( a ) Stable 293T in presence of pro - caspase - 1 , pro - IL - 1β ASC overexpression were transfected with control vector or BRLF1 with control vector , IFI16 , NLRP3 , RIG - I or AIM2 for 36 h . After were infected with HSV - 1 ( MOI = 1 ) for 12 h , were collected , lysates were analyzed by western blotting to determine of pro - caspase - 1 , caspase - 1 , pro - IL - 1β mature IL - 1β as indicated .

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(a) Stable 293T cells in the presence of pro-caspase-1, pro-IL-1β and ASC overexpression were transfected with control vector or BRLF1 with control vector, IFI16, NLRP3, RIG-I or AIM2 for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the cells were collected, and the cell lysates were analyzed by western blotting to determine the levels of pro-caspase-1, cleaved caspase-1, pro-IL-1β and mature IL-1β as indicated.


( b ) Scramble ( sc ) or shRNAs against RIG - I empty control vector ( ctr ) , BRLF1 - or BRLF1 L578A - expressing plasmids were co - transfected into EBV - ΔBRLF1 - harboring HNE1 . Thirty - six hours after transfection , pellets were collected , after which cleavage of caspase - 1 IL - 1β was measured as indicated .

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(b) Scramble (sc) or shRNAs against RIG-I and empty control vector (ctr), BRLF1- or BRLF1 L578A-expressing plasmids were co-transfected into EBV-ΔBRLF1-harboring HNE1 cells. Thirty-six hours after the transfection, cell pellets were collected, after which the cleavage of caspase-1 and IL-1β was measured as indicated.


( c ) of ASC oligomerization . ASC - expressing plasmid was transfected into 293T or HNE1 with empty vector or GFP - BRLF1 in presence of AIM2 - or RIG - I - expressing plasmid . Twelve hours after HSV - 1 infection ( MOI = 1 ) in 293T , or 48 h after EBV - ΔBRLF1 infection ( MOI = 1000 ) in HNE1 , pellets were collected , lysates were treated with DSS to induce chemical cross - linking analyzed by western blotting for ASC oligomerization .

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(c) Analysis of ASC oligomerization. ASC-expressing plasmid was transfected into 293T cells or HNE1 cells with empty vector or GFP-BRLF1 in the presence of AIM2- or RIG-I-expressing plasmid. Twelve hours after HSV-1 infection (MOI=1) in 293T cells, or 48 h after EBV-ΔBRLF1 infection (MOI=1000) in HNE1 cells, the cell pellets were collected, and cell lysates were treated with DSS to induce chemical cross-linking and analyzed by western blotting for ASC oligomerization.


( d ) EBV - ΔBRLF1 - harboring HNE1 were transfected with control vector , BRLF1 or BRLF1 L578A in absence or presence of BZLF1 for 48 h . total RNAs were extracted subjected to real - time PCR .

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(d) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis.


( e ) A549 were transfected with control vector , BRLF1 or BRLF1 L578A for 36 h . After were infected with HSV - 1 ( MOI = 1 ) for 12 h , total RNAs were extracted subjected to real - time PCR .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(e) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1 (MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis.


Jurkat ( a ) were infected with EBV - WT or EBV - ΔBRLF1 ( MOI = 500 ) for 36 h . Then , were collected , total RNAs were extracted , reverse - transcribed analyzed by real - time PCR . Color gradient depicts mean mRNA expression normalized to housekeeping gene GAPDH compared to mock group .

B-CELL_LINE O O O O O O B-ORGANISM O O O B-ORGANISM O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O

Jurkat cells (a) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group.


NK - 92MI ( b ) were infected with EBV - WT or EBV - ΔBRLF1 ( MOI = 500 ) for 36 h . Then , were collected , total RNAs were extracted , reverse - transcribed analyzed by real - time PCR . Color gradient depicts mean mRNA expression normalized to housekeeping gene GAPDH compared to mock group .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM O O O B-ORGANISM O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O

NK-92MI cells (b) were infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500) for 36 h. Then, the cells were collected, and the total RNAs were extracted, reverse-transcribed and analyzed by real-time PCR. Color gradient depicts the mean mRNA expression normalized to the housekeeping gene GAPDH and compared to the mock group.


Jurkat NK - 92MI were cultured with conditional medium from EBV - WT or EBV - ΔBRLF1 - infected Ramos ( paracrine ) , or Jurkat NK - 92MI were directly infected with EBV - WT or EBV - ΔBRLF1 ( autocrine ) . ( c ) pellets were collected whole extracts were analyzed by western blotting to detect TAK1 , IKKα / β IκB phosphorylation .

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Jurkat and NK-92MI cells were cultured with conditional medium from EBV-WT or EBV-ΔBRLF1-infected Ramos cells (paracrine), or Jurkat and NK-92MI cells were directly infected with EBV-WT or EBV-ΔBRLF1 viruses (autocrine). (c) The cell pellets were collected and the whole cell extracts were analyzed by western blotting to detect TAK1, IKKα/β and IκB phosphorylation.


killing activity of Jurkat ( d ) were measured by detection of LDH release from K562 target .

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The cell killing activity of Jurkat cells (d) were measured by the detection of the LDH release from the K562 target cells.


killing activity of NK - 92MI ( e ) were measured by detection of LDH release from K562 target .

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The cell killing activity of NK-92MI cells (e) were measured by the detection of the LDH release from the K562 target cells.


Anti - IL - 1β / or anti - IL - 18 neutralizing antibodies ( NA ) , TAT - Flag or TAT - N572 peptide were added into EBV - WT - or EBV - ΔBRLF1 - infected Jurkat or NK - 92MI for 24 h . ( f ) TAK1 , IKKα IκB phosphorylation were detected as indicated .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O

Anti-IL-1β and/or anti-IL-18 neutralizing antibodies (NA), TAT-Flag or TAT-N572 peptide were added into EBV-WT- or EBV-ΔBRLF1-infected Jurkat cells or NK-92MI cells for 24 h. (f) TAK1, IKKα and IκB phosphorylation were detected as indicated.


killing activity of Jurkat ( g ) were measured .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O

cell killing activity of Jurkat cells (g) were measured.


killing activity of NK - 92MI ( h ) were measured .

cell killing activity of NK-92MI cells (h) were measured.


( a - d ) PBMCs were isolated infected with EBV - WT or EBV - ΔBRLF1 ( MOI = 500 ) . Thirty - six hours after infection , were harvested total RNAs were extracted , reverse - transcribed subjected to real - time PCR . differential expression of receptors cytokines ( a ) , cytotoxic genes ( b ) , T - specific genes ( c ) NK - specific genes ( d ) are shown . values are shown as mean ± standard deviations of five analyses of PBMCs from five different health donors .

O O O O O B-CELL_TYPE O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O

(a-d) PBMCs were isolated and infected with EBV-WT or EBV-ΔBRLF1 virus (MOI=500). Thirty-six hours after infection, the cells were harvested and the total RNAs were extracted, reverse-transcribed and subjected to real-time PCR analysis. The differential expression of receptors and cytokines (a), cytotoxic genes (b), T cell-specific genes (c) and NK cell-specific genes (d) are shown. The values are shown as the mean ± standard deviations of five analyses of PBMCs from five different health donors.


