 + ] i concentrations during systole and diastole ( C ) Amplitude and tau decay of the [ Ca2 + ] i transient .

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

(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz. (B) Time to reach 50% of [Ca2+]i transient amplitude, t1/2; [Ca2+]i concentrations during systole and diastole (C) Amplitude and tau decay of the [Ca2+]i transient.


( A ) Representative traces of [ Ca2 + ] i transients in Indo - 1 AM - loaded hiPSC - CMs paced at 1 Hz , in the presence of caffeine ( caff ) or caffeine and NiCl2 . ( B ) Amplitude of [ Ca2 + ] i in the presence of caffeine and NiCl2and fractional SR release in hiPSC - CMs .

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(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz, in the presence of caffeine (caff) or caffeine and NiCl2. (B) Amplitude of [Ca2+]i in the presence of caffeine and NiCl2and fractional SR release in hiPSC-CMs.


( C ) Rate constants of SERCA - , NCX - and slow mechanisms - based [ Ca2 + ] i decay in hiPSC - CMs . ( D ) Relative contribution of SERCA , NCX and slow mechanisms to [ Ca2 + ] i extrusion in hiPSC - CMs .

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(C) Rate constants of SERCA-, NCX- and slow mechanisms-based [Ca2+]i decay in hiPSC-CMs. (D) Relative contribution of SERCA, NCX and slow mechanisms to [Ca2+]i extrusion in hiPSC-CMs.


( A ) AP illustrating the analysed parameters ( B ) Representative APs from control ( CTRL ) , heterozygous ( HET ) and homozygous ( HOM ) CMs . ( C ) RMP , dV / dtmax , APAmax , APAplat , APD20 , APD50 and APD90 of CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs .

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(A) AP illustrating the analysed parameters (B) Representative APs from control (CTRL), heterozygous (HET) and homozygous (HOM) CMs. (C) RMP, dV/dtmax , APAmax, APAplat, APD20, APD50 and APD90 of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs.


( A ) Representative AP traces and ( B ) averaged activity of triggered and spontaneous activity in hiPSC - CMs in the absence of NA . ( C ) Representative AP traces and ( D ) averaged activity of triggered and spontaneous activity in hiPSC - CMs in the presence of NA .

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(A) Representative AP traces and (B) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the absence of NA. (C) Representative AP traces and (D) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the presence of NA.


( A ) Representative AP trace of a TECRLHom - hiPSC - CM in the presence of NA alone or NA and flecainide . ( B ) Effect of flecainide on AP parameters of hiPSC - CMs . ( C - D ) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC - CMs . Note that the effect of flecainide on triggered activity of TECRLHet - and TECRLHom - hiPSC - CMs is more pronounced than its effect on their spontaneous activity .

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(A) Representative AP trace of a TECRLHom-hiPSC-CM in the presence of NA alone or NA and flecainide. (B) Effect of flecainide on AP parameters of hiPSC-CMs. (C-D) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC-CMs. Note that the effect of flecainide on triggered activity of TECRLHet- and TECRLHom-hiPSC-CMs is more pronounced than its effect on their spontaneous activity.


A , B Detection of γb palmitoylation through the biotin - switch assay in N . benthamiana . The γb - 3xFlag and γb3CS - 3xFlag proteins were treated with ( Hyd + ) or without ( Hyd - ) hydroxylamine , the thioester cleavage reagent . Lanes labelled ' Palmitoylation ' show γb - 3xFlag or γb3CS - 3xFlag amounts recovered from the neutravidin beads . The loading controls indicate sample loading onto the neutravidin beads . Data information : In ( A , representative data are shown and three biological replicates had similar results .

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A, B Detection of γb palmitoylation through the biotin-switch assay in N. benthamiana. The γb-3xFlag and γb3CS-3xFlag proteins were treated with (Hyd+) or without (Hyd-) hydroxylamine, the thioester cleavage reagent. Lanes labelled ' Palmitoylation ' show γb-3xFlag or γb3CS-3xFlag amounts recovered from the neutravidin beads. The loading controls indicate sample loading onto the neutravidin beads. Data information: In (A, representative data are shown and three biological replicates had similar results.


D Western blot detection of viral accumulation in BSMV - and BSMV3CS - infected N . benthamiana plants with anti - TGB1 or anti - γb antibodies . SL8 , eighth systemically infected leaf ; SL9 , ninth systemically infected leaf . Ino , agroinfiltrated leaves . Rubisco large subunit ( RbcL ) was used as a loading control . Data information : In representative data are shown and three biological replicates had similar results .

