Figure 4 ( A ) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30 ° C with increasing concentrations ( 5 - 10 nmol ) of a peptide corresponding to the BH3 domain of beclin 1 ( aa 108 - 127 ) and with the same peptide in which Thr 119 was substituted by alanine . An in vitro kinase assay was performed , and the reactions were applied to Whatman filters . Total levels of TCA ‐ insoluble counts were measured and plotted against substrate concentration . Data are the mean ± s . d . of three experiments . ( C ) Flag ‐ tagged DAPK ( 60 ng ) was incubated with GST - WT beclin 1 or with GST - T119A beclin 1 ( 1000 ng ) in the presence of Ca2 + , calmodulin and ATP for 30 min . GST ‐ beclin 1 levels were visualized by Ponceau S staining , and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody ( Western blot ) . ( D ) HEK293 cells were co ‐ transfected with Flag ‐ tagged beclin 1 with or without ΔCaM DAPK . After 24 h , beclin 1 was immunoprecipitated from cells , using Flag antibodies , and the immunoprecipitates were reacted with phosphoThr 119 antibodies . The Ponceau staining shows equal amounts of immunoprecipitated beclin 1 . The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney ; TCA , trichloro ‐ acetic acid .

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 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

Figure 4(A) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5-10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108-127) and with the same peptide in which Thr 119 was substituted by alanine. An in vitro kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments.(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).(D) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.


Figure 5 ( A ) HEK293 cells were transfected with Flag ‐ tagged beclin 1 and Bcl ‐ XL with or without haemagglutinin ‐ tagged DAPK . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies . ( B ) HEK293 cells were co ‐ transfected with Flag ‐ tagged T119A or T119E beclin 1 mutants and Bcl ‐ XL . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies . ( C ) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP - LC3 plasmid . After 24 h , cells were counted and lysates were prepared . ( a ) Representative GFP - LC3 staining . ( b ) The percentage of cells with punctate GFP - LC3 fluorescence per total GFP - LC3 ‐ positive cells was quantified . Data presented are the mean ± s . d . from a triplicate experiment with 100 transfected cells . The asterisks denote significance level : * P = 0 . 01 ; * * P = 0 . 005 . ( c ) Western blot analysis was performed using the indicated antibodies . ( D ) HEK293 cells were transfected with Bcl ‐ XL , Flag ‐ tagged beclin 1 ( WT ) or Flag ‐ tagged T119A beclin 1 mutant with or without haemagglutinin ‐ tagged activated DAPK ( ΔCaM ) . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies . Levels of Bcl ‐ XL were quantified using NIH image software , and the ratio between immunoprecipitated and expressed Bcl ‐ XL was calculated . ( E ) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl ‐ XL , Flag ‐ tagged beclin 1 and ΔCaM or pcDNA3 ‐ luciferase as a control . The images in ( c ) and ( d ) were taken at higher magnifications of the ΔCaM treatment ( see the scale bars ) . ' AV ' indicates autophagic vacuoles . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GFP , green fluorescent protein ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney ; NIH , National Institutes of Health .

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

Figure 5(A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.(B) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐XL. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.(C) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP-LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (a) Representative GFP-LC3 staining. (b) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: *P=0.01; **P=0.005. (c) Western blot analysis was performed using the indicated antibodies.(D) HEK293 cells were transfected with Bcl‐XL, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐XL were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐XL was calculated.(E) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐XL, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (c) and (d) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). 'AV' indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.


Figure 1 . MEFs from δWD and littermate control mice were incubated with HBSS , chloroquine or monensin for 2 hours as indicated . Cells were fixed and permeabilized and stained for LC3 . Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter . B . Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy . LC3 puncta were identified using spot function software to locate puncta ranging from 0 . 5 - 1 . 0mm diameter . LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter . MEFs from δWD and littermate control mice were incubated with HBSS , chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1 , δWD and LC3 as indicated . Control MEFS express α and β isoforms of ATG16L1 at 70 kDa , and MEFs from δWD mice express a truncated ATG16L1 at 30kDa . ( D ) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean ( ± SD ) of three replicate blots . E ) fluorescence images of phagosomes following engulfment of Zymosan ( red ) by bone marrow - derived macrophages ( BMDM ) from control and δWD mice . White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center . F shows examples of line profile analysis G , H . ( G ) shows the percentage of LC3 positive phagosomes per cell and ( H ) shows line profile analysis of data from 10 cells , bars represent the mean ± SD and were compared by Student ' s t - test ( * * * * P < 0 . 0001 ) .

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 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 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

Figure 1. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 hours as indicated. Cells were fixed and permeabilized and stained for LC3. Arrows indicate LC3 vacuoles where a ring of LC3 signal surrounds vacuoles ranging between 2 and 8 μm diameter. B. Numbers of fluorescent LC3 puncta and LC3 vacuoles in each cell were quantified by fluorescence microscopy. LC3 puncta were identified using spot function software to locate puncta ranging from 0.5-1.0mm diameter. LC3 vacuoles were identified by eye as rings of fluorescence ranging between 2 and 8 μm diameter. MEFs from δWD and littermate control mice were incubated with HBSS, chloroquine or monensin for 2 h as indicated and cell lysates analysed by western blot for ATG16L1, δWD and LC3 as indicated. Control MEFS express α and β isoforms of ATG16L1 at 70 kDa, and MEFs from δWD mice express a truncated ATG16L1 at 30kDa. (D) shows the level of conversion of LC3 to LC3II estimated by densitometry from a mean (±SD) of three replicate blots.E) fluorescence images of phagosomes following engulfment of Zymosan (red) by bone marrow-derived macrophages (BMDM) from control and δWD mice. White line indicates track used for line profile analysis to compare the LC3 signal on the limiting membrane of the phagosome with the center. F shows examples of line profile analysis G, H. (G) shows the percentage of LC3 positive phagosomes per cell and (H) shows line profile analysis of data from 10 cells, bars represent the mean ± SD and were compared by Student's t-test (****P<0.0001).


Figure 2Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( A ) Mice were monitored for weight loss at indicated time - points . ( n = 8 ) . Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( B ) Survival was assessed at indicated time points ( n = 15 ) . Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( C ) IAV titre in lungs was determined by plaque assay at 5 d . p . i . ( n = 6 ) . Littermate control and δWD mice were challenged intranasally with IAV strain X31 ( 103 pfu ) . ( D ) The presence of IAV antigen was assessed by IH at 7 d . p . i . ( representative images from n = 6 ) .

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 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 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

Figure 2Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points. (n = 8).Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (B) Survival was assessed at indicated time points (n = 15).Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).Littermate control and δWD mice were challenged intranasally with IAV strain X31 (103 pfu). (D) The presence of IAV antigen was assessed by IH at 7 d.p.i. (representative images from n = 6).