( e - h ) FACS of T NK activation in PBMCs in presence of EBV - WT or EBV - ΔBRLF1 infection . PBMCs were left uninfected or infected with EBV - WT or EBV - ΔBRLF1 for 36 h , EBV - ΔBRLF1 - infected lymphocytes were incubated with additional TAT - Flag or TAT - N572 peptide for 12 h . Immunofluorescent PE - CD3 APC - CD25 antibodies were used to detect T activation ( e , g ) , PE - CD56 APC - CD69 were used to detect NK activation ( f , h ) . Representative of FACS of T NK activation are shown ( e , f ) , percentages of activated T NK were calculated are shown as mean ± standard deviation ( g , h ) for lymphocytes from 5 different health donors ;

O O O O O B-EXP_ASSAY O O B-CELL_TYPE B-EXP_ASSAY O B-CELL_TYPE O O O B-ORGANISM O O O B-ORGANISM O O O O B-CELL_TYPE O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O B-ORGANISM O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O

(e-h) FACS analysis of T and NK cell activation in PBMCs in the presence of EBV-WT or EBV-ΔBRLF1 primary infection. The PBMCs were left uninfected or infected with EBV-WT or EBV-ΔBRLF1 virus for 36 h, and the EBV-ΔBRLF1-infected lymphocytes were incubated with additional TAT-Flag or TAT-N572 peptide for 12 h. Immunofluorescent PE-CD3 and APC-CD25 antibodies were used to detect T cell activation (e, g), and PE-CD56 and APC-CD69 were used to detect NK cell activation (f, h). Representative images of the FACS analysis of T cell and NK cell activation are shown (e, f), and the percentages of activated T cells and NK cells were calculated and are shown as the mean ± standard deviation (g, h) for lymphocytes from 5 different health donors;


B Representative showing co - expression of stage - specific markers Nestin , Tbr2 , Doublecortin ( DCX ) , Calbindin ( in green ) , with β - Galactosidase ( β - Gal , red ) reporter in 8 - week old BATGAL Axin2LacZ / + mice . Nuclei are counterstained with DAPI ( in blue ) . Scale bar = 10µm . Insets show a 1 . 5x magnification of selected ( position indicated by dashed box ) . Scale bar = 5µm .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O B-ORGANISM O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O 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 Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-Galactosidase (β-Gal, red) reporter in 8-week old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar= 10µm. Insets show a 1.5x magnification of selected cells (position indicated by dashed box). Scale bar= 5µm.


C Fraction of Nestin - , Tbr2 - , DCX - Calbindin - positive expressing β - Gal in BATGAL Axin2LacZ / + show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [ BATGAL : Tbr2 vs . DCX p = 0 . 0056 , DCX vs . Calbindin p < 0 . 0001 ; Axin2LacZ : Nestin vs . Tbr2 p = 0 . 0003 , Tbr2 vs . DCX p = 0 . 0115 , DCX vs . Calbindin p < 0 . 0001 ] ( n = 3 per mouse model marker ) .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O

C Fraction of Nestin-, Tbr2-, DCX- and Calbindin-positive cells expressing β-Gal in BATGAL and Axin2LacZ/+ animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [BATGAL: Tbr2 vs. DCX p=0.0056, DCX vs. Calbindin p<0.0001; Axin2LacZ: Nestin vs. Tbr2 p=0.0003, Tbr2 vs. DCX p=0.0115, DCX vs. Calbindin p<0.0001] (n= 3 animals per mouse model and marker).


D Detection of corrected total ( CTCF ) of β - Gal reporter in Nestin - , Tbr2 - , DCX - Calbindin - positive in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [ Nestin vs . Tbr2 p < 0 . 0001 , Tbr2 vs . DCX p = 0 . 0029 , DCX vs . Calbindin p < 0 . 0001 ] ( Nestin : n = 236 , Tbr2 : n = 134 , DCX : n = 200 , Calbindin : n = 300 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O

D Detection of the corrected total cell fluorescence (CTCF) of the β-Gal reporter in Nestin-, Tbr2-, DCX- and Calbindin-positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [Nestin vs. Tbr2 p <0.0001, Tbr2 vs. DCX p=0.0029, DCX vs. Calbindin p<0.0001] (Nestin: n= 236 cells, Tbr2: n= 134 cells, DCX: n= 200 cells, Calbindin: n= 300 cells).


F of BrdU + expressing β - Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [ 0 vs . 7dpi p = 0 . 0036 , 7 vs . 28dpi p = 0 . 0115 , 7 vs . 42dpi p = 0 . 0007 ] ( 0dpi time - point ; n = 4 ) , 3dpi ( n = 3 ) , 7dpi ( n = 3 ) , 14dpi ( n = 4 ) , 28dpi ( n = 6 ) 42dpi ( n = 6 ) .

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F Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [0 vs. 7dpi p=0.0036, 7 vs. 28dpi p=0.0115, 7 vs. 42dpi p=0.0007] (0dpi time-point; n= 4 animals), 3dpi (n= 3 animals), 7dpi (n= 3 animals), 14dpi (n= 4 animals), 28dpi (n= 6 animals) and 42dpi (n= 6 animals).


G Total of β - Gal in BrdU + indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [ 0 vs . 3dpi p < 0 . 0001 , 7 vs . 14dpi p < 0 . 0001 , 14 vs . 28dpi p < 0 . 0001 , 28 vs . 42dpi p = 0 . 0333 ] ( 0dpi n = 194 , 3dpi n = 245 , 7dpi n = 224 , 14dpi n = 72 , 28dpi n = 103 42dpi n = 114 )

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G Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [0 vs. 3dpi p<0.0001, 7 vs. 14dpi p<0.0001, 14 vs. 28dpi p<0.0001, 28 vs. 42dpi p=0.0333] (0dpi n= 194 cells, 3dpi n= 245 cells, 7dpi n= 224 cells, 14dpi n= 72 cells, 28dpi n= 103 cells and 42dpi n= 114 cells)


B Representative of transduced adult - born neurons at 17dpi . CAG - dnLEF - IRES - GFP ( dnLEF , green ) CAG - RFP ( control , red ) double transduced ( arrows ) CAG - RFP single transduced ( arrowheads ) . Scale bar = 20

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B Representative images of transduced adult-born neurons at 17dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar= 20 µm


C Representative reconstructions of control dnLEF neurons . Scale bar = 20

C Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µm


D of showed a reduction in dendritic in dnLEF transduced neurons [ p < 0 . 0001 ] , while number of branch points remained comparable [ p = 0 . 7914 ] . Sholl displayed a reduction in dendritic complexity in dnLEF transduced neurons indicated decreased growth of terminal dendritic branches [ p < 0 . 0001 ] ( control : n = 20 from 3 , dnLEF : n = 38 from 6 ) .