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D Western blot detection of viral accumulation in BSMV- and BSMV3CS-infected N. benthamiana plants with anti-TGB1 or anti-γb antibodies. SL8, eighth systemically infected leaf; SL9, ninth systemically infected leaf. Ino, agroinfiltrated leaves. Rubisco large subunit (RbcL) was used as a loading control. Data information: In representative data are shown and three biological replicates had similar results.


F Western blot analysis of protein accumulation in BSMV - and BSMV3CS - systemically infected barley leaves with anti - TGB1 or anti - γb antibodies . RbcL was used as a loading control . Data information : In F ) , representative data are shown and three biological replicates had similar results .

F Western blot analysis of protein accumulation in BSMV- and BSMV3CS- systemically infected barley leaves with anti-TGB1 or anti-γb antibodies. RbcL was used as a loading control. Data information: In F), representative data are shown and three biological replicates had similar results.


A Effects of γb palmitoylation on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ / RNAγ3CS ) was agroinfiltrated into N . benthamiana leaves . At 3 dpi , total protein and RNA were extracted for Western and Northern blot analyses . Antibodies and probes used for molecular analyses are indicated on the right of each panel . Actin was used to monitor equal protein loading , and methylene blue - stained rRNAs served as RNA loading controls . Data information : representative data are shown and three biological replicates had similar results .

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A Effects of γb palmitoylation on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin was used to monitor equal protein loading, and methylene blue-stained rRNAs served as RNA loading controls. Data information: representative data are shown and three biological replicates had similar results.


B Western blot analyses of chloroplast localization of γb and γb3CS . Transiently expressed αa - GFP in intact chloroplasts isolated from BSMVγb - 3xFlag - or BSMV3CS - 3xFlag - infected N . benthamiana leaves at 4 dpi . Detection of γb - 3xFlag and γb3CS - 3xFlag with an anti - Flag antibody ; TGB1 analysis with an anti - TGB1 antibody ; αa - GFP detection with an anti - GFP antibody . NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase ( PEPC ) was used to assess cytosolic contamination of isolated chloroplasts . Data information : , representative data are shown and three biological replicates had similar results .

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B Western blot analyses of chloroplast localization of γb and γb3CS. Transiently expressed αa-GFP in intact chloroplasts isolated from BSMVγb-3xFlag- or BSMV3CS-3xFlag-infected N. benthamiana leaves at 4 dpi. Detection of γb-3xFlag and γb3CS-3xFlag with an anti-Flag antibody; TGB1 analysis with an anti-TGB1 antibody; αa-GFP detection with an anti-GFP antibody. NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase (PEPC) was used to assess cytosolic contamination of isolated chloroplasts. Data information: , representative data are shown and three biological replicates had similar results.


C Subcellular localization of γb - GFP and γb3CS - GFP in N . benthamiana epidermal leaf cells . The first and second rows show subcellular localization of γb - GFP and γb3CS - GFP transient expression in leaves . The third and fourth rows show localization of γb - GFP and γb3CS - GFP in BSMVγb - GFP - and BSMV3CS - GFP - infected leaves at 3 dpi . Figures on the right indicate the normalized fluorescence intensities of the GFP ( green ) or the chloroplast autofluorescence ( red ) channels along the dashed white lines shown in the merged images of the left panel . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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C Subcellular localization of γb-GFP and γb3CS-GFP in N. benthamiana epidermal leaf cells. The first and second rows show subcellular localization of γb-GFP and γb3CS-GFP transient expression in leaves. The third and fourth rows show localization of γb-GFP and γb3CS-GFP in BSMVγb-GFP- and BSMV3CS-GFP-infected leaves at 3 dpi. Figures on the right indicate the normalized fluorescence intensities of the GFP (green) or the chloroplast autofluorescence (red) channels along the dashed white lines shown in the merged images of the left panel. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


D Subcellular localization of γb - GFP or γb3CS - GFP in isolated N . benthamiana protoplasts harvested at 3 dpi . The fifth and sixth columns show γb localization in BSMVγb - GFP - ( movement - deficient ) N . benthamiana protoplasts after treatment with 200 μM 2 - BP inhibitor . The released protoplasts were collected after 48 h incubation in DMSO or 2 - BP followed by confocal microscopy visualization . Figures at the bottom indicate the normalized fluorescence intensities of the GFP ( green ) and the chloroplast autofluorescence ( red ) channels along the dashed white lines in the merged images above . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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D Subcellular localization of γb-GFP or γb3CS-GFP in isolated N. benthamiana protoplasts harvested at 3 dpi. The fifth and sixth columns show γb localization in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts after treatment with 200 μM 2-BP inhibitor. The released protoplasts were collected after 48 h incubation in DMSO or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP (green) and the chloroplast autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


