Figure 5 ( A - B ) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments . ( A ) Cytokine levels of IL - 6 , IL - 1β and INF - λ were determined by ELISA ( n = 4 - 7 ) . Results represent two or more independent experiments . ( A - B ) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments . ( B ) mRNA expression levels of IL - 6 , IL - 12p35 and IL - 23p19 were assessed by quantitative RT - PCR and normalized to TBP ( n = 4 - 6 ) . Results represent two or more independent experiments .

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Figure 5(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β and INF-λ were determined by ELISA (n=4-7). Results represent two or more independent experiments.(A-B) Proteins and RNA samples of colon tissue were extracted from mice with the indicated treatments. (B) mRNA expression levels of IL-6, IL-12p35 and IL-23p19 were assessed by quantitative RT-PCR and normalized to TBP (n=4-6). Results represent two or more independent experiments.


Figure 6 ( A ) Flow cytometry analysis of colonic lamina propria CD11c - CD11b + LY6Chi MHCII - monocytes from steady - state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide , showing comparable monocytes infiltrates in to the colitic colon . Plots were pre gated on live CD45 + cells . Results are representative of three independent experiments ( n = 3 per group ) . ( B ) Graphical summary of monocytes presented as % out of CD11b + cells .

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Figure 6(A) Flow cytometry analysis of colonic lamina propria CD11c- CD11b+LY6Chi MHCII-monocytes from steady-state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocytes infiltrates in to the colitic colon. Plots were pre gated on live CD45+ cells. Results are representative of three independent experiments (n=3 per group). (B) Graphical summary of monocytes presented as % out of CD11b + cells.


Figure 7 ( B ) Weight loss of DSS challenged mice on day 6 . ( C ) Real - time PCR analysis showing mRNA expression levels of IL - 6 , IL - 12p35 and IL - 23p19 of FACS isolated monocytes collected from mice with the indicated treatments . Data are obtained from two to three independent experiments ( n = 6 - 7 mice pooled per group ) .

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Figure 7(B) Weight loss of DSS challenged mice on day 6.(C) Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35 and IL-23p19 of FACS isolated monocytes collected from mice with the indicated treatments. Data are obtained from two to three independent experiments (n=6-7 mice pooled per group).


Figure 1RT - qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF - β for 16h normalized to MoDC . Data represent mean values ± SD of triplicates from two donors . Subcellular localization analysis of S100A9 and S100A8 in MoLC , MoDC and MoDC - TGF - β . S100A9 mean fluorescence intensities ( MFI ) were normalized to MoLC and represented in the upper graph . The same was done for S100A9 cytoplasmic / nuclear MFI and shown in the lower graph . Scale bar : 5µm . Graphs show normalized percentages ± SD of data from four different donors for MoLC ( n = 4 ) and MoDC ( n = 4 ) and three different donors for TGF - β - treated MoDC ( n = 3 ) . When applicable , student t - test was performed to determine statistical significance * * P < 0 . 01 ) .

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Figure 1RT-qPCR analysis of mRNA expression of indicated genes from MoLC or MoDC treated with TGF-β for 16h normalized to MoDC. Data represent mean values ± SD of triplicates from two donors.Subcellular localization analysis of S100A9 and S100A8 in MoLC, MoDC and MoDC-TGF-β. S100A9 mean fluorescence intensities (MFI) were normalized to MoLC and represented in the upper graph. The same was done for S100A9 cytoplasmic/nuclear MFI and shown in the lower graph. Scale bar: 5µm. Graphs show normalized percentages ± SD of data from four different donors for MoLC (n=4) and MoDC (n=4) and three different donors for TGF-β-treated MoDC (n=3). When applicable, student t-test was performed to determine statistical significance **P<0.01).