Figure 3Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( A ) At the indicated time points , cytokine mRNA transcripts in lung tissue ( n = 5 ) were evaluated by qPCR . Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( B ) Representative lung sections from animals ( n = 6 ) taken at 3 d . p . i . were stained by IH for neutrophils ( Ly6G ) or neutrophil extracellular traps ( NET ; anti - H3 ) . Sections at 7 d . p . i . were stained for macrophages ( Iba - 1 ) . Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . C ) BAL ( n = 5 ) was taken at 2 d . p . i and evaluated by flow cytometry , with pre - gating on CD45 + . The percentage of neutrophils ( CD11b + , Ly6G + ) cells is shown ± SEM and were compared using Mann - Whitney U test ( * P < 0 . 05 ) . Littermate control and δWD mice ( n = 5 ) were challenged with IAV X31 ( 103 pfu ) . ( D ) Single cell suspensions were prepared from lungs taken at 5 d . p . i and evaluated by flow cytometry , with pre - gating on CD45 + . The percentage of macrophage / monocytes ( CD11b + , F4 / 80 + ) cells is shown ± SEM and were compared using Mann - Whitney U test ( * P < 0 . 05 ) . ( E ) δWD mice were treated with either anti - PCDA - 1 ( to deplete plasmacytoid DC ) or an isotype - matched control . Litter - mate control mice were used as comparator . Weight loss was measured at the indicated days p . i . ( n = 5 ) .

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 O O O O O O O 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 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 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

Figure 3Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (A) At the indicated time points, cytokine mRNA transcripts in lung tissue (n = 5) were evaluated by qPCR.Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (B) Representative lung sections from animals (n = 6) taken at 3 d.p.i. were stained by IH for neutrophils (Ly6G) or neutrophil extracellular traps (NET; anti-H3). Sections at 7 d.p.i. were stained for macrophages (Iba-1).Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). C) BAL (n = 5) was taken at 2 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of neutrophils (CD11b+, Ly6G+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05).Littermate control and δWD mice (n = 5) were challenged with IAV X31 (103 pfu). (D) Single cell suspensions were prepared from lungs taken at 5 d.p.i and evaluated by flow cytometry, with pre-gating on CD45+. The percentage of macrophage/monocytes (CD11b+, F4/80+) cells is shown ±SEM and were compared using Mann-Whitney U test (*P<0.05).(E) δWD mice were treated with either anti-PCDA-1 (to deplete plasmacytoid DC) or an isotype-matched control. Litter-mate control mice were used as comparator. Weight loss was measured at the indicated days p.i. (n = 5).


Figure 4A - D . Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for weight loss at indicated time - points . Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( B ) Survival was assessed at indicated time points . Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( C ) IAV titre in lungs was determined by plaque assay at 5 d . p . i . ( n = 6 ) . Bone marrow from wild - type ( Atg16L1 + / + ) was used to reconstitute irradiated littermate control mice ( B6 WT → control [ ● ] ) or δWD mice ( B6 WT → δWD [ ○ ] ) . Bone marrow from δWD mice was used to reconstitute irradiated δWD mice ( δWD → δWD [ ▲ ] ) . After 12 weeks , mice ( n = 5 per group ) were challenged with IAV X31 ( 103 pfu ) . ( D ) Lungs taken at 5 d . p . i . were analysed for neutrophils ( Ly6G ) , neutrophil extracellular traps ( NET ; anti - H3 ) and macrophages ( Iba - 1 ) .

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 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 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

Figure 4A-D. Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points.Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (B) Survival was assessed at indicated time points.Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (C) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).Bone marrow from wild-type (Atg16L1+/+) was used to reconstitute irradiated littermate control mice (B6 WT → control [●] ) or δWD mice (B6 WT → δWD [○] ). Bone marrow from δWD mice was used to reconstitute irradiated δWD mice (δWD → δWD [▲] ). After 12 weeks, mice (n = 5 per group) were challenged with IAV X31 (103 pfu). (D) Lungs taken at 5 d.p.i. were analysed for neutrophils (Ly6G), neutrophil extracellular traps (NET; anti-H3) and macrophages (Iba-1) .


Figure 5δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( A ) Mice were monitored for weight loss at indicated time - points . δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( B ) IAV titre in lungs was determined by plaque assay at 5 d . p . i . ( n = 6 ) . δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( C ) IL - 1β mRNA transcripts in lung at 5 d . p . i . were determined by qPCR . δWDphag mice lack non - canonical autophagy in myeloid ( LysMcre ) cells Offspring negative for LysMcre were used as littermate controls . Mice ( n = 6 per group ) were challenged intranasally with IAV X31 ( 103 pfu ) . ( D ) Precision - cut lung slices from control and δWD mice were infected with IAV . Virus titres were determined at indicated time points .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (A) Mice were monitored for weight loss at indicated time-points.δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (B) IAV titre in lungs was determined by plaque assay at 5 d.p.i. (n = 6).δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (C) IL-1β mRNA transcripts in lung at 5 d.p.i. were determined by qPCR.δWDphag mice lack non-canonical autophagy in myeloid (LysMcre) cells Offspring negative for LysMcre were used as littermate controls. Mice (n = 6 per group) were challenged intranasally with IAV X31 (103 pfu). (D) Precision-cut lung slices from control and δWD mice were infected with IAV. Virus titres were determined at indicated time points.


Figure 6A ) . The WD domain of ATG16L1 reduces IAV replication in MEFs . Cells were infected with IAV and virus titres were estimated at 8 hours post infection by plaque assay . B ) . IAV binding is similar between control and δWD MEFs . Cells were bound with IAV X31 labeled with AF488 on ice for 1 h , and fixed immediately . The cells were stained with anti - HA ( H3SKE , red ) without permeabilisation to visualise extracellular IAV particles , counter stained with Hoechst ( nuclei ) and WGA - AF647 to label the cell membrane . Cells were imaged by automated confocal microscopy using a 40x objective , and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell . The results and means ( horizontal line ) of n = 3 independent experiments ( 50 - 100 cells per experiment ) are shown . C ) IAV infection following acid bypass is comparable between control and δWD MEFs . IAV was bound on ice for 1 h , and warmed at either neutral pH ( 6 . 8 ) or low pH ( pH 5 . 0 ) for 2 min , followed by incubation in STOP medium containing NH4Cl for 18 hours . Cells were fixed and stained for NP and Hoechst , imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity . The results from N = 3 independent experiments ( 5 , 000 - 10 , 000 cells quantified per experiment ) , and the means are shown . Scale bar ; 500 µm . D ) . IAV fusion with endosomes is increased in δWD MEFs . Cells were infected with dual labeled ( R18 / SP - DiOC18 ) IAV for 1 h , fixed , counterstained with Hoechst ( nuclei blue ) and WGA - AF647 ( cell perimeter in white ) . Cells were imaged by automated confocal microscopy and the number of fusion events per cell ( represented by number of SP - DiOC18 puncta ) was quantified . Fused and non - fused viral particles are shown as green and red spots respectively . The results and means ( horizontal lines ) of n = 3 independent experiments ( 30 - 60 cells per experiment ) are shown . Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right . E , F ) Numbers of fusion positive cells is increased for δWD MEFs . MEFs from δWD ( white circles ) or littermate control mice ( black circles ) were incubated with dual - labelled ( SP - DiOC18 / R18 ) IAV at 40C for 45 min and warmed to 37 ° C for 30 ad 60 minutes . Cells were harvested by trypsinisation , fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion ( E ) and median fluorescence intensity ( MFI ) of dequenched SP - DiOC18 signa ( F ) . Graphs show individual replicates ( n = 3 ) with a horizontal line at the mean and were compared using two - way ANOVA with Bonferroni post - tests * * P < 0 . 01 , * * * P < 0 . 001 ) . ( G , H ) Endocytosis of IAV increases in δWD MEFs . MEFs from δWD or littermate control mice were incubated with AlexFluor 488 - labelled IAV at 40C for 1hr and warmed to 37 ° C for 15 Cells were fixed and permeabilised and stained with antibody against HA ( red ) . The blue line indicates the plasma membrane of the cell . Green arrows indicate examples of viruses only labelled with green fluorescence . Double - labelled virus particles ( green - red ) represent non - endocytosed particles bound at the cell surface , whereas green particles represent endocytosed particles . The results and means of n = 3 independent experiments ( 50 - 100 cells per experiment ) are shown . ( I , J ) Interferon signalling increases in δWD MEFs . MEFs from δWD or littermate control mice were infected with IAV . At the indicated time points , mRNA transcripts were evaluated by qPCR for IFIT1 ( I ) and ISG15 ( J ) .