A Effects of γb palmitoylation on virus cell - to - cell movement at 3 dpi with the BSMV duplex fluorescence ( dfBSMV ) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci , whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci . Fluorescence was evaluated by confocal microscopy . Scale bars , 100 μm . B Quantification of virus movement in Panel A . To determine the movement capacity of the viruses depicted in each panel , the mCherry and GFP fluorescent intensities were first determined by ImageJ software . Then , the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci ( n = 10 ) . The Y - axis indicates the relative sizes of the green areas in comparison to that of the red - colored areas . Data information : In ( A , representative data are shown and three biological replicates had similar results . In ( B ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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A Effects of γb palmitoylation on virus cell-to-cell movement at 3 dpi with the BSMV duplex fluorescence (dfBSMV) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci, whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci. Fluorescence was evaluated by confocal microscopy. Scale bars, 100 μm. B Quantification of virus movement in Panel A. To determine the movement capacity of the viruses depicted in each panel, the mCherry and GFP fluorescent intensities were first determined by ImageJ software. Then, the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci (n = 10). The Y-axis indicates the relative sizes of the green areas in comparison to that of the red-colored areas. Data information: In (A, representative data are shown and three biological replicates had similar results. In (B), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


D Biotin - switch assay to access palmitoylation of γb in BSMV - infected N . benthamiana leaves at different timepoints . BSMVγb - 3xFlag was treated with the hydroxylamine ( Hyd + ) thioester cleavage reagent . Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti - Flag antibody . Data information : In representative data are shown and three biological replicates had similar results .

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D Biotin-switch assay to access palmitoylation of γb in BSMV-infected N. benthamiana leaves at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine (Hyd+) thioester cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti-Flag antibody. Data information: In representative data are shown and three biological replicates had similar results.


E Subcellular localization of BSMVγb - GFP and BSMV3CS - GFP in N . benthamiana leaf epidermal cells . The mCherry - HDEL and mTalin - RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above . Scale bars , 10 μm . Data information : In E ) , representative data are shown and three biological replicates had similar results .

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E Subcellular localization of BSMVγb-GFP and BSMV3CS-GFP in N. benthamiana leaf epidermal cells. The mCherry-HDEL and mTalin-RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above. Scale bars, 10 μm. Data information: In E), representative data are shown and three biological replicates had similar results.


F , G Western blot analysis of subcellular fractions extracted from N . benthamiana leaf tissues at 3 dpi . S3 indicates the supernatant separated by centrifugation at 3 , 000 g ; and S30 is the 30 , 000 g supernatant ; P30 is the 30 , 000 g pellet . The association of γb with membranes was determined by 1 % Triton X - 100 extraction .

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F, G Western blot analysis of subcellular fractions extracted from N. benthamiana leaf tissues at 3 dpi. S3 indicates the supernatant separated by centrifugation at 3,000 g; and S30 is the 30,000 g supernatant; P30 is the 30,000 g pellet. The association of γb with membranes was determined by 1% Triton X-100 extraction.


A BiFC co - localization analyses of αa and TGB1 in N . benthamiana leaf cells at 3 dpi . YFPn and YFPc was fused to the TGB1 and the αa proteins , respectively . Combinations of γb , γb3CS and the TBSV P19 control are shown above the micrographs . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color . Scale bars , 30 μm . B Quantification of the numbers of YFP foci per visual field ( n = 10 ) . Data information : In ( A , , representative data are shown and three biological replicates had similar results . In ( B , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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A BiFC co-localization analyses of αa and TGB1 in N. benthamiana leaf cells at 3 dpi. YFPn and YFPc was fused to the TGB1 and the αa proteins, respectively. Combinations of γb, γb3CS and the TBSV P19 control are shown above the micrographs. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color. Scale bars, 30 μm. B Quantification of the numbers of YFP foci per visual field (n = 10). Data information: In (A, , representative data are shown and three biological replicates had similar results. In (B, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


C Co - IP assays to determine in vivo interactions between TGB1 and γb or γb3CS . N . benthamiana leaf tissues co - agroinfiltrated with RNAα , RNAγγb - GFP or RNAγ3CS - GFP , and TGB1 - 3xFlag plasmids were harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Input and immunoprecipitated proteins ( IP ) were analyzed by Western blotting with anti - GFP or anti - Flag antibodies .