Figure 2MoLC from six different donors ( n = 6 ) were left untreated ( NT ) or incubated with LPS for 48h and analysed for S100A9 expression by RT - qPCR ( left graph ) . MoLC treated as before were then transduced with HIV - GFP vectors for 48h and GFP + cells were analysed by flow cytometry and data were graphically represented as fold relative to non - treated MoLC ( right graph ) . Black horizontal line correspond to the mean . When applicable , student t - test was performed to determine statistical significance ( * * * P < 0 . 001 ) .

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Figure 2MoLC from six different donors (n=6) were left untreated (NT) or incubated with LPS for 48h and analysed for S100A9 expression by RT-qPCR (left graph). MoLC treated as before were then transduced with HIV-GFP vectors for 48h and GFP+ cells were analysed by flow cytometry and data were graphically represented as fold relative to non-treated MoLC (right graph). Black horizontal line correspond to the mean. When applicable, student t-test was performed to determine statistical significance (***P<0.001).


Figure 3Cells transfected as above were pre - treated or not with nevirapine and transduced with HIV - GFP vectors for 48h . The percentage of GFP + MoLC was analysed by flow cytometry . Data represent means values ± SD from two different donors .

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Figure 3Cells transfected as above were pre-treated or not with nevirapine and transduced with HIV-GFP vectors for 48h. The percentage of GFP+ MoLC was analysed by flow cytometry. Data represent means values ± SD from two different donors.


Figure 4MoLC , dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis . Scale bar : 5µm . Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV - Luc . When indicated cells were pre - treated with 10μM Nevirapine for 30 min before viral challenge . Luciferase activities measured in duplicates for each condition from two ( infection with 20 ng P24 ) or three ( infection with 200 ng P24 ) different skin samples were represented on a graph as means of relative luminescence units ( RLU ) ± SD .

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Figure 4MoLC, dermal and epidermal cells were subjected to confocal immunofluorescence microscopy for langerin and S100A9 expression analysis. Scale bar: 5µm.Human epidermal cells treated as before were challenged for 48h with 20 and 200ng of p24 of HIV-Luc. When indicated cells were pre-treated with 10μM Nevirapine for 30 min before viral challenge. Luciferase activities measured in duplicates for each condition from two (infection with 20 ng P24) or three (infection with 200 ng P24) different skin samples were represented on a graph as means of relative luminescence units (RLU) ± SD.


Figure 5The experiment shown is representative of four donors for HIV - 1 - R5 ( n = 4 ) and three donors for HIV - 1 primary isolates ( n = 3 ) . Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV - 1 - R5 and HIV - 1 primary isolates . Nevirapine ( Nev ) ( 10μM ) was used as control when indicated and data were represented as means ± SD of HIV - Gag + cells from at least 2 ( for siS100A9 - MoLC infected by HIV - 1 primary isolates ) to 3 donors . When applicable , student t - test was performed to determine statistical significance ( * P < 0 . 1 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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Figure 5The experiment shown is representative of four donors for HIV-1-R5 (n=4) and three donors for HIV-1 primary isolates (n=3). Data collected from all donors were compiled and represented as bar charts for cells challenged with HIV-1-R5 and HIV-1 primary isolates. Nevirapine (Nev) (10μM) was used as control when indicated and data were represented as means ± SD of HIV-Gag+ cells from at least 2 (for siS100A9-MoLC infected by HIV-1 primary isolates) to 3 donors. When applicable, student t-test was performed to determine statistical significance (*P<0.1;**P<0.01;***P<0.001).


Figure 6E2C - and S100A9 - expressing HEK293T cells were infected with the indicated MOI of HIV - GFP for 48h and GFP + cells were analysed by flow cytometry . Mean values ± SD from four independent experiments ( n = 4 ) were normalized to the E2C - HEK293T control and represented in graph .

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Figure 6E2C- and S100A9-expressing HEK293T cells were infected with the indicated MOI of HIV-GFP for 48h and GFP+ cells were analysed by flow cytometry. Mean values ± SD from four independent experiments (n=4) were normalized to the E2C-HEK293T control and represented in graph.