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 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 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 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 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 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 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

Figure 6A). The WD domain of ATG16L1 reduces IAV replication in MEFs. Cells were infected with IAV and virus titres were estimated at 8 hours post infection by plaque assay.B). IAV binding is similar between control and δWD MEFs. Cells were bound with IAV X31 labeled with AF488 on ice for 1 h, and fixed immediately. The cells were stained with anti-HA (H3SKE, red) without permeabilisation to visualise extracellular IAV particles, counter stained with Hoechst (nuclei) and WGA-AF647 to label the cell membrane. Cells were imaged by automated confocal microscopy using a 40x objective, and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell. The results and means (horizontal line) of n = 3 independent experiments (50-100 cells per experiment) are shown.C) IAV infection following acid bypass is comparable between control and δWD MEFs. IAV was bound on ice for 1 h, and warmed at either neutral pH (6.8) or low pH (pH 5.0) for 2 min, followed by incubation in STOP medium containing NH4Cl for 18 hours. Cells were fixed and stained for NP and Hoechst, imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity. The results from N=3 independent experiments (5,000-10,000 cells quantified per experiment), and the means are shown. Scale bar; 500 µm.D). IAV fusion with endosomes is increased in δWD MEFs. Cells were infected with dual labeled (R18/SP-DiOC18) IAV for 1 h, fixed, counterstained with Hoechst (nuclei blue) and WGA-AF647 (cell perimeter in white). Cells were imaged by automated confocal microscopy and the number of fusion events per cell (represented by number of SP-DiOC18 puncta) was quantified. Fused and non-fused viral particles are shown as green and red spots respectively. The results and means (horizontal lines) of n = 3 independent experiments (30-60 cells per experiment) are shown. Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right.E, F) Numbers of fusion positive cells is increased for δWD MEFs. MEFs from δWD (white circles) or littermate control mice (black circles) were incubated with dual-labelled (SP-DiOC18/R18) IAV at 40C for 45 min and warmed to 37°C for 30 ad 60 minutes. Cells were harvested by trypsinisation, fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion (E) and median fluorescence intensity (MFI) of dequenched SP-DiOC18 signa (F). Graphs show individual replicates (n = 3) with a horizontal line at the mean and were compared using two-way ANOVA with Bonferroni post-tests **P<0.01, ***P<0.001).(G, H) Endocytosis of IAV increases in δWD MEFs. MEFs from δWD or littermate control mice were incubated with AlexFluor 488-labelled IAV at 40C for 1hr and warmed to 37°C for 15 Cells were fixed and permeabilised and stained with antibody against HA (red). The blue line indicates the plasma membrane of the cell. Green arrows indicate examples of viruses only labelled with green fluorescence. Double-labelled virus particles (green-red) represent non-endocytosed particles bound at the cell surface, whereas green particles represent endocytosed particles. The results and means of n = 3 independent experiments (50-100 cells per experiment) are shown.(I, J) Interferon signalling increases in δWD MEFs. MEFs from δWD or littermate control mice were infected with IAV. At the indicated time points, mRNA transcripts were evaluated by qPCR for IFIT1 (I) and ISG15 (J).


Figure 1 ( A ) Microchamber that allows the isolation of axons within a corticostriatal network and live - cell imaging of axonal BDNF - mCherry transduced into mouse primary cortical neurons . Data information : Scale bars = 20 µm . ( B ) Representative kymographs and quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry - containing vesicles in cortical neurons transfected with siMecp2 ( n = 753 vesicles / 81 axons ) or siControl ( siCtl ) ( n = 894 vesicles / 94 axons ) , inducing significant silencing of Mecp2 ( 3 independent experiments ; unpaired t - test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 Data are presented as the means ± SEM . ( C ) Quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry trafficking into cortical neurons obtained from WT ( n = 753 vesicles / 81 axons ) , HTTSA ( n = 812 vesicles / 81 axons ) , or HTTSD ( n = 787 vesicles / 82 axons ) homozygous knock - in mice in which S421 of HTT was replaced by an alanine ( HTTS421A / S421A or HTTSA ) , mimicking the absence of phosphorylation , or by an aspartic acid ( HTTS421D / S421D or HTTSD ) , mimicking constitutive phosphorylation ( 3 independent experiments ; one - way ANOVA test with Tukey ' s multiple comparisons ) . Data information : , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . Data are presented as the means ± SEM . ( D ) Cell viability in WT ( n = 3 ) , HTTSA ( n = 1 ) , or HTTSD ( n = 2 ) neurons measured by MTT assay . No differences were observed between the different groups ( 3 independent experiments ; one - way ANOVA Kruskal - Wallis test , p = 0 . 5701 Data information : ns = not significant . Data are presented as the means ± SEM . ( E ) Kymographs and quantification of anterograde , retrograde , mean velocity , and linear flow rate of BDNF - mCherry axonal trafficking into WT ( n = 894 vesicles / 94 axons ) , HTTSA ( n = 812 vesicles / 82 axons ) , or HTTSD ( n = 787 vesicles / 94 axons ) cortical neurons transfected with siCtl or siMecp2 , which significantly silenced Mecp2 ( 3 independent experiments ; one - way ANOVA with Tukey ' s multiple comparisons ) . ( F ) Cell viability in WT , HTTSA , or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay ( WT siCtl : n = 3 ; WT siMecp2 : n = 3 ; HTTSD siMecp2 : n = 3 ; HTTSA siMecp2 : n = 2 ) . No differences were observed between the different groups ( 3 independent experiments ; one - way ANOVA Kruskal - Wallis test , p = 0 . 5701 ) Data information : Scale bars = 20 µm . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . Data are presented as the means ± SEM .