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C Co-IP assays to determine in vivo interactions between TGB1 and γb or γb3CS. N. benthamiana leaf tissues co-agroinfiltrated with RNAα, RNAγγb-GFP or RNAγ3CS-GFP, and TGB1-3xFlag plasmids were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blotting with anti-GFP or anti-Flag antibodies.


D Confocal microscopy analyses showing RFP - TGB1 localization at the chloroplast periphery in N . benthamiana leaves agroinfiltrated with αa - GFP and CFP - γb plasmids . CFP - γb or CFP - γb3CS were co - expressed with RFP - TGB1 and αa - GFP in N . benthamiana leaves . Chloroplast autofluorescence was displayed in false pink color . The third and fourth rows show leaves treated with 400 μM 2 - BP inhibitor or the DMSO control at 1 dpi . Two days later , confocal microscopy observations of RFP - TGB1 , αa - GFP , and CFP - γb were performed . Figures on the right indicate the normalized fluorescence intensity of GFP , RFP , and CFP channels along the white dashed line in the merged confocal images . Scale bars , 10 μm . Data information : In , representative data are shown and three biological replicates had similar results .

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D Confocal microscopy analyses showing RFP-TGB1 localization at the chloroplast periphery in N. benthamiana leaves agroinfiltrated with αa-GFP and CFP-γb plasmids. CFP-γb or CFP-γb3CS were co-expressed with RFP-TGB1 and αa-GFP in N. benthamiana leaves. Chloroplast autofluorescence was displayed in false pink color. The third and fourth rows show leaves treated with 400 μM 2-BP inhibitor or the DMSO control at 1 dpi. Two days later, confocal microscopy observations of RFP-TGB1, αa-GFP, and CFP-γb were performed. Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP channels along the white dashed line in the merged confocal images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.


E Statistical analyses of the GFP and RFP co - localization in panel D . Pearson correlation coefficient ( Pearson ' s r ) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool ( n = 8 ) . F Relative percentages of RFP - TGB1 foci that co - localized with αa - GFP and CFP - γb at the periphery of the chloroplast images shown in panel D ( n = 8 ) . Data information : In E and F ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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E Statistical analyses of the GFP and RFP co-localization in panel D. Pearson correlation coefficient (Pearson's r) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool (n = 8). F Relative percentages of RFP-TGB1 foci that co-localized with αa-GFP and CFP-γb at the periphery of the chloroplast images shown in panel D (n = 8). Data information: In E and F), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


G Chloroplast extraction experiment for detection of TGB1 chloroplast localization . Plasmids harboring αa - GFP and RFP - TGB1 were co - infiltrated into N . benthamiana along with CFP - γb or CFP - γb3CS . Intact chloroplast preparations isolated from infiltrated N . benthamiana leaves at 4 dpi were subjected to Western blot analyses . RFP - TGB1 was analyzed with an anti - TGB1 antibody . The αa - GFP , CFP - γb , and CFP - γb3CS proteins were detected with an anti - GFP antibody . NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase ( PEPC ) served as a control to assess cytosolic contamination of isolated chloroplasts . Data information : In G ) , representative data are shown and three biological replicates had similar results .

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G Chloroplast extraction experiment for detection of TGB1 chloroplast localization. Plasmids harboring αa-GFP and RFP-TGB1 were co-infiltrated into N. benthamiana along with CFP-γb or CFP-γb3CS. Intact chloroplast preparations isolated from infiltrated N. benthamiana leaves at 4 dpi were subjected to Western blot analyses. RFP-TGB1 was analyzed with an anti-TGB1 antibody. The αa-GFP, CFP-γb, and CFP-γb3CS proteins were detected with an anti-GFP antibody. NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase (PEPC) served as a control to assess cytosolic contamination of isolated chloroplasts. Data information: In G), representative data are shown and three biological replicates had similar results.


B Analysis of γb palmitoylation in NbPAT15 - or NbPAT21 - silenced N . benthamiana plants . NbPAT15 and NbPAT21 were silenced by intron - spliced hairpin RNA - mediated RNAi approaches The γb - 3xFlag plasmid was agroinfiltrated into the leaves , followed by biotin - switch assays at 3 dpi . Data information : In ( B representative data are shown and three biological replicates had similar results .

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B Analysis of γb palmitoylation in NbPAT15- or NbPAT21-silenced N. benthamiana plants. NbPAT15 and NbPAT21 were silenced by intron-spliced hairpin RNA-mediated RNAi approaches The γb-3xFlag plasmid was agroinfiltrated into the leaves, followed by biotin-switch assays at 3 dpi. Data information: In (B representative data are shown and three biological replicates had similar results.


