Figure 7 A Representative tile scan 3D images with α - SMA , Isolectin , FABP4 and Emcn in the pancreas from 17 - weeks - old Gja1i ∆ EC mutant and control mice . Insets show higher magnifications of regions . B Box whiskers , and scatter plot ( left ) shows the quantification of CD31 + Emcnhi vessel density in Gja1i ∆ EC mutant and control pancreatic islets . ( n = 7 ) , P - value , and two - tailed unpaired t - test . Combo plot ( right ) shows the quantification of P - Histone H3 + EC numbers per islet ( f . u ) counted on thick murine pancreatic sections . ( n = 5 ) , P - value , and two - tailed unpaired t - test . C 3D images show CD31 and Emcn immunostaining in the Gja1i ∆ EC mutant and control pancreas . The scale bars are 50 µm . D Combined violin , scatter and sample distribution plot shows the quantification of β - cell mass ( % ) in pancreatic islets from Gja1i ∆ EC and control mice . Data represent mean ± s . d . ( n = 140 islets from 7 pancreas ) , P - value , and two - tailed unpaired t - test . E Representative 3D images with immunostaining of α - SMA , Insulin and VE - Cadherin in the pancreatic islets from Gja1i ∆ EC and control mice . The scale bars are 50 µm . F Combo plot ( left ) shows insulin measurement in lysate of pancreas by ELISA . Combo plot ( right ) shows the percentage of Insulin + β - cells per islet in Gja1i ∆ EC mutant and control mice . ( n = 5 ) , P - values , and two - tailed unpaired t - tests . Data information : The dashed lines in panel A represent the outlines of pancreas . The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : DAPI Is : pancreatic islet . f . u : fluorescence unit . The line on the right side of the combo plots represent the sample distribution . Scale bars are 200 µm for tile scan images of longitudinal thick sections across the entire glands , and 50 µm for the higher magnification insets images . G Representative 3D images of ESM - 1 and Caveolin - 1 in pancreatic islets from Gja1i ∆ EC and control mice . Insets ( * and # ) show higher magnifications with surface rendering for ESM - 1 and Caveolin - 1 immunostaining . Combo plot with the quantification of ESM - 1 + cell numbers per islet ( f . u ) . ( n = 5 ) , P - value , and two - tailed unpaired t - test . The scale bars for the 3D images are 50 µm , and 5 µm for the higher magnifications with single - cell resolution images . The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO - 3 as indicated . Is : pancreatic islet . f . u : fluorescence unit . H 3D images with CXCR4 and Emcn immunostaining in Gja1i ∆ EC and control pancreas . The scale bars are 50 µm . I Representative 3D images show CXCR4 , Ki67 and VE - Cadherin immunostaining in Gja1i ∆ EC and control pancreas . The scale bars are 50 µm . J Combo plots show the percentage of CXCR4 + β - cells per islet and Ki67 + β - cells normalized to the total number of β - cells per islet in Gja1i ∆ EC mutant versus the littermate control mice . ( n = 5 ) , P - values , and two - tailed unpaired t - tests . K Box whiskers , and scatter plot shows the quantification of CD31 + Emcnhi vessel density in aged Gja1i ∆ EC mutant and control pancreatic islets . Data represent mean ± s . d . ( n = 4 ) , P - value , and two - tailed unpaired t - test . Combo plot ( below ) shows the quantification of β - cells on thick murine pancreatic sections . Data represent mean ± s . d . ( n = 4 ) , P - value , and two - tailed unpaired t - test . Data information : The dashed lines in panel C , E , G , H and I represent the outlines of pancreatic islets . Two - tailed Student ' s t - test were performed for the statistical analysis . * * : P < 0 . 01 ; * * * * : P < 0 . 0001 . Nuclei : TO - PRO -