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 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 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 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 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 O O O O O O O O O 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) Microchamber that allows the isolation of axons within a corticostriatal network and live-cell imaging of axonal BDNF-mCherry transduced into mouse primary cortical neurons. Data information: Scale bars = 20 µm.(B) Representative kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 (n= 753 vesicles/ 81 axons) or siControl (siCtl) (n= 894 vesicles/ 94 axons), inducing significant silencing of Mecp2 (3 independent experiments; unpaired t-test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Data are presented as the means ± SEM.(C) Quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry trafficking into cortical neurons obtained from WT (n= 753 vesicles/ 81 axons), HTTSA (n= 812 vesicles/ 81 axons), or HTTSD (n= 787 vesicles/ 82 axons) homozygous knock-in mice in which S421 of HTT was replaced by an alanine (HTTS421A/S421A or HTTSA), mimicking the absence of phosphorylation, or by an aspartic acid (HTTS421D/S421D or HTTSD), mimicking constitutive phosphorylation (3 independent experiments; one-way ANOVA test with Tukey's multiple comparisons). Data information: , ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.(D) Cell viability in WT (n=3), HTTSA (n=1), or HTTSD (n=2) neurons measured by MTT assay. No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701 Data information: ns = not significant. Data are presented as the means ± SEM.(E) Kymographs and quantification of anterograde, retrograde, mean velocity, and linear flow rate of BDNF-mCherry axonal trafficking into WT (n= 894 vesicles/ 94 axons), HTTSA (n= 812 vesicles/ 82 axons), or HTTSD (n=787 vesicles/ 94 axons) cortical neurons transfected with siCtl or siMecp2, which significantly silenced Mecp2 (3 independent experiments; one-way ANOVA with Tukey's multiple comparisons). (F) Cell viability in WT, HTTSA, or HTTSD neurons transfected with siCtl or siMecp2 measured by MTT assay (WT siCtl: n=3; WT siMecp2: n=3; HTTSD siMecp2: n=3; HTTSA siMecp2: n=2). No differences were observed between the different groups (3 independent experiments; one-way ANOVA Kruskal-Wallis test, p=0.5701) Data information: Scale bars = 20 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Data are presented as the means ± SEM.


Figure 2 ( A ) Left : The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day - old WT ( n = 8 ) , Mecp2 KO ( n = 6 ) , Mecp2 KO / HTTSD ( mimicking the absence of phosphorylation ) ( n = 4 ) and Mecp2 KO / HTTSA mice ( mimicking constitutive phosphorylation ) ( n = 5 ) . Since striatal BDNF depends only on BDNF transport from the cortex , this ratio reflects BDNF transport through corticostriatal pathway ( Mann Whitney test ) . Right : Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting . The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio . ( n = 6 mice per group ) ( Mann Whitney test ) Middle : Quantitative analysis of PSD - 95 protein level in striatum of Mecp2 KO / HTT WT mice and KO / HTTSD mice by immunoblotting . The relative expression levels of PSD - 95 were normalized against GAPDH and are presented as the ratio . ( n = 6 mice per group ) ( Mann Whitney test ) Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . ( B ) Left : We investigated the behavior of Mecp2 KO mice at 35 , 45 , and 55 days of age and assessed their survival . Right : Mecp2 KO / HTTSD mice ( n = 30 ) and WT mice ( n = 17 ) had a significantly longer lifespan than KO ( n = 22 ) or KO / HTTSA mice ( n = 21 ) ( Kaplan - Meier survival test ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . ( C ) Body weight of 10 WT , 24 KO , 32 KO / HTTSD , and 24 KO / HTTSA mice at P30 and P50 ( one - way ANOVA with Tukey ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . ( D ) Frequency of apnea of 9 WT , 10 KO , 14 KO / HTTSD , and 9 KO / HTTSA mice at P35 and P55 . ( Kruskal - Wallis test with Dunn ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . ( E ) Mecp2 KO / HTTSD mice ( n = 19 ) performed as well as WT ( n = 17 ) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO ( n = 16 ) and Mecp2 KO / HTTSA ( n = 13 ) at P55 ( one - way ANOVA with Fisher ' s LSD test ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant . ( F ) Time before the onset of spontaneous locomotor activity of 17 WT , 20 KO , 19 KO / HTTSD , and 17 KO / HTTSA mice at P55 . ( Kruskal - Wallis test with Dunn ' s comparison ) . Data information : Data are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ns = not significant .

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

Figure 2(A) Left: The ratio between the striatal and the cortical BDNF determined by quantifying the level of BDNF proteins at the cortex and the striatum of 55 day-old WT (n=8), Mecp2 KO (n=6), Mecp2 KO/HTTSD (mimicking the absence of phosphorylation) (n=4) and Mecp2 KO/HTTSA mice (mimicking constitutive phosphorylation) (n=5). Since striatal BDNF depends only on BDNF transport from the cortex, this ratio reflects BDNF transport through corticostriatal pathway (Mann Whitney test). Right: Quantitative analysis of phospho TrkB protein level in striatum of KO WT mice and KO HTTSD mice by immunoblotting. The relative expression levels of phospho TrkB were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Middle: Quantitative analysis of PSD-95 protein level in striatum of Mecp2 KO/HTT WT mice and KO/HTTSD mice by immunoblotting. The relative expression levels of PSD-95 were normalized against GAPDH and are presented as the ratio. (n = 6 mice per group) (Mann Whitney test) Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.(B) Left: We investigated the behavior of Mecp2 KO mice at 35, 45, and 55 days of age and assessed their survival. Right: Mecp2 KO/HTTSD mice (n = 30) and WT mice (n = 17) had a significantly longer lifespan than KO (n=22) or KO/HTTSA mice (n = 21) (Kaplan-Meier survival test). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.(C) Body weight of 10 WT, 24 KO, 32 KO/HTTSD, and 24 KO/HTTSA mice at P30 and P50 (one-way ANOVA with Tukey's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.(D) Frequency of apnea of 9 WT, 10 KO, 14 KO/HTTSD, and 9 KO/HTTSA mice at P35 and P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.(E) Mecp2 KO/HTTSD mice (n=19) performed as well as WT (n=17) at P35 on the accelerating rotarod test and continued to outperform Mecp2 KO (n=16) and Mecp2 KO/ HTTSA (n=13) at P55 (one-way ANOVA with Fisher's LSD test). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.(F) Time before the onset of spontaneous locomotor activity of 17 WT, 20 KO, 19 KO/HTTSD, and 17 KO/HTTSA mice at P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ns = not significant.