O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [p<0.0001], while the number of branch points remained comparable [p=0.7914]. Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


E DnLEF neurons displayed basal dendrites [ p < 0 . 0001 ] ( control : n = 20 from 3 , dnLEF : n = 38 from 6 ) .

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E DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


G Representative depicting CAG - GFP - IRES - Cre transduced adult - born neurons in control β - catex3 mice at 17dpi . Scale bar =

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G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µm


H Representative reconstructions of control β - catex3 neurons . Scale bar =

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H Representative reconstructions of control and β-catex3 neurons. Scale bar= 20µm


I showed decreased dendritic of β - catex3 neurons [ p < 0 . 0001 ] ; no difference was apparent in number of branch points [ p = 0 . 3256 ] . Sholl displayed a less dendritic tree of β - catex3 neurons [ p < 0 . 0001 ] ( control : n = 20 from 5 , β - catex3 : n = 20 from 5 ) .

O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

I Quantification showed decreased dendritic length of β-catex3 neurons [p<0.0001]; no difference was apparent in number of branch points [p=0.3256]. Sholl analysis displayed a less complex dendritic tree of β-catex3 neurons [p<0.0001] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).


J number of basal dendrites was increased in β - catex3 neurons [ p = 0 . 0003 ] ( control : n = 20 from 5 , β - catex3 : n = 20 from 5 ) .

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J The number of basal dendrites was increased in β-catex3 neurons [p=0.0003] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).


B Representative reconstructions of control β - catex3 iDCX neurons at 3dpT . Scale bar = 20µm .

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B Representative reconstructions of control and β-catex3 iDCX neurons at 3dpT. Scale bar= 20µm.


C of showed increased dendritic in β - catex3 iDCX neurons at 3dpT [ p = 0 . 0320 ] . number of branch points was unaltered [ p = 0 . 0651 ] . Sholl indicated increased growth of terminal dendrite branches in neurons of β - catex3 iDCX mice [ p < 0 . 0001 ] ( control : n = 25 from 9 ; β - catex3 iDCX : n = 25 from 8 ) . D of showed no difference in dendritic [ p = 0 . 1029 ] branch point number [ p = 0 . 3726 ] between experimental groups at 13dpT . Sholl indicated a decrease in of terminal dendrites in neurons of β - catex3 iDCX mice [ p < 0 . 01 ] ( control : n = 23 from 9 , β - catex3 iDCX : n = 26 from 8 ) .

O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

C Analysis of morphology showed increased dendritic length in β-catex3 iDCX neurons at 3dpT [p=0.0320]. The number of branch points was unaltered [p=0.0651]. Sholl analysis indicated increased growth of terminal dendrite branches in neurons of β-catex3 iDCX mice [p<0.0001] (control: n = 25 cells from 9 animals; β-catex3 iDCX: n = 25 cells from 8 animals). D Analysis of morphology showed no difference in dendritic length [p=0.1029] and branch point number [p=0.3726] between experimental groups at 13dpT. Sholl analysis indicated a decrease in length of terminal dendrites in neurons of β-catex3 iDCX mice [p<0.01] (control: n= 23 cells from 9 animals, β-catex3 iDCX: n= 26 cells from 8 animals).


E Spine densities were comparable between experimental groups at 3dpT [ iML p = 0 . 1512 , oML p = 0 . 3841 ] . 13dpT β - catex3 iDCX neurons showed significantly increased spine density in inner molecular layer [ iML p < 0 . 0001 , oML p = 0 . 0402 ] ( 3dpT : control : n = 20 from 3 , β - catex3 iDCX : n = 20 from 4 ; 13dpT control : n = 20 from 5 , β - catex3 iDCX : n = 20 from 7 ) . Representative showing dendritic segments in inner molecular layer of DG at 13dpT . Scale bars = 1µm .

O B-SUBCELLULAR O 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-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-SUBCELLULAR O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O O O O

E Spine densities were comparable between experimental groups at 3dpT [iML p=0.1512, oML p=0.3841]. 13dpT β-catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [iML p<0.0001 , oML p=0.0402] (3dpT: control: n= 20 cells from 3 animals, β-catex3 iDCX: n= 20 cells from 4 animals; 13dpT control: n= 20 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 7 animals). Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT. Scale bars= 1µm.


F Representative of recombined ( GFP + ) neurons co - expressing Prox1 ( grey ) as a marker for neuronal fate stage specific markers DCX ( red ) for immature neurons Calbindin ( red ) for mature neurons . Arrows arrowheads indicate marker negative marker positive , respectively . Scale bar = 10µm . G Fraction of DCX expressing neurons is slightly reduced in β - catex3 iDCX at 3dpT [ p = 0 . 0338 ] , while fraction of Calbindin expressing is slightly increased in β - catex3 iDCX at 3dpT [ p = 0 . 0345 ] . At 13dpT no difference is visible in stage specific marker expression [ DCX p = 0 . 8564 , Calbindin p = 0 . 0841 ] ( 3dpT : control : n = 12 , β - catex3 iDCX : n = 12 , 13dpT : control : n = 10 , β - catex3 iDCX : n = 14 ) .

O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O B-CELL_TYPE B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

F Representative images of recombined (GFP+) neurons co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar = 10µm. G Fraction of DCX expressing neurons is slightly reduced in β-catex3 iDCX at 3dpT [p=0.0338], while fraction of Calbindin expressing cells is slightly increased in β-catex3 iDCX at 3dpT [p=0.0345]. At 13dpT no difference is visible in stage specific marker expression [DCX p=0.8564, Calbindin p=0.0841] (3dpT: control: n= 12 animals, β-catex3 iDCX: n= 12 animals, 13dpT: control: n= 10 animals, β-catex3 iDCX: n= 14 animals).


B Expression of DCX Calbindin in BrdU + neurons was comparable between experimental groups at 3dpT 13dpT ( control : n = 5 , β - catex3 iDCX : n = 5 ) .

O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

B Expression of DCX and Calbindin in BrdU+ neurons was comparable between experimental groups at 3dpT and 13dpT (control: n= 5 animals, β-catex3 iDCX: n= 5 animals).


C Representative reconstructions of control β - catex3 iDCX neurons expressing RFP at 3dpT . Scale bar = 20µm .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O O O

C Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 3dpT. Scale bar= 20µm.


D of dendritic [ p = 0 . 0003 ] , branch points [ p = 0 . 0433 ] , Sholl [ p < 0 . 0001 ] showed a higher dendritic complexity of β - catex3 iDCX neurons at 3dpT ( control : n = 22 from 4 , β - catex3 iDCX : n = 19 from 5 ) .

O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

D Quantification of dendritic length [p=0.0003], branch points [p=0.0433], and Sholl analysis [p<0.0001] showed a higher dendritic complexity of β-catex3 iDCX neurons at 3dpT (control: n= 22 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


E Representative reconstructions of control β - catex3 iDCX neurons expressing RFP at 13dpT . Scale bar = 20µm .

E Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 13dpT. Scale bar= 20µm.