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

Figure 7 A Representative tile scan 3D images with α-SMA, Isolectin, FABP4 and Emcn in the pancreas from 17-weeks-old Gja1i∆EC mutant and control mice. Insets show higher magnifications of regions. B Box whiskers, and scatter plot (left) shows the quantification of CD31+ Emcnhi vessel density in Gja1i∆EC mutant and control pancreatic islets. (n=7), P-value, and two-tailed unpaired t-test. Combo plot (right) shows the quantification of P-Histone H3+ EC numbers per islet (f.u) counted on thick murine pancreatic sections. (n=5), P-value, and two-tailed unpaired t-test. C 3D images show CD31 and Emcn immunostaining in the Gja1i∆EC mutant and control pancreas. The scale bars are 50 µm. D Combined violin, scatter and sample distribution plot shows the quantification of β-cell mass (%) in pancreatic islets from Gja1i∆EC and control mice. Data represent mean ± s.d. (n=140 islets from 7 pancreas), P-value, and two-tailed unpaired t-test. E Representative 3D images with immunostaining of α-SMA, Insulin and VE-Cadherin in the pancreatic islets from Gja1i∆EC and control mice. The scale bars are 50 µm. F Combo plot (left) shows insulin measurement in lysate of pancreas by ELISA. Combo plot (right) shows the percentage of Insulin+ β-cells per islet in Gja1i∆EC mutant and control mice. (n=5), P-values, and two-tailed unpaired t-tests. Data information: The dashed lines in panel A represent the outlines of pancreas. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: DAPI Is: pancreatic islet. f.u: fluorescence unit. The line on the right side of the combo plots represent the sample distribution. Scale bars are 200 µm for tile scan images of longitudinal thick sections across the entire glands, and 50 µm for the higher magnification insets images. G Representative 3D images of ESM-1 and Caveolin-1 in pancreatic islets from Gja1i∆EC and control mice. Insets (* and #) show higher magnifications with surface rendering for ESM-1 and Caveolin-1 immunostaining. Combo plot with the quantification of ESM-1+ cell numbers per islet (f.u). (n=5), P-value, and two-tailed unpaired t-test. The scale bars for the 3D images are 50 µm, and 5 µm for the higher magnifications with single-cell resolution images. The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3 as indicated. Is: pancreatic islet. f.u: fluorescence unit.  H 3D images with CXCR4 and Emcn immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. I Representative 3D images show CXCR4, Ki67 and VE-Cadherin immunostaining in Gja1i∆EC and control pancreas. The scale bars are 50 µm. J Combo plots show the percentage of CXCR4+ β-cells per islet and Ki67+ β-cells normalized to the total number of β-cells per islet in Gja1i∆EC mutant versus the littermate control mice. (n=5), P-values, and two-tailed unpaired t-tests. K Box whiskers, and scatter plot shows the quantification of CD31+ Emcnhi vessel density in aged Gja1i∆EC mutant and control pancreatic islets. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Combo plot (below) shows the quantification of β-cells on thick murine pancreatic sections. Data represent mean±s.d. (n=4), P-value, and two-tailed unpaired t-test. Data information: The dashed lines in panel C, E, G, H and I represent the outlines of pancreatic islets. Two-tailed Student's t-test were performed for the statistical analysis. **: P < 0.01; ****: P < 0.0001. Nuclei: TO-PRO-3