Figure 7The percentage of HIV - Gag p24 + cells from four independent experiments for CEM ( n = 4 ) and two for Jurkat ( n = 2 ) analysed by flow cytometry were graphically represented as means of HIV - Gag + cells ± SD . When applicable , student t - test was performed to determine statistical significance ( * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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Figure 7The percentage of HIV-Gag p24+ cells from four independent experiments for CEM (n=4) and two for Jurkat (n=2) analysed by flow cytometry were graphically represented as means of HIV-Gag+ cells ± SD. When applicable, student t-test was performed to determine statistical significance (**P<0.01;***P<0.001).


Figure 8E2C - and S100A9 - expressing CHO cells were challenged with indicated MOI of MMLV - YFP vectors for 24h or 48h . The percentages of YFP + CHO infected cells analysed by flow cytometry and data were represented on graphs .

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Figure 8E2C- and S100A9-expressing CHO cells were challenged with indicated MOI of MMLV-YFP vectors for 24h or 48h. The percentages of YFP+ CHO infected cells analysed by flow cytometry and data were represented on graphs.


Figure 9E2C - or S100A9 - expressing THP1 were left uninfected ( / ) or infected for 3 h with the indicated p24 amounts of HIV - 1 NL4 - 3 / Nef - IRES - Renilla viruses containing BlaM - VPR . Cells were processed for Blam - Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry . The mock condition corresponds to unstained E2C - expressing cells . Dot - plots from a representative experiment are shown .

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Figure 9E2C- or S100A9-expressing THP1 were left uninfected (/) or infected for 3 h with the indicated p24 amounts of HIV-1 NL4-3/Nef-IRES-Renilla viruses containing BlaM-VPR. Cells were processed for Blam-Vpr assay and the percentage of cleaved CCF2 was analysed by flow cytometry. The mock condition corresponds to unstained E2C-expressing cells. Dot-plots from a representative experiment are shown.


Figure 10Schematic description of fate - of - capsid assay is displayed above . HEK293T expressing E2C or S100A9 were transduced with HIV - Luc ( 10ng p24 ) for 4 h . Cells were then subjected to the fate of capsid assay ( see methods ) in order to pull - down intact cores ( CA cores ) ( recovered in the pellet ) . Equal volumes from the soluble ( top fraction ) and pelleted ( down fraction ) materials were analyzed by western blotting in order to evaluate the presence of p24 ( CA ) , CypA , S100A9 and E2C proteins .

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Figure 10Schematic description of fate-of-capsid assay is displayed above. HEK293T expressing E2C or S100A9 were transduced with HIV-Luc (10ng p24) for 4 h. Cells were then subjected to the fate of capsid assay (see methods) in order to pull-down intact cores (CA cores) (recovered in the pellet). Equal volumes from the soluble (top fraction) and pelleted (down fraction) materials were analyzed by western blotting in order to evaluate the presence of p24 (CA), CypA, S100A9 and E2C proteins.


Figure 11Colorimetric HIV - 1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations . Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C - containing conditions and shown on graphs .

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Figure 11Colorimetric HIV-1 RTase activity was performed with 10µg of immunoprecipitated and eluted E2C or S100A9 in presence of increasing MgCl2 concentrations. Means ± SD colorimetric data obtained from at least three independent experiments were normalized to E2C-containing conditions and shown on graphs.


Figure 1Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in ( A ) . n = 3 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed Student ' s t test . Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine . Protein expression was analyzed on day 6 . n = 3 biological replicates . Brown preadipocytes were infected by lentiviral shRNAs against Asns . Cells were differentiated in culture media with or without 1mM asparagine . Western blot was performed on day 6 .

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Figure 1Gene expression analyzed by quantitative PCR in brown adipocytes that were fed with or without 1mM asparagine as in (A). n=3 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.Preadipocytes bearing the shRNA targeting Pgc1α were induced differentiation with or without 1mM asparagine. Protein expression was analyzed on day 6. n=3 biological replicates.Brown preadipocytes were infected by lentiviral shRNAs against Asns. Cells were differentiated in culture media with or without 1mM asparagine. Western blot was performed on day 6.