F of dendritic [ p < 0 . 0001 ] , branch points [ p = 0 . 0495 ] , Sholl [ p < 0 . 0001 ] showed a lower dendritic complexity of β - catex3 iDCX neurons at 13dpT ( control : n = 20 from 4 , β - catex3 iDCX : n = 19 from 5 ) .

F Quantification of dendritic length [p<0.0001], and branch points [p=0.0495], and Sholl analysis [p<0.0001] showed a lower dendritic complexity of β-catex3 iDCX neurons at 13dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


A Representative overview of β - Galactosidase reporter signal in DG of 8 - week old , 24 - week old 36 - week old BATGAL mice . Note decrease in reporter signal with increasing ( n = 4 per , 200 - 300 per ) [ 8 vs 24 p = 0 . 0268 , 8 vs 36 p = 0 . 0268 ] . Scale bar = 100µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative overview images and quantification of the β-Galactosidase reporter signal in the DG of 8-week old, 24-week old and 36-week old BATGAL mice. Note the decrease in reporter signal with increasing age (n= 4 animals per age, 200-300 cells per animal) [8 weeks vs 24 weeks p=0.0268, 8 weeks vs 36 weeks p=0.0268]. Scale bar= 100µm.


B Representative of BrdU + ( red ) expressing DCX ( grey ) or β - Galactosidase ( green ) at 14 days post BrdU injection ( dpBrdU ) , 28dpBrdU 42dpBrdu in 8 - week old 24 - week old BATGAL mice . Scale bar = 10µm . C of BrdU + expressing DCX in 8 - week old 24 - week old BATGAL mice ( 14dpi : n = 3 , 28dpi : n = 3 , 42dpi : n = 3 ) . Note prolonged expression of DCX in 24 - week old mice [ 28dpBrdU p = 0 . 0155 ] . D of BrdU + expressing β - Galactosidase reporter in 8 - week old 24 - week old BATGAL mice ( 14dpi : n = 3 , 28dpi : n = 3 , 42dpi : n = 3 ) . Note reduced reporter expression in 24 - week old mice [ 28dpBrdU p = 0 . 0117 , 42dpBrdU p = 0 . 0034 ] .

O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-ORGANISM 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-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O

B Representative images of BrdU+ cells (red) expressing DCX (grey) or β-Galactosidase (green) at 14 days post BrdU injection (dpBrdU), 28dpBrdU and 42dpBrdu in 8-week old and 24-week old BATGAL mice. Scale bar= 10µm. C Percentage of BrdU+ cells expressing DCX in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the prolonged expression of DCX in 24-week old mice [28dpBrdU p=0.0155]. D Percentage of BrdU+ cells expressing the β-Galactosidase reporter in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the reduced reporter expression in 24-week old mice [28dpBrdU p=0.0117, 42dpBrdU p=0.0034].


B Representative reconstructions of control β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O

B Representative reconstructions of control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar = 20µm.


C Dendritic [ p < 0 . 0001 ] number of branch points [ p < 0 . 0001 ] was increased in 24 - week - old β - catex3 iDCX compared to 24 - week - old controls at 3dpT ( control : n = 21 from 5 , β - catex3 iDCX : n = 20 from 6 ) . At 13dpT dendritic [ p = 0 . 5788 ] number of branch points [ p = 0 . 4978 ] were comparable ( control : n = 21 from 5 , β - catex3 iDCX : n = 20 from 8 ) .

O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

C Dendritic length [p<0.0001] and number of branch points [p<0.0001] was increased in 24-week-old β-catex3 iDCX compared to 24-week-old controls at 3dpT (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 6 animals). At 13dpT dendritic length [p=0.5788] and number of branch points [p=0.4978] were comparable (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 8 animals).


D Representative showing dendritic segment of inner molecular layer of DG in 24 - week old mice at 13dpT . of number of spines showed increased spine density in inner molecular outer molecular layer in 24 - week old β - catex3 iDCX mice at 13dpT [ iML p = 0 . 0047 , oML p < 0 . 0001 ] ( control n = 19 from 4 , β - catex3 iDCX : n = 20 from 4 ) . Scale bars = 1µm .

O O O B-SUBCELLULAR O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-ORGANISM O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

D Representative images showing dendritic segment of the inner molecular layer of the DG in 24-week old mice at 13dpT. Quantification of the number of spines showed increased spine density in inner molecular and outer molecular layer in 24-week old β-catex3 iDCX mice at 13dpT [iML p=0.0047, oML p<0.0001] (control n=19 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals). Scale bars = 1µm.


E Representative of recombined ( GFP + , green ) neurons in 24 - week old at 13dpT co - expressing Prox1 ( grey ) as a marker for neuronal fate stage specific markers DCX ( red ) for immature neurons Calbindin ( red ) for mature neurons . Arrows arrowheads indicate marker negative marker positive , respectively . Scale bar = 10µm . F of marker expression . fraction of Calbindin expressing was increased in recombined population in β - catex3 iDCX at 3dpT [ p = 0 . 0081 ] 13dpT [ p = 0 . 0259 ] ( 3dpT : control : n = 4 , β - catex3 iDCX : n = 9 , 13dpT : control : n = 8 , β - catex3 iDCX : n = 10 ) .

O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O B-CELL_TYPE B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

E Representative images of recombined (GFP+, green) neurons in 24-week old animals at 13dpT co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. F Quantification of marker expression. The fraction of Calbindin expressing cells was increased in the recombined population in β-catex3 iDCX at 3dpT [p=0.0081] and 13dpT [p=0.0259] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 9 animals, 13dpT: control: n= 8 animals, β-catex3 iDCX: n= 10 animals).


B Representative of recombined ( GFP + , green ) BrdU + ( grey ) neurons in 24 - week old control β - catex3 iDCX at 13dpT expressing stage specific markers DCX ( red ) for immature neurons Calbindin ( red ) for mature neurons . Arrows arrowheads indicate marker negative marker positive , respectively . Scale bar = 10µm . C of DCX Calbindin expression in BrdU + recombined . At 13dpT , labeled neurons in β - catex3 iDCX show a shift towards a more mature marker profile [ DCX p = 0 . 0238 , Calbindin p = 0 . 0714 ] ( 3dpT : control : n = 4 , β - catex3 iDCX : n = 8 ; 13dpT control : n = 5 , β - catex3 iDCX : n = 5 ) .

O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

B Representative images of recombined (GFP+, green) BrdU+ (grey) neurons in 24-week old control and β-catex3 iDCX animals at 13dpT expressing the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm. C Quantification of DCX and Calbindin expression in BrdU+ recombined cells. At 13dpT, labeled neurons in β-catex3 iDCX animals show a shift towards a more mature marker profile [DCX p=0.0238, Calbindin p=0.0714] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 8 animals; 13dpT control: n= 5 animals, β-catex3 iDCX: n= 5 animals).


D Representative reconstructions of RFP - birthdated control β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm .

O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O

D Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar= 20µm.


E of dendritic [ p = 0 . 0007 ] , branch points [ p = 0 . 3909 ] , Sholl [ p = 0 . 0001 ] showed increased complexity of birthdated β - catex3 iDCX at 3dpT ( control : n = 20 from 4 , β - catex3 iDCX : n = 19 from 5 ) .