C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell - to - cell movement . Infiltrated leaves were observed by confocal microscopy at 3 dpi . Red fluorescence produced from the mCherry expression cassette shows the primary infection foci , and GFP fluorescence outside the red regions shows virus spread into secondary infected cells . Scale bars , 100 μm . Data information : In C ) , representative data are shown and three biological replicates had similar results .

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C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell-to-cell movement. Infiltrated leaves were observed by confocal microscopy at 3 dpi. Red fluorescence produced from the mCherry expression cassette shows the primary infection foci, and GFP fluorescence outside the red regions shows virus spread into secondary infected cells. Scale bars, 100 μm. Data information: In C), representative data are shown and three biological replicates had similar results.


D Quantification of BSMV movement . The green and red fluorescent areas were measured by ImageJ software ( n = 10 ) . Data information : In ( D ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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D Quantification of BSMV movement. The green and red fluorescent areas were measured by ImageJ software (n = 10). Data information: In (D), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


A , B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N . benthamiana at 3 dpi . PAT15 - YFPn and PAT21 - YFPn were co - expressed with BSMVγb - YFPc . Chloroplast autofluorescence is depicted as a false red color and mCherry - HDEL provides the ER marker . Figures on the right show the normalized fluorescence intensities of GFP , mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images . Scale bars , 20 μm . Data information : In ( A , B , representative data are shown and three biological replicates had similar results .

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A, B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N. benthamiana at 3 dpi. PAT15-YFPn and PAT21-YFPn were co-expressed with BSMVγb-YFPc. Chloroplast autofluorescence is depicted as a false red color and mCherry-HDEL provides the ER marker. Figures on the right show the normalized fluorescence intensities of GFP, mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 20 μm. Data information: In (A, B, representative data are shown and three biological replicates had similar results.


C Co - localization analyses of αa - GFP , RFP - TGB1 , and CFP - γb in N . benthamiana leaves at 3 dpi . Mixtures of A . tumefaciens containing αa - GFP , RFP - TGB1 , and CFP - γb were infiltrated into NbPATs - silenced N . benthamiana leaves or the non - silenced TRV : GUS control . Chloroplast autofluorescence is displayed as a false pink color . Figures on the right show the normalized fluorescence intensities of GFP , RFP , CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images . Scale bars , 10 μm . Data information : In , representative data are shown and three biological replicates had similar results .

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C Co-localization analyses of αa-GFP, RFP-TGB1, and CFP-γb in N. benthamiana leaves at 3 dpi. Mixtures of A. tumefaciens containing αa-GFP, RFP-TGB1, and CFP-γb were infiltrated into NbPATs-silenced N. benthamiana leaves or the non-silenced TRV:GUS control. Chloroplast autofluorescence is displayed as a false pink color. Figures on the right show the normalized fluorescence intensities of GFP, RFP, CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.


D Co - IP assays of interactions between γb and TGB1 in NbPAT15 - and NbPAT21 - silenced N . benthamiana leaves , TRV : GUS and TRV : GUS combined with the 2 - BP inhibitor provided positive and negative controls , respectively . The silenced leaves were inoculated with BSMV3xFlag - TGB1 / γb - GFP mixtures ( RNAα + RNAβ3xFlag - TGB1 + RNAγγb - GFP ) and harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Input and IP proteins were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . E Average amounts of RFP - TGB1 foci co - localizing with αa - GFP and CFP - γb at the chloroplast peripheries shown in panel C ( n = 12 ) . Data information : In E ) , representative data are shown and three biological replicates had similar results . In ( E ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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D Co-IP assays of interactions between γb and TGB1 in NbPAT15- and NbPAT21-silenced N. benthamiana leaves, TRV:GUS and TRV:GUS combined with the 2-BP inhibitor provided positive and negative controls, respectively. The silenced leaves were inoculated with BSMV3xFlag-TGB1/γb-GFP mixtures (RNAα + RNAβ3xFlag-TGB1 + RNAγγb-GFP) and harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and IP proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. E Average amounts of RFP-TGB1 foci co-localizing with αa-GFP and CFP-γb at the chloroplast peripheries shown in panel C (n = 12). Data information: In E), representative data are shown and three biological replicates had similar results. In (E), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


B Fractionation assay to detect γb membrane - associations . Separation of total protein ( T ) extracted from BSMV infected leaves into soluble ( S ) and membrane ( M ) fractions by ultracentrifugation at 100 , 000 g . PEPC and BiP , respectively , provide soluble and membrane fraction markers .