Figure 1 ( a ) THP - 1 cells were transiently infected with BRLF1 - expressing or empty control lentivirus . Twenty - four hours later , the control or BRLF1 - expressing THP - 1 cells were primed with 40 ng / ml TPA overnight and then were either uninfected or infected with HSV - 1 ( MOI = 1 ) in serum - free medium for 12 h . The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro - caspase - 1 ( p45 ) , cleaved caspase - 1 ( p20 ) , pro - IL - 1β , mature IL - 1β , pro - IL - 18 and mature IL - 18 as indicated . The release of mature IL - 1β ( b ) from THP - 1 cells were measured by enzyme - linked immunosorbent assay ( ELISA ) following HSV - 1 infection . The release of IL - 18 ( c ) from THP - 1 cells were measured by enzyme - linked immunosorbent assay ( ELISA ) following HSV - 1 infection . Ramos cells were infected with wild type EBV - WT or BRLF1 - deficient EBV - ΔBRLF1 virus at a high titer ( MOI = 100 ) . After being transduced with empty control or BZLF1 - expressing lentivirus , the supernatants and cell pellets of EBV - WT - or EBV - ΔBRLF1 - harboring Ramos cells were collected after 12 h in serum - free culture , and then the levels of EBNA1 , BRLF1 , BZLF1 , pro - caspase - 1 ( p45 ) , cleaved caspase - 1 ( p20 ) , pro - IL - 1β , mature IL - 1β , pro - IL - 18 , mature IL - 18 , GSDMD and cleaved N - GSDMD in the supernatants and whole - cell lysates were analyzed as indicated ( d ) . The release of mature IL - 1β ( e ) from Ramos cells were measured by enzyme - linked immunosorbent assay ( ELISA ) . The release of mature IL - 18 ( f ) from Ramos cells were measured by enzyme - linked immunosorbent assay ( ELISA ) . ( g ) After being transduced with control or BZLF1 - expressing lentivirus for 48 h , EBV - WT - and EBV - ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry . Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(a) THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI=1) in serum-free medium for 12 h. The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18 and mature IL-18 as indicated.The release of mature IL-1β (b) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection.The release of IL-18 (c) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection.Ramos cells were infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (d).The release of mature IL-1β (e) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA).The release of mature IL-18 (f) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA).(g) After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate.


Figure 2 ( a ) GST - tagged BRLF1 - expressing plasmid or control vector was transfected into A549 cells . After HSV - 1 infection ( MOI = 1 ) for 12 h , the cells were collected , and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads . The immunoprecipitated complexes were then separated by SDS - PAGE , and the image obtained from silver staining is shown . ( b ) Scramble ( sc ) or shRNAs against POLR3F and POLR3G with empty control vector ( ctr ) or BRLF1 - expressing plasmid were transfected into EBV - ΔBRLF1 - harboring HNE1 cells and A549 cells , followed by HSV - 1 infection . Thirty - six hours after the transfection of the HNE1 cells , and 12 h after HSV - 1 infection ( MOI = 1 ) of the A549 cells , the cell pellets were collected and then the cleavage of caspase - 1 and IL - 1β were measured as indicated . The release of mature IL - 1β and IL - 18 from HNE - 1 and A549 cells were measured by enzyme - linked immunosorbent assay ( ELISA ) after ultrafiltration and concentration with Amicon ® Ultra - 0 . 5 centrifugal filter devices . ( c ) GFP - tagged BRLF1 wild type , BRLF1 L578A or empty vector was transfected into 293T cells with GST - tagged POLR3F or POLR3G expressing plasmids for 48 h . Cells were collected and immunoprecipitation with GST - affinity beads was performed , and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated . ( d ) GFP - tagged empty vector or GFP - BRLF1 - or GFP - BRLF1 L578A - expressing plasmids were transfected into A549 cells , after which the cells were then infected with HSV - 1 , or transfected into HNE - 1 - EBV - ΔBRLF1 cells . The cells were then collected and lysed . Immunoprecipitation with GST - affinity beads was subsequently performed , and then whole - cell lysates and immunoprecipitated complexes were analyzed as indicated . ( e ) P3HR - 1 cells were induced by TPA plus NaB into lytic infection , and the cells were then collected . Immunoprecipitation with anti - mouse IgG or BRLF1 antibody was performed , after which whole - cell lysates and immunoprecipitated complexes were analyzed as indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(a) GST-tagged BRLF1-expressing plasmid or control vector was transfected into A549 cells. After HSV-1 infection (MOI=1) for 12 h, the cells were collected, and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads. The immunoprecipitated complexes were then separated by SDS-PAGE, and the image obtained from silver staining is shown.(b) Scramble (sc) or shRNAs against POLR3F and POLR3G with empty control vector (ctr) or BRLF1-expressing plasmid were transfected into EBV-ΔBRLF1-harboring HNE1 cells and A549 cells, followed by HSV-1 infection. Thirty-six hours after the transfection of the HNE1 cells, and 12 h after HSV-1 infection (MOI=1) of the A549 cells, the cell pellets were collected and then the cleavage of caspase-1 and IL-1β were measured as indicated. The release of mature IL-1β and IL-18 from HNE-1 and A549 cells were measured by enzyme-linked immunosorbent assay (ELISA) after ultrafiltration and concentration with Amicon® Ultra-0.5 centrifugal filter devices.(c) GFP-tagged BRLF1 wild type, BRLF1 L578A or empty vector was transfected into 293T cells with GST-tagged POLR3F or POLR3G expressing plasmids for 48 h. Cells were collected and immunoprecipitation with GST-affinity beads was performed, and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated.(d) GFP-tagged empty vector or GFP-BRLF1- or GFP-BRLF1 L578A-expressing plasmids were transfected into A549 cells, after which the cells were then infected with HSV-1, or transfected into HNE-1-EBV-ΔBRLF1 cells. The cells were then collected and lysed. Immunoprecipitation with GST-affinity beads was subsequently performed, and then whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.(e) P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.