O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

E Quantification of dendritic length [p=0.0007], branch points [p=0.3909], and Sholl analysis [p=0.0001] showed increased complexity of birthdated β-catex3 iDCX cells at 3dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).


F Representative reconstructions of RFP - birthdated control β - catex3 iDCX neurons in 24 - week old mice at 13dpT . Scale bar = 20µm .

F Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 13dpT. Scale bar= 20µm.


G of dendritic [ p = 0 . 1857 ] , branch points [ p = 0 . 1331 ] , Sholl [ p = 0 . 1501 ] showed no difference between β - catex3 iDCX control at 13dpT ( control : n = 18 from 4 , β - catex3 iDCX : n = 20 from 4 )

O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

G Quantification of dendritic length [p=0.1857], branch points [p=0.1331], and Sholl analysis [p=0.1501] showed no difference between β-catex3 iDCX and control cells at 13dpT (control: n= 18 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals)


Luciferase - imaging of drug response in Vegfr3Luc ; Tyr : CreERT2 ; BrafV600E ; Ptenflox / flox mice . Panels labeled as " basal " " induced " correspond to bioluminescence of prior 5 after administration of 4OH - tamoxifen ( 5 mM , topical administration , 3 consecutive days ) for induction of melanomas . ( Right panels ) Treatment with anti - PD - L1 antibody ( αPD - L1 ; clone 10F . 9G2 , 3 ) or corresponding control IgG ( 200 ug / dose , twice per week , 3 ) ; vemurafenib ( Vem , 50 mg / Kg , oral once per day , 3 ) or BO - 110 ( BO , 0 . 8 mg / kg , twice per week , 3 ) . Scale : p / s / cm2 / sr ( x106 ) .

B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Luciferase-based imaging of drug response in Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) Treatment with anti-PD-L1 antibody (αPD-L1; clone 10F.9G2, 3 weeks) or the corresponding control IgG (200 ug/dose, twice per week, 3 weeks); vemurafenib (Vem, 50 mg/Kg, oral once per day, 3 weeks) or BO-110 (BO, 0.8 mg/kg, twice per week, 3 weeks). Scale: p/s/cm2/sr (x106).


Treatment with BO - 110 of human - derived xenografts ( PDX ) implanted in Vegfr3Luc nu / nu . 42 days after implantation ( when systemic luciferase was detected ) , were randomized for treatment with vehicle ( V ) or with 0 . 8 mg / kg BO - 110 ( BO , twice per week ) , luciferase emission was acquired at indicated times . Scale , p / s / cm2 / sr ( x106 ) .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Treatment with BO-110 of human patient-derived xenografts (PDX) implanted in Vegfr3Luc nu/nu. 42 days after implantation (when systemic luciferase was detected), animals were randomized for treatment with vehicle (V) or with 0.8 mg/kg BO-110 (BO, twice per week), and luciferase emission was acquired at the indicated times. Scale, p/s/cm2/sr (x106).


qRT - PCR of relative mRNA of MDA5 16 h after treatment of HLEC with 0 . 5 ug / ml BO - 110 ( BO ) , 10 µM vemurafenib ( Vem ) , or vehicle control ( V ) . Data correspond to mean ± SD of 3 biological replicates . Statistical significance was determined by t - . qRT - PCR of relative mRNA of VEGFR3 16 h after treatment of HLEC with 0 . 5 or 1 ug / ml BO - 110 ( VO ) , 10 µM vemurafenib ( Vem ) , or corresponding vehicle control ( V ) . Data correspond to mean ± SD of 3 biological replicates . Statistical significance was determined by ANOVA .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml BO-110 (BO), 10 µM vemurafenib (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.   qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.


Luciferase signal driven by FLT4 ( VEGFR3 ) - promoter transduced into HLEC treated with vehicle ( v ) or BO - 110 ( BO ) at indicated doses ( ug / ml ) as indicated in methods . Results were normalized to vehicle control . N = 4 biological replicates . Error bars correspond to mean ± SD . Statistical significance was determined by ANOVA .

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Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA.


of apoptotic at indicated time points . HLEC were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for indicated time points . were collected apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to mean ± SD of 3 experiments . Statistical significance was determined by t - .

O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Relative mRNA of VEGFC VEGFD in indicated melanoma lines 8 h after treatment with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) , as determined by qRT - PCR . Data correspond to mean ± SD of 3 experiments . Statistical significance was determined by t - .

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Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Inhibitory effect of indicated doses of BO - 110 ( in µg / ml ) or vehicle ( V ) on MDK mRNA expression determined by qRT - PCR in SK - Mel - 147 ( 16 h after treatment ) . Data correspond to average mRNA in three experiments with technical replicates normalized to vehicle control ± SD . Statistical significance was determined by ANOVA . qRT - PCR of relative mRNA of MDA5 16 h after treatment of SK - Mel - 147 with indicated doses of BO - 110 ( in µg / ml ) ( BO ) . Data correspond to mean ± SD of three experiments with three technical replicates . Statistical significance was determined by ANOVA .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA expression determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.   qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.


of apoptotic at indicated time points . SK - Mel - 147 were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for indicated time points . were collected apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to mean ± SD of 3 experiments . Statistical significance was determined by t - .

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Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Immunohistochemical of MDK repression ( pink ) in SK - Mel - 147 xenografts PDX lesions after treatment with BO - 110 ( BO , 0 . 8 mg / kg , 2 doses / week ) . Histological in tumors extracted from treated with vehicle control ( V ) are included as a reference . Nuclei were counterstained in with hematoxylin .

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Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained in with hematoxylin.


Type I IFN mRNA induction ( IFNA2 IFNB1 ) in SK - Mel - 147 melanoma treated for 16 h with indicated amounts of BO - 110 ( in mg / ml ) . Data correspond to mean ± SD of three experiments with three technical replicates normalized to vehicle control . Statistical significance was determined by ANOVA .

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Type I IFN mRNA induction (IFNA2 and IFNB1) in SK-Mel-147 melanoma cells treated for 16 h with the indicated amounts of BO-110 (in mg/ml). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Statistical significance was determined by ANOVA.


depicting expression changes in interferon - related genes ( GO : 0034340 ) in SK - Mel - 147 melanoma ( left panel ) HLEC ( right panel ) treated with vehicle or 0 . 5 g / ml of BO - 110 for 10 hours . CD274 , LAG3 PDCD1 genes were also included as a reference .

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Heatmap depicting expression changes in interferon-related genes (GO:0034340) in SK-Mel-147 melanoma cells (left panel) and HLEC (right panel) treated with vehicle or 0.5 g/ml of BO-110 for 10 hours. CD274, LAG3 and PDCD1 genes were also included as a reference.


IFNB1 mRNA induction analyzed by qPCR at indicated times after BO - 110 treatment ( 0 . 5 µg / ml ) of SK - Mel - 147 melanoma or HLEC ( left right graphs , respectively ) . Data correspond to mean ± SD of three experiments with three technical replicates normalized to vehicle control .