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B Fractionation assay to detect γb membrane-associations. Separation of total protein (T) extracted from BSMV infected leaves into soluble (S) and membrane (M) fractions by ultracentrifugation at 100,000 g. PEPC and BiP, respectively, provide soluble and membrane fraction markers.


C Aqueous two - phase partitioning of γb PM localization . The microsomal pellet from BSMVγb - GFP - infected leaves was obtained by ultracentrifugation at 100 , 000 g , and the two - phase fractionation procedure was used to separate the plasma membrane ( upper phase ) from other cellular membranes ( lower phase ) . H + - ATPase is a PM marker .

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C Aqueous two-phase partitioning of γb PM localization. The microsomal pellet from BSMVγb-GFP-infected leaves was obtained by ultracentrifugation at 100,000 g, and the two-phase fractionation procedure was used to separate the plasma membrane (upper phase) from other cellular membranes (lower phase). H+-ATPase is a PM marker.


D PM associations of γb - GFP and γb3CS - GFP in N . benthamiana protoplasts . The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb - GFP and γb3CS - GFP . The third and fourth columns show γb localization in BSMVγb - GFP and BSMV3CS - GFP - infected leaves . PIP2A provided a PM marker ( Nelson et al . , 2007 ) . Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images . Scale bars , 10 μm . Data information : In D , , representative data are shown and three biological replicates had similar results .

D PM associations of γb-GFP and γb3CS-GFP in N. benthamiana protoplasts. The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb-GFP and γb3CS-GFP. The third and fourth columns show γb localization in BSMVγb-GFP and BSMV3CS-GFP-infected leaves. PIP2A provided a PM marker (Nelson et al., 2007). Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In D, , representative data are shown and three biological replicates had similar results.


E Western blot analysis of subcellular fractions of BSMV infected N . benthamiana leaves at 3 dpi . The S3 and S30 fractions are the 3000 and 30000 g supernatants , and P30 is the 30000 g pellet . Membrane associated γb was released by 1 % Triton X - 100 treatment .

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E Western blot analysis of subcellular fractions of BSMV infected N. benthamiana leaves at 3 dpi. The S3 and S30 fractions are the 3000 and 30000 g supernatants, and P30 is the 30000 g pellet. Membrane associated γb was released by 1% Triton X-100 treatment.


F Western blot detection of BSMV accumulation in N . benthamiana plants . A . tumefaciens mixtures harboring RNAα and RNAβmTGB2 , along with RNAγ , RNAγγb - ROP , or RNAγγb - mROP , were agroinfiltrated into N . benthamiana leaves . Western blotting to detect virus accumulation at 3 dpi used anti - TGB1 or anti - CP antibodies . Actin provided loading controls .

F Western blot detection of BSMV accumulation in N. benthamiana plants. A. tumefaciens mixtures harboring RNAα and RNAβmTGB2, along with RNAγ, RNAγγb-ROP, or RNAγγb-mROP, were agroinfiltrated into N. benthamiana leaves. Western blotting to detect virus accumulation at 3 dpi used anti-TGB1 or anti-CP antibodies. Actin provided loading controls.


G Analysis of the effects of enhanced PM association of γb on cell - to - cell virus movement with the BSMV duplex fluorescence ( dfBSMV ) reporter system . Mixtures of A . tumefaciens containing dfBSMV , dfBSMVγb - GFP - ROP , and dfBSMVγb - GFP - mROP were infiltrated into N . benthamiana leaves . Infiltrated leaves were observed by confocal microscope at 3 dpi . Scale bars , 100 μm . ( n = 6 ) . Data information : In G ) , representative data are shown and three biological replicates had similar results . In ( G ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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G Analysis of the effects of enhanced PM association of γb on cell-to-cell virus movement with the BSMV duplex fluorescence (dfBSMV) reporter system. Mixtures of A. tumefaciens containing dfBSMV, dfBSMVγb-GFP-ROP, and dfBSMVγb-GFP-mROP were infiltrated into N. benthamiana leaves. Infiltrated leaves were observed by confocal microscope at 3 dpi. Scale bars, 100 μm. (n = 6). Data information: In G), representative data are shown and three biological replicates had similar results. In (G), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


B Co - IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N . benthamiana leaf tissues infiltrated with A . tumefaciens mixtures containing movement - deficient BSMV plasmids ( RNAα + RNAγγb - GFP or RNAγ3CS - GFP ) and NbREM1 - 3xFlag . Total proteins were extracted at 3 dpi and immunoprecipitated with anti - Flag beads . Input and immunoprecipitated proteins ( IP ) were analyzed by Western blot analyses with an anti - GFP or anti - Flag antibodies . Data information : In B , , representative data are shown and three biological replicates had similar results .