Figure 3 ( A ) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl ( siCtl ) and treated with 1 µM FK506 or vehicle for 1 h , and quantification of pS421 HTT ( n = 9 per group ) . Data are presented as means ± SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test . ( B ) Representative kymographs showing axonal trafficking of BDNF - mCherry - containing vesicles in cortical neurons transfected with siMecp2 or siControl ( siCtl ) and treated with 1 µM FK506 or vehicle for 1 h . Scale bar = 20 µm . ( C ) Quantification of anterograde , retrograde , mean velocity , linear flow rate and number of BDNF - mCherry - containing vesicles from the data in ( A ) ( siMecp2 + Vehicle : n = 801 vesicles / 95 axons ; siMecp2 + FK506 : n = 1020 vesicles / 116 axons ; siCtl + Vehicle : n = 780 vesicles / 83 axons ; siCtl + FK506 : n = 1029 vesicles / 102 axons ) . Data are presented as the mean ± SEM of at least three independent experiments ( one - way ANOVA with Tukey ' s comparison ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns = not significant .

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 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 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

Figure 3(A) Western blot analysis of DIV 5 cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h, and quantification of pS421 HTT (n=9 per group). Data are presented as means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test.(B) Representative kymographs showing axonal trafficking of BDNF-mCherry-containing vesicles in cortical neurons transfected with siMecp2 or siControl (siCtl) and treated with 1 µM FK506 or vehicle for 1 h. Scale bar = 20 µm.(C) Quantification of anterograde, retrograde, mean velocity, linear flow rate and number of BDNF-mCherry-containing vesicles from the data in (A) (siMecp2+Vehicle: n=801 vesicles/95 axons; siMecp2+FK506: n=1020 vesicles/116 axons; siCtl+Vehicle: n=780 vesicles/83 axons; siCtl+FK506: n=1029 vesicles/102 axons). Data are presented as the mean ± SEM of at least three independent experiments (one-way ANOVA with Tukey's comparison). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.


Figure 4 ( A ) Western blot of HTT S421 phosphorylation . We treated 30 - day - old Mecp2 KO mice intraperitoneally with FK506 ( 5 mg / kg ) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting , 2 h after administration , using an anti - phospho - HTT - S421 specific antibody . The D7F7 antibody recognizes total HTT . The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio ( KO FK506 n = 4 , KO Vehicle n = 4 ) . Data are presented as means ± SEM , * p < 0 . 05 , Mann Whitney test . ) We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( B ) FK506 - treated KO mice lived longer than vehicle - treated KO mice and FK506 - treated KO HTTSA mice ( Kaplan - Meier survival test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM . We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( C ) Body weight of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice at P35 and P55 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM . We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( D ) Frequency of apnea of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice at P35 and P55 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM . We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( E ) Motor coordination of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice on the accelerating rotarod test at P30 and P50 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM . We treated 30 - day - old Mecp2 KO mice ( n = 10 ) and Mecp2 KO / HTTSA mice ( n = 10 ) with 5mg / kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests . ( F ) Forelimb strength of FK506 - treated KO , FK506 - treated KO HTTSA and vehicle - treated KO mice assessed by the grip strength test at P40 and P60 ( Mann - Whitney test ) . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant . Data are means ± SEM .

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 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 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 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 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 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

Figure 4(A) Western blot of HTT S421 phosphorylation. We treated 30-day-old Mecp2 KO mice intraperitoneally with FK506 (5 mg/kg) or vehicle and analyzed brain extracts for endogenous HTT phosphorylation by western blotting, 2 h after administration, using an anti-phospho-HTT-S421 specific antibody. The D7F7 antibody recognizes total HTT. The relative protein level of phospho HTT was normalized on total HTT protein level and are presented as the ratio (KO FK506 n = 4, KO Vehicle n = 4). Data are presented as means ± SEM, *p < 0.05, Mann Whitney test.)We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (B) FK506-treated KO mice lived longer than vehicle-treated KO mice and FK506-treated KO HTTSA mice (Kaplan-Meier survival test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (C) Body weight of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (D) Frequency of apnea of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice at P35 and P55 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (E) Motor coordination of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice on the accelerating rotarod test at P30 and P50 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.We treated 30-day-old Mecp2 KO mice (n=10) and Mecp2 KO/HTTSA mice (n=10) with 5mg/kg FK506 three times a week by intraperitoneal injection and assessed them in various behavioral tests. (F) Forelimb strength of FK506-treated KO, FK506-treated KO HTTSA and vehicle-treated KO mice assessed by the grip strength test at P40 and P60 (Mann-Whitney test). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are means ± SEM.


Figure 1 ( A ) . A FRET assay was used to measure the difference in melting temperature ( ΔTm ) of ( G4C2 ) 4 RNA or DNA G - Qs in the presence of 2 μM of 138 different small molecules . An increase in ΔTm indicates stabilisation of the G - Q . Small molecules are ranked on the x axis according to their increasing thermal stabilisation of the DNA ( G4C2 ) 4 G - Q . Small molecules that preferentially stabilise RNA over DNA ( G4C2 ) 4 G - Qs reside in the upper part of the scatter plot , above the blue curve . An arbitrary ΔTm threshold of 13 ° C greater than vehicle ( grey line ) and a differential binding to RNA over DNA ( ΔTmRNA - ΔTmDNA ≥ 5 ° C ) were used to select candidate small molecules . ( C ) . FRET dose - response of DB1246 , DB1247 and DB1273 on stabilisation of RNA or DNA ( G4C2 ) 4 G - Qs . ( D ) . Temperature unfold CD spectra for ( G4C2 ) 4 RNA alone ( which shows a characteristic G - Q structure with minima at 237 nm , maxima at 264 nm and no additional signal ) , or in the presence of 2 μM DB1246 , DB1247 or DB1273 . A characteristic induced CD spectrum , in the 350 nm - 550 nm region , is observed only in the presence of each small molecule , confirming that each of these three compounds are binding to ( G4C2 ) 4 RNA G - Qs . Data in A represent mean ± s . d . , n = 1 with 3 technical replica . Data in C represent mean ± s . d . , n = 3 independent experiments . Data in D represent mean ± s . d . , n = 3 independent experiments .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). A FRET assay was used to measure the difference in melting temperature (ΔTm) of (G4C2)4 RNA or DNA G-Qs in the presence of 2 μM of 138 different small molecules. An increase in ΔTm indicates stabilisation of the G-Q. Small molecules are ranked on the x axis according to their increasing thermal stabilisation of the DNA (G4C2)4 G-Q. Small molecules that preferentially stabilise RNA over DNA (G4C2)4 G-Qs reside in the upper part of the scatter plot, above the blue curve. An arbitrary ΔTm threshold of 13 °C greater than vehicle (grey line) and a differential binding to RNA over DNA (ΔTmRNA-ΔTmDNA ≥ 5 °C) were used to select candidate small molecules.(C). FRET dose-response of DB1246, DB1247 and DB1273 on stabilisation of RNA or DNA (G4C2)4 G-Qs.(D). Temperature unfold CD spectra for (G4C2)4 RNA alone (which shows a characteristic G-Q structure with minima at 237 nm, maxima at 264 nm and no additional signal), or in the presence of 2 μM DB1246, DB1247 or DB1273. A characteristic induced CD spectrum, in the 350 nm - 550 nm region, is observed only in the presence of each small molecule, confirming that each of these three compounds are binding to (G4C2)4 RNA G-Qs. Data in A represent mean ± s.d., n=1 with 3 technical replica. Data in C represent mean ± s.d., n=3 independent experiments. Data in D represent mean ± s.d., n=3 independent experiments.