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IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control.


of impact of BO - 110 as single agent or in presence of indicated blocking antibodies for type I interferon ( IFNB1 or IFNAR1 ) . Upper graphs show effect of these agents on MDK mRNA in SK - Mel - 147 melanoma . Similar treatments were performed on HLEC for of VEGFR3 mRNA ( middle graphs ) tube formation capacity ( lower graphs ) . Data correspond to mean ± SD of 3 biological replicates in triplicate .

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Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.


Growth of B16 melanoma xenografts in siblings of Ifnar1 + / + , Ifnar1 + / - or Ifnar1 - / - mice . Treatment started 10 days after implantation . BO - 110 was administered at 0 . 8 mg / kg , every third day for 2 . N = 6 mice per condition . Graphs show mean ± SD at each time point . Statistical significance was determined by Two - Way ANOVA .

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Growth of B16 melanoma xenografts in siblings of Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA.


Impact of BO - 110 on - bearing mice implanted with syngeneic B16R2L ( 5 x 105 ) . When tumors have an average on 100 mm3 , mice were randomized into two groups treated with BO - 110 ( BO ) or vehicle ( V ) , every second day for 2 . arrow indicates start of treatment . was measured with a caliper at indicated time points . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM O O O B-CELL_LINE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O

Impact of BO-110 on tumor-bearing mice implanted with syngeneic B16R2L (5 x 105 cells). When tumors have an average size on 100 mm3, mice were randomized into two groups and treated with BO-110 (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points. , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Vegfr3Luc emission in inguinal brachial lymph nodes of mice in experiment ( A ) . N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment (A). N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


G . ELISA of Mdk blood in mice in experiment ( A ) . H . ELISA of mouse Ifn - β blood in mice in experiment ( A ) . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

O O B-EXP_ASSAY O B-GENEPROD B-TISSUE O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

G. ELISA analysis of Mdk blood levels in mice in experiment (A). H. ELISA analysis of mouse Ifn-β blood levels in mice in experiment (A). , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Efficacy of BO - 110 as an adjuvant ( preventing relapse after surgical removal of lesion ) . Shown are representative examples of Vegfr3Luc mice implanted with mCherry - SK - Mel - 147 imaged for luciferase emission ( prior to after removal . were left to recover from surgery ( 4 days ) then treated for 2 ( 4 dosis ) with 0 . 8 mg / Kg BO - 110 or vehicle control ( n = 8 for control n = 10 for treatment arm ) . Scale , p / s / cm2 / sr x106 . mCherry emission from of in ( A ) . Scale , p / s / cm2 / sr x109 .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O

Efficacy of BO-110 as an adjuvant (preventing relapse after surgical removal of the primary lesion). Shown are representative examples of Vegfr3Luc mice implanted with mCherry-SK-Mel-147 and imaged for luciferase emission (prior to and after tumor removal. Animals were left to recover from surgery (4 days) and then treated for 2 weeks (4 dosis) with 0.8 mg/Kg BO-110 or vehicle control (n= 8 for control and n=10 for treatment arm). Scale, p/s/cm2/sr x106.   mCherry emission from tumor cells of the animals in (A). Scale, p/s/cm2/sr x109.


Kaplan - Meier survival curves of treated as in ( A ) . 8 / 8 treated with vehicle ( V ) control had to be sacrificed for humane reasons 110 days after surgery . 9 / 10 in BO - 110 arm ( BO ) remained - free 8 months after stopping treatment . grey box marks period of treatment with BO - 110 . Statistical significance was determined by logrank .

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Kaplan-Meier survival curves of animals treated as in (A). 8/8 animals treated with vehicle (V) control had to be sacrificed for humane reasons 110 days after surgery. 9/10 animals in the BO-110 arm (BO) remained tumor-free 8 months after stopping treatment. The grey box marks the period of treatment with BO-110. Statistical significance was determined by logrank test.


( a ) Different VPS34 complexes were immunoprecipitated ( IP ) from MEFs in presence ( N ) or absence ( − A ) of using indicated antibodies assayed for kinase activity ( left , top panel ) . Inputs were immunoblotted using antibodies indicated ( left , lower panels ) . of VPS34 activity is from 3 biological repeats ( right panel ; data shown are mean + s . d . ) . AR , autoradiography .

O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O 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-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(a) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography.


( b ) Immunoprecipitation of three VPS34 complexes , normalized for VPS34 , was performed using indicated antibodies under nutrient - rich ( N ) or starvation ( - A ) conditions . VPS34 - binding partners were analysed by western blotting .

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(b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting.


( c ) ATG14L - containing VPS34 complexes were immunopurified from WT , ULK1 − / − 2KD ( ULK def . ) FIP200 − / − MEFs , measured for lipid kinase activity as in a .

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(c) ATG14L-containing VPS34 complexes were immunopurified from WT, ULK1−/− 2KD (ULK def.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a.


( d ) VPS34 was immunopurified from WT , ULK - deficient FIP200 − / − MEFs . A kinase was performed as in a .

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(d) VPS34 was immunopurified from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.


( e ) Immunoprecipitation of Beclin - 1 - containing VPS34 complexes from WT , ULK - deficient FIP200 − / − MEFs . A kinase was performed as in a .

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(e) Immunoprecipitation of Beclin-1-containing VPS34 complexes from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.


( f ) LC3B PtdIns ( 3 ) P were analysed with anti - LC3B biotin - 2XFYVE domain probe . Representative immunofluorescence of LC3B 2XFYVE domain binding are shown ( scale bars , 10 μm ) . ( g ) of LC3B from f . Details of are provided in Methods ; Data shown in g - i are mean + s . d . from a minimum of 6 unique fields of view from a representative experiment ( see statistical source data in Supplementary Table S1 ) . ( h ) Total PtdIns ( 3 ) P was quantified from experiment in f . Error bars were calculated as in g . ( i ) of PtdIns ( 3 ) P that co - localizes with LC3B on - acid withdrawal from experiment described in f .

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(f) LC3B puncta and PtdIns(3)P levels were analysed with anti-LC3B and the biotin-2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g-i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3)P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3)P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f.


( j ) VPS34 activity with or without ULK1 overexpression was assayed . A representative experiment of four repeats is shown .

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(j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown.


( a ) HEK293 were transfected with ATG14L , VPS34 Beclin - 1 . ATG14L - containing VPS34 complexes were immunopurified subjected to an in vitro ULK1 kinase in presence of γ - 32P [ ATP ] . Bound ATG14L complexes soluble ULK1 were separated phosphorylation was detected by autoradiography ( AR , left panels ) . Western blotting was performed ( right panels ) . Results are representative of two unique experiments .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

(a) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ-32P[ATP]. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blotting was performed (right panels). Results are representative of two unique experiments.


( b ) Full - murine GST - Beclin - 1 various truncations ( as labelled ) were subjected to an in vitro HA - ULK1 kinase . GST - Beclin - 1 6 ( S / T ) A has serine / threonine residues 4 , 7 , 10 , 14 , 29 42 mutated to alanine . ULK1 inputs were determined by western blotting , Beclin - 1 inputs by Coomassie ( Coom ) target phosphorylation by autoradiography .