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B Co-IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N. benthamiana leaf tissues infiltrated with A. tumefaciens mixtures containing movement-deficient BSMV plasmids (RNAα + RNAγγb-GFP or RNAγ3CS-GFP) and NbREM1-3xFlag. Total proteins were extracted at 3 dpi and immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blot analyses with an anti-GFP or anti-Flag antibodies. Data information: In B, , representative data are shown and three biological replicates had similar results.


D Analysis of the cleaved 5 ( 6 ) - carboxyfluorescein ( CF ) movement in N . benthamiana leaves . RNAα + RNAγ / RNAγ3CS were co - infiltrated into transiently overexpressed NbREM1 N . benthamiana leaves , and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi . The areas of dye diffusion are marked by dashed white circles . Scale bars , 100 μm . Data information : In D , representative data are shown and three biological replicates had similar results .

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D Analysis of the cleaved 5(6)-carboxyfluorescein (CF) movement in N. benthamiana leaves. RNAα + RNAγ/RNAγ3CS were co-infiltrated into transiently overexpressed NbREM1 N. benthamiana leaves, and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi. The areas of dye diffusion are marked by dashed white circles. Scale bars, 100 μm. Data information: In D, representative data are shown and three biological replicates had similar results.


E Quantification of CF movement . Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software ( n = 6 ) . Data information : In ( E , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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E Quantification of CF movement. Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software (n = 6). Data information: In (E, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


F Aniline blue staining of callose in N . benthamiana leaf tissues . Leaves were infiltrated with mixtures of different A . tumefaciens constructs indicated beside the panels . At 3 dpi , the 0 . 1 % aniline blue fluorochrome was infiltrated into N . benthamiana leaves . After incubation at 5 min in the dark , the leaves were observed by confocal microscopy at 405 nm . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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F Aniline blue staining of callose in N. benthamiana leaf tissues. Leaves were infiltrated with mixtures of different A. tumefaciens constructs indicated beside the panels. At 3 dpi, the 0.1% aniline blue fluorochrome was infiltrated into N. benthamiana leaves. After incubation at 5 min in the dark, the leaves were observed by confocal microscopy at 405 nm. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


G Quantification of callose staining intensity as shown in panel F . The relative callose staining intensity was set to 1 in the REM samples ( n = 10 ) . Data information : In , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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G Quantification of callose staining intensity as shown in panel F. The relative callose staining intensity was set to 1 in the REM samples (n = 10). Data information: In , the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants . Scale bars , 20 μm . Data information : In H ) , representative data are shown and three biological replicates had similar results .

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H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants. Scale bars, 20 μm. Data information: In H), representative data are shown and three biological replicates had similar results.


I Quantification of callose staining intensity as shown in panel H . The relative callose staining intensity was set as 1 in the samples of EV ( n = 8 ) . Data information : In I ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) .

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I Quantification of callose staining intensity as shown in panel H. The relative callose staining intensity was set as 1 in the samples of EV (n = 8). Data information: In I), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


( B ) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT - PCR . Ribosomal protein 49 ( rp49 ) was used as an internal control ; n = 3 . Bars indicate mean ± s . d .

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(B) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT-PCR. Ribosomal protein 49 (rp49) was used as an internal control; n=3. Bars indicate mean±s.d.


( C ) Images of the larvae of denoted genotypes at 3 days ( top ) and 6 days ( middle ) after egg ‐ laying ( AEL ) , and quantification of the larvae that developed into the mid - late third instar stage of each genotype ( bottom ) ; n = 3 . Bars indicate mean ± s . d . Fifteen larvae of each genotype were examined in each experiment .

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(C) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (middle) after egg‐laying (AEL), and quantification of the larvae that developed into the mid-late third instar stage of each genotype (bottom); n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment.


( D ) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1 . The fat body images of the second / early third instar larvae of denoted genotypes under fully fed conditions .

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(D) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1. The fat body images of the second/early third instar larvae of denoted genotypes under fully fed conditions.


( E ) Images of the larval salivary glands of denoted genotypes . Hoechst33342 ( pseudo - coloured green ) and phalloidin - TRITC ( red ) were used to visualize the nuclei and cell boundary of larval salivary gland cells , respectively .