Figure 2 ( A ) Representative images of C9orf72 iPSC - cortical neurons treated with DMSO ( vehicle control ) or 1 µM of DB1246 , DB1247 or DB1273 , for 4 days . RNA foci are shown in red and nuclei ( DAPI ) in blue . Scale bar represents 10 µm . ( B ) Quantification shows RNA foci are significantly reduced by all three compounds in iPSC - cortical neurons and by DB1246 and DB1273 in iPSC - motor neurons . Data are shown as the average percentage of neurons containing RNA foci relative to vehicle ( DMSO ) . N = 3 independent C9orf72 patient lines with 2 - 3 inductions per line and at least 70 neurons counted per induction . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one sample two - tailed t - test versus normalised control . For cortical neurons , p = 0 . 0124 ( DB1246 ) , p = 0 . 0065 ( DB1247 ) , p = 0 . 0096 ( DB1273 ) . For motor neurons , p = 0 . 0004 ( DB1246 ) , p = 0 . 0030 ( DB1273 ) .

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 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

Figure 2(A) Representative images of C9orf72 iPSC-cortical neurons treated with DMSO (vehicle control) or 1 µM of DB1246, DB1247 or DB1273, for 4 days. RNA foci are shown in red and nuclei (DAPI) in blue. Scale bar represents 10 µm.(B) Quantification shows RNA foci are significantly reduced by all three compounds in iPSC-cortical neurons and by DB1246 and DB1273 in iPSC-motor neurons. Data are shown as the average percentage of neurons containing RNA foci relative to vehicle (DMSO). N=3 independent C9orf72 patient lines with 2-3 inductions per line and at least 70 neurons counted per induction. *p <0.05, **p <0.01, ***p <0.001, one sample two-tailed t-test versus normalised control. For cortical neurons, p=0.0124 (DB1246), p=0.0065 (DB1247), p=0.0096 (DB1273). For motor neurons, p=0.0004 (DB1246), p=0.0030 (DB1273).


Figure 3 ( A ) Poly ( GP ) levels were measured by MSD immunoassay in C9orf72 patient iPSC - motor neurons treated for 7 days with 8 - 16 µM of the G4C2 repeat G - Q binding small molecules DB1247 , DB1247 and DB1273 . Treatment with DB1246 or DB1273 leads to a significant reduction in poly ( GP ) levels relative to vehicle - treated controls . Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines , with 1 - 6 differentiations per line . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one sample two - tailed t - test versus normalised control . p = 0 . 0068 ( DB1246 , 12 µm ) , p = 0 . 0417 ( DB1246 , 16 µm ) , p = 0 . 0196 ( DB1247 , 8 µm ) , p = 0 . 0002 ( DB1273 , 8 µm ) , p = 0 . 0194 ( DB1273 , 12 µm ) , p = 0 . 0062 ( DB1273 , 16 µm ) ( B ) C9orf72 patient iPSC - motor neurons were treated for 7 days with 16 µM of each small molecule and the expression levels of MCM2 and the three C9orf72 transcript variants ( V1 - V3 ) measured by quantitative RT - PCR . Data are shown as the mean and s . d . of 3 independent iPSC - motor neuron lines ( one induction per line ) relative to vehicle ( DMSO ) treated controls . No significant changes in gene expression were observed , one way ANOVA , Dunnett ' s post hoc test , p > 0 . 05 . ( C ) XTT cell death assay for C9orf72 iPSC - motor neurons treated for 7 days with 0 - 40 µM of DB1246 , DB1247 , DB1273 or cisplatin as a positive control . Data are shown as the mean and SEM of two independent iPSC - motor neuron lines . Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM . ( D ) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273 , and the number reaching the pupal stage of development counted after 7 days . DB1273 treatment ( n = 1200 flies ) significantly improves survival compared to vehicle ( n = 1150 flies ) , p = 0 . 0320 , chi - squared test . Data are shown as proportion reaching the pupal stage , with numbers within each group indicated on the bars . Genotype was w1118 ; ( GGGGCC ) 36 / + ; daGAL4 / + ( daGAL4 > 36R ) . ( E ) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats ( 36R ) were treated with vehicle or 1 mM DB1273 for 5 days , until the third instar stage ( L3 ) , and poly ( GR ) measured by MSD immunoassay . Poly ( GR ) was also measured in control ( w1118 ) larvae that do not express G4C2 repeats . DB1273 treatment significantly reduced poly ( GR ) expression . n = 8 for 36R groups , n = 7 for control group . * * p < 0 . 01 , * * * p < 0 . 001 , one way ANOVA with Dunnett ' s post hoc test . p = 0 . 0001 ( w1118 versus 36R 0 mM larvae ) , p = 0 . 0019 ( 0 mM versus 1 mM 36R larvae ) . Genotypes were w1118 ; ; and w1118 ; ( GGGGCC ) 36 / + ; daGAL4 / + ( daGAL4 > 36R ) .