O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O

(b) Full-length murine GST-Beclin-1 and various truncations (as labelled) were subjected to an in vitro HA-ULK1 kinase assay. GST-Beclin-1 6(S/T)A has serine/threonine residues 4, 7, 10, 14, 29 and 42 mutated to alanine. ULK1 inputs were determined by western blotting, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by autoradiography.


( c ) GST - Beclin - 1 ( 1 - 85 ) was subjected to an in vitro ULK1 kinase reaction analysed by mass spectrometry . Ser 14 , Ser 15 in humans ( outlined ) , is main phosphorylation site identified ( for mass spectrometry data , see Supplementary Fig . S2b , c ) . Mass spectrometry was performed on a single experiment .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(c) GST-Beclin-1 (1-85) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Ser 14, Ser 15 in humans (outlined), is the main phosphorylation site identified (for mass spectrometry data, see Supplementary Fig. S2b,c). Mass spectrometry was performed on a single experiment.


( d ) Beclin - 1 Ser 14 is main in vitro ULK1 phosphorylation site . Beclin - 1 WT , S4A S14A mutants were subjected to an in vitro ULK1 kinase . reaction was developed by autoradiography stained for Beclin - 1 input by Coomassie blue . Results are representative of two unique experiments .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(d) Beclin-1 Ser 14 is the main in vitro ULK1 phosphorylation site. Beclin-1 WT, and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay. The reaction was developed by autoradiography and stained for Beclin-1 input levels by Coomassie blue. Results are representative of two unique experiments.


( e ) HEK293 were transfected with indicated plasmids under nutrient - rich conditions . Beclin - 1 was immunoprecipitated immunoblotted with pBeclin - 1 ( Ser 14 ) , or anti - Beclin - 1 as a loading control . ULK1 inputs are included below immunoprecipitated samples .

O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O

(e) HEK293 cells were transfected with the indicated plasmids under nutrient-rich conditions. Beclin-1 was immunoprecipitated and immunoblotted with pBeclin-1(Ser 14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below immunoprecipitated samples.


( f ) Purified GST - Beclin - 1 ( 1 - 85 ) was subjected to in vitro phosphorylation by GST - ULK1 ( top panel ) GST - ULK2 ( bottom panel ) . Reactions were immunoblotted with indicated antibodies .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O

(f) Purified GST-Beclin-1 (1-85) was subjected to in vitro phosphorylation by GST-ULK1 (top panel) and GST-ULK2 (bottom panel). Reactions were immunoblotted with the indicated antibodies.


( g ) HEK293 were transfected with ATG14L , VPS34 Beclin - 1 grown under nutrient - rich conditions . ATG14L - containing VPS34 complexes were immunoprecipitated lipid kinase activity was assayed as described in Fig . 1j . Inputs were immunoblotted with indicated antibodies . Representative of four unique experiments .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O

(g) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1 and grown under nutrient-rich conditions. ATG14L-containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig. 1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments.


( h ) Stable lines containing Beclin - 1 ( WT or S14A ) were used for Beclin - 1 immunoprecipitation . Binding partners were determined by SDS - PAGE western blot using indicated antibodies . Uncropped of blots are shown in Supplementary Fig . S4 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

(h) Stable lines containing Beclin-1(WT or S14A) were used for Beclin-1 immunoprecipitation. Binding partners were determined by SDS-PAGE analysis and western blot using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) WT MEFs were cultured with or without . ATG14L - associated Beclin - 1 was immunoprecipitated treated with lambda phosphatase treatment ( PPase ) as indicated . Western blotting ( immunoblotting , IB ) was performed with indicated antibodies . Beclin - 1 Ser 14 phosphorylation was quantified ( shown under top panel ) normalized to total Beclin - 1 .

O O O O B-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1.


( b ) WT MEFs were starved for indicated times . Beclin - 1 was immunopurified by ATG14L immunoprecipitation immunoblotted as indicated ( top panels ) . Whole - lysates were immunoblotted with pULK1 S757 ( mTORC1 - target site ) , pS6K ULK1 antibodies ( bottom panels ) . Two unique experiments were performed .

O O O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O

(b) WT MEFs were starved for the indicated times. Beclin-1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated (top panels). Whole-cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed.


( c ) WT or FIP200 − / − MEFs were incubated under nutrient - rich , - acid - deprived or Torin - 1 ( + T , an mTOR inhibitor ) conditions . Beclin - 1 was purified immunoblotted as in Fig . 3b .

O O O O O B-GENEPROD O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O

(c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b.


( d ) WT or ULK - deficient MEFs were incubated with or without . Beclin - 1 was purified immunoblotted as in a . Two unique experiments were performed . Uncropped of blots are shown in Supplementary Fig . S4 .

O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) Beclin - 1 alone ( lanes 1 - 4 ) or Beclin - 1 ATG14L ( lanes 5 - 8 ) were overexpressed in HEK293 . Beclin - 1 was purified either by direct immunoprecipitation ( lanes 1 - 4 ) or by ATG14L immunoprecipitation ( lanes 5 - 8 ) . Immunoprecipitated samples were subjected to an in vitro ULK1 kinase with increasing amounts of ULK1 . Reactions were immunoblotted with indicated antibodies . Black denotes discontinuous lanes from same gel . Two unique experiments were performed .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(a) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed.


( b ) Beclin - 1 alone or bound to ATG14L was purified as described in a . Equal amounts of ULK1 were added to each reactions were quenched at indicated time points . Western blotting was performed with indicated antibodies .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(b) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies.


( c ) An ATG14L - FLAG - 6His - inducible U2OS was induced for 16 h in presence of . Endogenous Beclin - 1 was immunoprecipitated immunoblotted as in Fig . 3a . ATG14L input were detected by immunoblotting . Two unique experiments were performed .

O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O

(c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed.


( d ) HEK293 transfected with either ATG14L or Beclin - 1 , or both , in conjunction with ULK1 , were immunoprecipitated as indicated blotted with indicated antibodies .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O

(d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies.


( e ) HEK293 were transfected with Beclin - 1 ULK1 in presence of ATG14L WT or ATG14LΔCCD , which is defective in Beclin - 1 binding , under nutrient - rich conditions . Lysates were resolved by SDS - PAGE blotted with indicated antibodies .

O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

(e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


( f ) HEK293 were transfected with ULK1 Beclin - 1 in conjunction with either ATG14L WT , or one of two mutants ( ΔBATS , ΔN ) that are defective in phagophore localization . Samples were handled as in e . Uncropped of blots are shown in Supplementary Fig . S4 . Lysates were resolved by SDS - PAGE blotted with indicated antibodies .

O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


( a ) HEK293 were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated in presence of . Lysates were immunoblotted with indicated antibodies . A representative experiment of three repeats is shown .

O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown.