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(E) Images of the larval salivary glands of denoted genotypes. Hoechst33342 (pseudo-coloured green) and phalloidin-TRITC (red) were used to visualize the nuclei and cell boundary of larval salivary gland cells, respectively.


( F ) Quantitative analysis of cell and nuclear sizes in Fig 1E ; n = 5 . Bars indicate mean ± s . d . DmATG1 , Drosophila Autophagy ‐ specific gene 1 ; dTOR , Drosophila TOR ; qRT - PCR , quantitative real ‐ time reverse transcriptase - PCR .

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(F) Quantitative analysis of cell and nuclear sizes in Fig 1E; n=5. Bars indicate mean±s.d. DmATG1, Drosophila Autophagy‐specific gene 1; dTOR, Drosophila TOR; qRT-PCR, quantitative real‐time reverse transcriptase-PCR.


( A ) Quantification of adults that survived after eclosion for denoted genotypes ; n = 3 . Bars indicate mean ± s . d . Fifteen larvae of each genotype were examined in each experiment .

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(A) Quantification of adults that survived after eclosion for denoted genotypes; n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment.


( B , C ) Activation of dS6K in DmATG1 mutants . Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes . Tubulinimmunoblot was used as a loading control . Rheb expressing flies ( hs > Rheb ) were used as a positive control ( C ) . hs > Rheb larvae were subjected to heat shock at 37 ° C for 1 h and then incubated at 30 ° C for 3 h before sample preparation to induce Rheb expression . For quantification , the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop , and normalized to the tubulin levels . Results are expressed as a fold change compared with the wild ‐ type controls ( w1118 ) . DmATG1 , Drosophila Autophagy ‐ specific gene 1 ; dS6k , Drosophila S6 kinase .

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(B,C) Activation of dS6K in DmATG1 mutants. Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes. Tubulinimmunoblot was used as a loading control. Rheb expressing flies (hs>Rheb) were used as a positive control (C). hs>Rheb larvae were subjected to heat shock at 37°C for 1 h and then incubated at 30°C for 3 h before sample preparation to induce Rheb expression. For quantification, the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop, and normalized to the tubulin levels. Results are expressed as a fold change compared with the wild‐type controls (w1118). DmATG1, Drosophila Autophagy‐specific gene 1; dS6k, Drosophila S6 kinase.


( A , B ) HEK 293T cells transfected with denoted plasmids were ( A ) deprived of nutrients for 90 min , and then replenished with DMEM for 30 min or ( B ) stimulated with 50 ng / ml epidermal growth factor ( EGF ) for 30 min . The S6K immune complexes were analysed by immunoblot using appropriate antibodies ( top three panels ) . Flag ( ATG1α ) blots were completed from the same cell lysates ( bottom panel ) .

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(A,B) HEK 293T cells transfected with denoted plasmids were (A) deprived of nutrients for 90 min, and then replenished with DMEM for 30 min or (B) stimulated with 50 ng/ml epidermal growth factor (EGF) for 30 min. The S6K immune complexes were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel).


( C ) HEK 293T cells transfected with control siRNA , ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min , and then the phosphorylation and protein levels of endogenous S6K ( top two panels ) and S6 ( bottom two panels ) were determined .

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(C) HEK 293T cells transfected with control siRNA, ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min, and then the phosphorylation and protein levels of endogenous S6K (top two panels) and S6 (bottom two panels) were determined.


( D ) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min , and then replenished with DMEM for 30 min . The phosphorylation and protein levels of endogenous S6K were examined . For quantification , the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop , and normalized to the S6K protein levels . Results are expressed as a fold change compared with the first indicated sample .

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(D) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The phosphorylation and protein levels of endogenous S6K were examined. For quantification, the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop, and normalized to the S6K protein levels. Results are expressed as a fold change compared with the first indicated sample.


( E ) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP ‐ C1 ( a transfection marker , green ) , and nutrient ‐ starved for 90 min , fixed and stained with phosphospecific S6 antibody ( red ) . ATG1 , Autophagy ‐ specific gene 1 ; HA , haemagglutinin ; KI , ATG1α Lys 46 Ile ( kinase ‐ dead ) mutant ; siRNA , short interfering RNA ; S6K , S6 kinase ; WT , wild type .

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(E) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP‐C1 (a transfection marker, green), and nutrient‐starved for 90 min, fixed and stained with phosphospecific S6 antibody (red). ATG1, Autophagy‐specific gene 1; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; siRNA, short interfering RNA; S6K, S6 kinase; WT, wild type.