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 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 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 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 3(A) Poly(GP) levels were measured by MSD immunoassay in C9orf72 patient iPSC-motor neurons treated for 7 days with 8-16 µM of the G4C2 repeat G-Q binding small molecules DB1247, DB1247 and DB1273. Treatment with DB1246 or DB1273 leads to a significant reduction in poly(GP) levels relative to vehicle-treated controls. Data are shown as the mean and SEM of 3 independent C9orf72 iPSC lines, with 1-6 differentiations per line. *p<0.05, **p<0.01, *** p<0.001, one sample two-tailed t-test versus normalised control. p=0.0068 (DB1246, 12 µm), p=0.0417 (DB1246, 16 µm), p=0.0196 (DB1247, 8 µm), p=0.0002 (DB1273, 8 µm), p=0.0194 (DB1273, 12 µm), p=0.0062 (DB1273, 16 µm)(B) C9orf72 patient iPSC-motor neurons were treated for 7 days with 16 µM of each small molecule and the expression levels of MCM2 and the three C9orf72 transcript variants (V1-V3) measured by quantitative RT-PCR. Data are shown as the mean and s.d. of 3 independent iPSC-motor neuron lines (one induction per line) relative to vehicle (DMSO) treated controls. No significant changes in gene expression were observed, one way ANOVA, Dunnett's post hoc test, p>0.05.(C) XTT cell death assay for C9orf72 iPSC-motor neurons treated for 7 days with 0 - 40 µM of DB1246, DB1247, DB1273 or cisplatin as a positive control. Data are shown as the mean and SEM of two independent iPSC-motor neuron lines. Toxicity is only observed at the highest dose of 40 µM and not at the penultimate dose of 20 µM.(D) Drosophila first instar larvae expressing 36 G4C2 repeats were placed on food containing either vehicle or 1 mM DB1273, and the number reaching the pupal stage of development counted after 7 days. DB1273 treatment (n=1200 flies) significantly improves survival compared to vehicle (n=1150 flies), p=0.0320, chi-squared test. Data are shown as proportion reaching the pupal stage, with numbers within each group indicated on the bars. Genotype was w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).(E) Drosophila first instar larvae ubiquitously expressing 36 G4C2 repeats (36R) were treated with vehicle or 1 mM DB1273 for 5 days, until the third instar stage (L3), and poly(GR) measured by MSD immunoassay. Poly(GR) was also measured in control (w1118) larvae that do not express G4C2 repeats. DB1273 treatment significantly reduced poly(GR) expression. n=8 for 36R groups, n=7 for control group. **p<0.01, *** p<0.001, one way ANOVA with Dunnett's post hoc test. p=0.0001 (w1118 versus 36R 0 mM larvae), p=0.0019 (0 mM versus 1 mM 36R larvae). Genotypes were w1118; ; and w1118; (GGGGCC)36/+; daGAL4/+ (daGAL4>36R).


Figure 1A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( A ) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine ( CQ ) . Cell lysates were evaluated by immunoblotting . A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( B ) Cells were infected , for 1 and 4 hours , with K . pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment . Cell lysates were plated in LB agar for 18 hours and CFU were determined . Data information : the triangles and dots represent the individual test ( three technical replicates per individual test ) on control and Atg7 knock down cells . The bars represent the mean CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . Statistical significance between assays were calculated using two - tailed student ' s t test with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( D - E ) Cells were infected , for 1 - 4 hours , with WT ( D ) or ΔpscD P . aeruginosa ( E ) at MOI of 10 . All infections were followed by 1 hour of gentamycin treatment . Cell lysates were plated in LB agar for 18 hours and CFU were determined . Data information : , the triangles and dots represent the individual test ( three technical replicates per individual test ) on control and Atg7 knock down cells . The bars represent the mean CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . Statistical significance between assays were calculated using two - tailed student ' s t test with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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 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 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 1A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (A) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine (CQ). Cell lysates were evaluated by immunoblotting. A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (B) Cells were infected, for 1 and 4 hours, with K. pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01. A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (D-E) Cells were infected, for 1-4 hours, with WT (D) or ΔpscD P. aeruginosa (E) at MOI of 10. All infections were followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: , the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


Figure 2 ( A ) A549 cells were infected with WT P . aeruginosa for various time points and cell lysates were evaluated by immunoblotting . ( B ) A549 cells infected with WT or ΔpscD P . aeruginosa for 4 hours or left uninfected ( NI ) in the presence of lysosomal inhibitor CQ ( 50 nM ) . The cell lysates were evaluated by immunoblotting . ( C ) A549 cells stably expressing GFP - LC3 were left non - infected or infected , for 4 hours , with WT or ΔpscD P . aeruginosa , and evaluated by fluorescence microscopy . Cell nuclei were counterstained with DAPI ( blue ) and autophagosome were visualized as green puncta . Graph represents quantitative analysis of GFP - LC3 positive autophagosome per cell , scale bars : 10 µm . Each bar represents mean value of three independent experiments , error bars represent standard deviation . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison Test . * * p ≤ 0 . 01 . ( D - E ) A549 cells were treated for 16 hours with rapamycin 0 . 8 μg / ml or DMSO , then infected with either WT ( D ) or ΔpscD P . aeruginosa and followed by 1 hour of gentamycin treatment ( E ) . The circles and squares represent the individual test ( three technical replicates per individual test ) with DMSO and rapamycin treatment . The bars represent the means of CFU from three independent experiments , error bars represent standard deviation . The significance of differences between different drug treatment was determined using two - tailed student ' s t test with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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 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 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 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

Figure 2(A) A549 cells were infected with WT P. aeruginosa for various time points and cell lysates were evaluated by immunoblotting.(B) A549 cells infected with WT or ΔpscD P. aeruginosa for 4 hours or left uninfected (NI) in the presence of lysosomal inhibitor CQ (50 nM). The cell lysates were evaluated by immunoblotting.(C) A549 cells stably expressing GFP-LC3 were left non-infected or infected, for 4 hours, with WT or ΔpscD P. aeruginosa, and evaluated by fluorescence microscopy. Cell nuclei were counterstained with DAPI (blue) and autophagosome were visualized as green puncta. Graph represents quantitative analysis of GFP-LC3 positive autophagosome per cell, scale bars: 10 µm. Each bar represents mean value of three independent experiments, error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. **p ≤ 0.01.(D-E) A549 cells were treated for 16 hours with rapamycin 0.8 μg/ml or DMSO, then infected with either WT (D) or ΔpscD P. aeruginosa and followed by 1 hour of gentamycin treatment (E). The circles and squares represent the individual test (three technical replicates per individual test) with DMSO and rapamycin treatment. The bars represent the means of CFU from three independent experiments, error bars represent standard deviation. The significance of differences between different drug treatment was determined using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


Figure 3A549 cells were infected with one of several P . aeruginosa mutants . Cell lysates were evaluated by immunoblotting . ( B ) primary human bronchial epithelial cells were infected with one of several P . aeruginosa mutants . Cell lysates were evaluated by immunoblotting . ( C ) A549 cells , stably expressing GFP - LC3 , were infected with WT or P . aeruginosa mutants and evaluated by fluorescence microscopy , scale bars : 10 µm . GFP - LC3 puncta representing the autophagosomes was quantified and presented in the graph . Each bar represents mean value from three independent experiments and error bars represent standard deviation . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison Test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . P . aeruginosa mutant strains legend : ΔSTY ( mutant deficient for the three cytotoxins ) , WT ( contains all cytotoxin ) , ΔS ( deficient for ExoS but still express ExoT and ExoY ) , ΔTY ( express ExoS but deficient for ExoT and ExoY ) . ( D , E ) A549 cells , stably expressing GFP - LC3 , were infected for 4 hours with WT or with one of P . aeruginosa mutants ( ΔSTY , ΔS and ΔTY ) and evaluated by fluorescence microscopy ( D ) . P . aeruginosa were visualized using specific antibody ( red ) and the colocalized bacteria signals with GFP - LC3 puncta ( green ) were merged and presented as yellow . Scale bars : 10 µm . ( E ) Quantitative analysis of co - localization of internalized P . aeruginosa with GFP - LC3 positive puncta . The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells . Data represent mean ± SD of three independent experiments . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison Test . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . ( F ) Representative transmission electron microscopy images of A549 cells infected with P . aeruginosa containing ExoS ( ΔTY ) or deficient for the three T3SS effector proteins ( ΔSTY ) for 4 hours . ΔSTY P . aeruginosa ( white star ) was engulfed by multiple membrane autophagosomes ( black arrow ) . ΔTY P . aeruginosa ( white star ) were found in cytoplasm with single membrane surrounded , scale bars : 0 . 5 µm . ( G ) A549 cells , stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA , were infected with WT or P . aeruginosa mutants ( ΔSTY , ΔTY and ΔS ) at MOI of 10 . All infections were followed by 1 hours of gentamycin treatment . Cell lysates were plated in LB agar for 18 hours and CFU were determined . Data information : The triangles and dots represent the individual test ( three technical replicates per individual test ) on control and Atg7 knock down cells from three biological replicates . The bars represent the means of CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . The significance of differences between different assay was determined using two - tailed student ' s t test with Welch ' s correction , NS : not significant ; * * p ≤ 0 . 01 .