Figure 3 ( a ) After infected with wild type EBV - WT or BRLF1 - deficient EBV - ΔBRLF1 virus at a high titer ( MOI = 100 ) , POLR3 - dependent RNA transcripts in Ramos cells were detected by RNA - seq analysis . The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock ( mock group vs . mock group ) , EBV - WT ( EBV - WT group vs . mock group ) and EBV - ΔBRLF1 ( EBV - ΔBRLF1 group vs . mock group ) comparisons . ( b ) 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 . ( c ) 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 . ( d ) EBER1 5 ' - pppRNA detection using splint - ligation . EBV - WT - or EBV - ΔBRLF1 - harboring HNE1 cells were cotransfected with empty vector , BRLF1 or BRLF1 L578A , BRLF1 plus BZLF1 , or BRLF1 L578A plus BZLF1 for 48 h . The total RNAs were extracted , and 5 μg of each RNA was subjected to splint - ligation with FAM - labeled probe to quantify the 5 ′ - monophosphorylated EBER1 and total EBER1 RNA . The ligation products were separated using urea - PAGE , visualized by a fluorescent scanner and analyzed using ImageJ software . The ratio of 5 ' - pRNA density to total RNA density and the relative 5 ' - pppRNA level were calculated for three independent experiments . ( e ) EBV - WT - harboring HNE1 cells were transfected with control vector , BRLF1 or BRLF1 L578A - expressing plasmid in the presence of GST - tagged RNA polymerase subunit POLR3F for 48 h . The cells were harvested and ChIP assay was performed with anti - GST affinity beads . The POLR3F - binding promoter fragments of BSRF1 , BMLF1 , EBER1 , EBER2 , EBNA1 , 7SK , RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real - time PCR assays .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(a) After infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100), POLR3-dependent RNA transcripts in Ramos cells were detected by RNA-seq analysis. The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock (mock group vs. mock group), EBV-WT (EBV-WT group vs. mock group) and EBV-ΔBRLF1 (EBV-ΔBRLF1 group vs. mock group) comparisons.(b) 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.(c) 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.(d) EBER1 5'-pppRNA detection using splint-ligation. EBV-WT- or EBV-ΔBRLF1-harboring HNE1 cells were cotransfected with empty vector, BRLF1 or BRLF1 L578A, BRLF1 plus BZLF1, or BRLF1 L578A plus BZLF1 for 48 h. The total RNAs were extracted, and 5 μg of each RNA was subjected to splint-ligation with FAM-labeled probe to quantify the 5′-monophosphorylated EBER1 and total EBER1 RNA. The ligation products were separated using urea-PAGE, visualized by a fluorescent scanner and analyzed using ImageJ software. The ratio of 5'-pRNA density to total RNA density and the relative 5'-pppRNA level were calculated for three independent experiments.(e) EBV-WT-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A-expressing plasmid in the presence of GST-tagged RNA polymerase subunit POLR3F for 48 h. The cells were harvested and ChIP assay was performed with anti-GST affinity beads. The POLR3F-binding promoter fragments of BSRF1, BMLF1, EBER1, EBER2, EBNA1, 7SK, RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real-time PCR assays.