( b ) UVRAG bridges interaction between Beclin - 1 ULK1 . HEK293 were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated . Lysates were immunoprecipitated with anti - HA ( Beclin - 1 ) antibody blotted with indicated antibodies . A representative experiment of three repeats is shown . Uncropped of blots are shown in Supplementary Fig . S4 .

O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY 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) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) HEK293 were transfected with indicated plasmids . were grown in presence of treated with NH4Cl to block autophagic turnover where indicated . Lysates were immunoblotted with indicated antibodies .

O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O

(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies.


( b ) HEK293 transfected with Beclin - 1 ATG14L were grown in presence or absence of . Lysates were immunoblotted with indicated antibodies ( left panel ) quantified by densitometry ( right panel ) . Data represent mean + s . d . of three unique experiments .

O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O 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) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.


( c ) Beclin - 1 - shRNA reconstituted lines ( WT or mutant ) controls ( scramble shRNA or Beclin - 1 shRNA ) were grown with or without assessed for autophagy ( left panel ) Beclin - 1 ( right panel ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel).


( d ) Autophagosome ( denoted by arrowheads ) generation on - acid withdrawal was analysed by electron microscopy . lines conditions from c were used representative from indicated - acid - starved lines are shown ; scale bars , 0 . 4 μm .

O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O

(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.


( d ) Autophagosome ( denoted by arrowheads ) generation on - acid withdrawal was analysed by electron microscopy . lines conditions from c were used representative from indicated - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( e ) of d . Fold induction was determined by arbitrarily making nutrient - rich condition 1 ( solid bars ) for each . Error bars represent s . d . of mean value over an average of 20 fields of view within a representative experiment . NS , not significant .

O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.


( f ) HA - Beclin - 1 WT or S14D was transiently expressed in FIP200 − / − MEFs grown under nutrient - rich conditions . Indirect immunofluorescence was performed using antibodies against endogenous LC3B HA - Beclin - 1 . Scale bars , 20 μm . ( g ) of LC3B from confocal in f . In HA - Beclin - 1 - or HA - Beclin - 1 - S14D - transfected samples , only HA - positive were counted for LC3B . Error bars were processed as in e . Mean value shown ; P values determined by Student ' s t - using 10 unique fields of view from f . Uncropped of blots are shown in Supplementary Fig . S4 .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) Bec - 1C . elegans were reconstituted with either WT or mutant GFP - BEC - 1 . Stable worm lines with Bec - 1 rescue were obtained were stained with anti - PGL - 1 antibody . Arrowheads indicate normal PGL - 1 in germline . ( b ) of PGL - 1 outside germline ( left panel ) . Error bars represents s . d . between 3 unique in a representative experiment . Reconstituted Bec - 1 ( WT mutant ) in Bec − / − stable worms were compared by western blotting . Mean value presented .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(a) Bec-1C. elegans were reconstituted with either WT or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented.


( c ) Spectrum of defects in PGL granule degradation in bec - 1 mutant rescue . Mutant exhibited either high of diffuse PGL - 1 ( middle - left panel ; one - third of ) , or large punctuate PGL - 1 structures in somatic ( bottom - left panel ; two - thirds of ) . Both diffuse or punctuate PGL - 1 in somatic have been described in autophagy - deficient .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O

(c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos.


( d ) from lines described in a were labelled with anti - LGG - 1 , along with WT unc - 51 worms . Representative at ∼ 100 stage are shown . ( e ) of LGG - 1 per from labelling in d . Error bars generated as in b . Mean value presented . Uncropped of blots are shown in Supplementary Fig . S4 . Scale bar , 10 μm .

O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.


( F ) Single tube qPCR experiments showing down - regulation of synaptic genes NLGN3 , SLC6A , TRIM9 , SYT1 , SYNGR1 , SNAP91 in ALSC9orf72 MN ( Welch ' s t - ) . n = 3 independent cultures for each hiPSC . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Single tube qPCR experiments showing the down-regulation of the synaptic genes NLGN3, SLC6A, TRIM9, SYT1, SYNGR1, and SNAP91 in ALSC9orf72 MN (Welch's t-test). n=3 independent cultures for each hiPSC line. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( G - H ) Time course of pCREBS133 MN survival in ALSC9orf72 Healthy cultures ( two - way ANOVA ) . n = 3 independent cultures for each hiPSC ( for each time point ) . Scale bar : 25 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-H) Time course analysis of nuclear pCREBS133 levels and MN survival in ALSC9orf72 and Healthy cultures (two-way ANOVA). n=3 independent cultures for each hiPSC line (for each time point). Scale bar: 25 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( A ) Both K + channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing neurite of mutant MN ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . n = 3 independent treatments with each hiPSC . Scale bar : 50 µm Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-DISEASE O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Both K+ channel blockers exert a neuroprotective effect in ALSC9orf72 cultures by increasing the neurite length of mutant MN (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 50 µm Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( C ) Apamin XE991 decrease of aberrant aggresomes , confirming enhanced autophagy degradation ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . n = 3 independent treatments with ALSC9orf72 I . Scale bar : 5 Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O 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-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Apamin and XE991 decrease the size of aberrant aggresomes, confirming enhanced autophagy degradation (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with the ALSC9orf72 I line. Scale bar: 5 Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) load of aggregated SQSTM1 / p62 is also reduced by treatments in ALSC9orf72 cultures ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . n = 3 independent treatments for each . Scale bar : 20 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The load of aggregated SQSTM1/p62 is also reduced by the treatments in ALSC9orf72 cultures (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments for each cell line. Scale bar: 20 µm. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( F ) Treatment with K + channel blockers reduces number of GGGGCC toxic foci in ALSC9orf72 MN . n = 3 independent treatments with each hiPSC ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . Scale bar : 5 µm . Dashed represents soma . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Error bars represent . Arrow indicates structure displayed at higher magnification . Exact p - values are reported in Appendix Table S1 .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Treatment with the K+ channel blockers reduces the number of GGGGCC toxic RNA foci in ALSC9orf72 MN. n=3 independent treatments with each hiPSC line (one-way ANOVA followed by Dunnett's multiple comparison test). Scale bar: 5 µm. Dashed line represents the cell soma. Data information: *p<0.05; **p<0.01; ***p<0.001. Error bars represent SEM. Arrow indicates the structure displayed at higher magnification. Exact p-values are reported in Appendix Table S1.


( E ) Apamin XE991 increase of pCREBS133 in ALSC9orf72 lines ( one - way ANOVA followed by Dunnett ' s multiple comparison ) . n = 3 independent treatments with each hiPSC . Scale bar : 10 µm . Data information : * p < 0 . 05 ; * * p < 0 . 01 . Error bars represent . Exact p - values are reported in Appendix Table S1 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Apamin and XE991 increase the levels of pCREBS133 in all the ALSC9orf72 lines (one-way ANOVA followed by Dunnett's multiple comparison test). n=3 independent treatments with each hiPSC line. Scale bar: 10 µm. Data information: *p<0.05; **p<0.01. Error bars represent SEM. Exact p-values are reported in Appendix Table S1.