O B-PANEL_START 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A549 cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.(B) primary human bronchial epithelial cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.(C) A549 cells, stably expressing GFP-LC3, were infected with WT or P. aeruginosa mutants and evaluated by fluorescence microscopy, scale bars: 10 µm. GFP-LC3 puncta representing the autophagosomes was quantified and presented in the graph. Each bar represents mean value from three independent experiments and error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01. P. aeruginosa mutant strains legend: ΔSTY (mutant deficient for the three cytotoxins), WT (contains all cytotoxin), ΔS (deficient for ExoS but still express ExoT and ExoY), ΔTY (express ExoS but deficient for ExoT and ExoY).(D,E) A549 cells, stably expressing GFP-LC3, were infected for 4 hours with WT or with one of P. aeruginosa mutants (ΔSTY, ΔS and ΔTY) and evaluated by fluorescence microscopy (D). P. aeruginosa were visualized using specific antibody (red) and the colocalized bacteria signals with GFP-LC3 puncta (green) were merged and presented as yellow. Scale bars: 10 µm. (E) Quantitative analysis of co-localization of internalized P. aeruginosa with GFP-LC3 positive puncta. The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells. Data represent mean ± SD of three independent experiments. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01.(F) Representative transmission electron microscopy images of A549 cells infected with P. aeruginosa containing ExoS (ΔTY) or deficient for the three T3SS effector proteins (ΔSTY) for 4 hours. ΔSTY P. aeruginosa (white star) was engulfed by multiple membrane autophagosomes (black arrow). ΔTY P. aeruginosa (white star) were found in cytoplasm with single membrane surrounded, scale bars: 0.5 µm.(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01.


Figure 4 ( A ) Primary airway epithelial cells were isolated from tracheas of Atg7 + / + and Atg7 - / - mouse and were evaluated by immunoblotting . ( B ) Sections of tracheas from Atg7 + / + and Atg7 - / - mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI ( Blue ) , ciliated epithelial cell marker β - tubulin IV ( green ) and Atg7 ( red ) . Scale bars : 20 µm . ( C , D ) Atg7 + / + and Atg7 - / - mice were intranasally injected with 3 x107 CFU of WT , ΔS , ΔTY or ΔSTY P . aeruginosa . After 24 hours , lungs ( C ) , spleens and livers ( D ) were harvested , homogenized and diluted for CFU counting . Each data point represents bacterial count ( Log10 CFU / g ) from individual mouse . The horizontal lines represent the mean value from each group ( 6 mice each group ) , error bars represent standard deviation . The significance of differences between different drug treatment was determined using One - way ANOVA and Dunn ' s multiple comparisons test . , NS : not significant ; * p ≤ 0 . 05 ( E , F ) Atg7 + / + and Atg7 - / - mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY ( E ) ΔSTY ( F ) . Mice survival was monitored for 10 days post infection . Kaplan - Meyer survival curves were made based on the observation of 12 - 18 mice / group from two independent experiments . The P - values were calculated using the log - rank test ( ΔTYAtg7 + / + vs ΔTYAtg7 - / - , P = 0 . 2186 ; ΔSTYAtg7 + / + vs ΔSTYAtg7 - / - , P = 0 . 0145 ) .

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 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 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

Figure 4(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting.(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05(E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).


Figure 5 ( A A549 cells were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in the GTPase activating domain ( SG - A + ) , in the ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting . ( B ) A549 cells were transfected , for 24hours , with a plasmid expressing empty vector ( EV ) , WT GFP - ExoS , a GFP - ExoS mutant for ADPRT domain ( SG + A - ) or a GFP - ExoS mutant deficient in both GAP and ADPRT domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting . ( C ) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected ( NI ) or infected with P . aeruginosa containing ExoS with ADPRT activity only ( SG - A + ) . Cell lysates were evaluated by immunoblotting . A549 cells were infected for 4 hours with WT or P . aeruginosa mutants ( ΔpscD Cell lysates were evaluated by immunoblotting . A549 cells were infected for 4 hours with WT or P . aeruginosa mutants STY , ΔS and ΔTY ) . Cell lysates were evaluated by immunoblotting . F ) A549 cells were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in the GTPase activating domain ( SG - A + ) , in the ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

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

Figure 5(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting.(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD Cell lysates were evaluated by immunoblotting.A549 cells were infected for 4 hours with WT or P. aeruginosa mutants STY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


Figure 6 ( A ) A549 cells were transfected with empty vector ( VE ) and vector contains Ras - G12V for 24 hours , followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . Cell lysates were evaluated by immunoblotting . ( B ) A549 cells were transfected , for 24 hours , with empty vector ( VE ) and vectors contain Ras - G12V or Ras - G12V & R41K followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . Cell lysates were evaluated by immunoblotting .

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 O

Figure 6(A) A549 cells were transfected with empty vector (VE) and vector contains Ras-G12V for 24 hours, followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.(B) A549 cells were transfected, for 24 hours, with empty vector (VE) and vectors contain Ras-G12V or Ras-G12V&R41K followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


Figure 7 ( A ) A549 cells were transfected , for 48 hours , with a mCherry - DFCP1 plasmid and then infected , for 4 hours , with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY , SG - A - , SG - A + or SG + A - ) and the mCherry puncta number per cell was analyzed by fluorescence microscopy . Representative immunofluorescence images were showed , and scale bars represent 10 µm . ( B ) Quantitative analysis of mCherry - DFCP1 puncta by visually count more than 100 cells for each sample . Each bar represents mean value of from three independent experiments and error bars represent standard deviation . The significance of differences between assays was determined using ANOVA with Dunnett multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

Figure 7(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.