Figure 4 ( 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 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 ) 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 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 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) 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 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 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.


Figure 5Jurkat 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 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 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 . The cell killing activity of Jurkat cells ( d ) were measured by the detection of the LDH release from the K562 target cells . 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 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 . cell killing activity of Jurkat cells ( g ) were measured . cell killing activity of NK - 92MI cells ( h ) were measured .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O

Figure 5Jurkat 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 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 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.The cell killing activity of Jurkat cells (d) were measured by the detection of the LDH release from the K562 target cells.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 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.cell killing activity of Jurkat cells (g) were measured.cell killing activity of NK-92MI cells (h) were measured.


Figure 6 ( 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 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 ;

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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;


Figure 1B 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 - 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 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 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 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 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B 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- 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 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 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 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)


Figure 2B 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 µmC Representative reconstructions of control and dnLEF neurons . Scale bar = 20 µmD 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 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) . G Representative images depicting CAG - GFP - IRES - Cre transduced adult - born neurons in control and β - catex3 mice at 17dpi . Scale bar = 20µmH Representative reconstructions of control and β - catex3 neurons . Scale bar = 20µmI 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B 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 µmC Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µmD 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 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µmH Representative reconstructions of control and β-catex3 neurons. Scale bar= 20µmI 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 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).


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

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Figure 4B 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 and β - catex3 iDCX neurons expressing RFP at 3dpT . Scale bar = 20µm . 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 and β - catex3 iDCX neurons expressing RFP at 13dpT . Scale bar = 20µm . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B 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 and β-catex3 iDCX neurons expressing RFP at 3dpT. Scale bar= 20µm.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 and β-catex3 iDCX neurons expressing RFP at 13dpT. Scale bar= 20µm.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).


Figure 5A 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 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 ] .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A 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 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].


Figure 6B Representative reconstructions of control and β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm . 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 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 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 ) .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B Representative reconstructions of control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar = 20µm.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 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 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).


Figure 7B 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 and β - catex3 iDCX neurons in 24 - week old mice at 3dpT . Scale bar = 20µm . 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 and β - catex3 iDCX neurons in 24 - week old mice at 13dpT . Scale bar = 20µm . 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 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B 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 and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar= 20µm.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 and β-catex3 iDCX neurons in 24-week old mice at 13dpT. Scale bar= 20µm.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)


Figure 1Luciferase - 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Luciferase-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 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).


Figure 2qRT - 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 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 . 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 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 .

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Figure 2qRT-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 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.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 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.


Figure 3Inhibitory 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 . 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 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 .

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Figure 3Inhibitory 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.   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 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.


Figure 4Type 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 . 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 .

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Figure 4Type 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.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.


Figure 5IFNB1 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 . 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 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 .

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Figure 5IFNB1 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.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 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.


Figure 6Impact 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 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 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 .

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Figure 6Impact 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 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 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.


Figure 7Efficacy 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 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 .

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Figure 7Efficacy 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 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.


figf1 ( 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 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 . ) and FIP200 − / − MEFs , and measured for lipid kinase activity as in a . ( 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 and FIP200 − / − MEFs . A kinase assay was performed as in a . ( 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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figf1(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 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.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a.(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 and FIP200−/− MEFs. A kinase assay was performed as in a.(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.


figf2 ( 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 - 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 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 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 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 ) and GST - ULK2 ( bottom panel ) . Reactions were immunoblotted with the indicated antibodies . ( 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 analysis and western blot using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S4 .

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figf2(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-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 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 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 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) and GST-ULK2 (bottom panel). Reactions were immunoblotted with the indicated antibodies.(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 analysis and western blot using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.


figf3 ( 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 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 , 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(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 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, 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 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.