Figure 2UCP1 and PPARγ protein levels in BAT and inguinal WAT ( iWAT ) from mice in ( E ) . I , J . Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal ( I ) and CL316 , 243 - stimulated ( J ) states . n = 5 mice each group . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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Figure 2UCP1 and PPARγ protein levels in BAT and inguinal WAT (iWAT) from mice in (E).I,J. Mice supplemented with asparagine in drinking water for 3 weeks were subjected to the indirect calorimetry analysis at both basal (I) and CL316,243-stimulated (J) states. n=5 mice each group. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


Figure 3Rectal temperature in mice of ( A ) during cold exposure . n = 5 mice each group . BAT and inguinal WAT ( iWAT ) gene expression analysis in mice of ( A ) . Hematoxylin and eosin staining of BAT , inguinal WAT ( iWAT ) and epididymal WAT ( eWAT ) of mice in ( A ) .

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Figure 3Rectal temperature in mice of (A) during cold exposure. n=5 mice each group.BAT and inguinal WAT (iWAT) gene expression analysis in mice of (A).Hematoxylin and eosin staining of BAT, inguinal WAT (iWAT) and epididymal WAT (eWAT) of mice in (A).


Figure 4Glucose tolerance test was performed after a high - fat diet feeding for 17 weeks in mice of ( A ) . n = 5 mice each group . Serum insulin levels in mice of ( A ) . n = 5 mice each group . Serum triglyceride levels in mice of ( A ) . n = 5 mice each group . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed Student ' s t test . BAT and inguinal WAT ( iWAT ) UCP1 and PPARγ protein levels in mice of ( A ) .

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Figure 4Glucose tolerance test was performed after a high-fat diet feeding for 17 weeks in mice of (A). n=5 mice each group.   Serum insulin levels in mice of (A). n=5 mice each group.   Serum triglyceride levels in mice of (A). n=5 mice each group.   Data information: Data represent means±SEM. *P<0.05; **P<0.01; unpaired two-tailed Student's t test.BAT and inguinal WAT (iWAT) UCP1 and PPARγ protein levels in mice of (A).


Figure 5Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine . Immunoblot detection of the glucose transporters , glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation . Immunoblot detection of the glucose transporters , glycolysis and TCA key enzymes in immortalized brown fat cell line ( E ) , BAT from mice fed asparagine ( F ) and injected asparaginase ( G ) . Asparagine was supplemented to cells during differentiation .

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Figure 5Heat map of metabolite relative levels in brown fat cells treated with or without 1mM asparagine.Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in primary brown fat cell line Asparagine was supplemented to cells during differentiation.Immunoblot detection of the glucose transporters, glycolysis and TCA key enzymes in immortalized brown fat cell line (E), BAT from mice fed asparagine (F) and injected asparaginase (G). Asparagine was supplemented to cells during differentiation.


Figure 6Intracellular abundance of 13C - isotopologue metabolites downstream of glycolysis in brown adipocytes . n = 5 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test . C , D . Quantification of glucose ( C ) and lactate ( D ) concentration from media . The media was collected after incubated with mature brown adipocytes for 48 h . n = 3 independent experiments in ( C ) , n = 6 independent experiments in ( D ) . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test .

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Figure 6Intracellular abundance of 13C-isotopologue metabolites downstream of glycolysis in brown adipocytes. n=5 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.C,D. Quantification of glucose (C) and lactate (D) concentration from media. The media was collected after incubated with mature brown adipocytes for 48 h. n=3 independent experiments in (C), n=6 independent experiments in (D). Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.


Figure 7Quantification of intracellular amino acid levels in brown fat cells ( day 6 ) treated with or without 1mM asparagine . n = 4 independent experiments . Data information : Data represent means ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; unpaired two - tailed Student ' s t test . B - D . mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E - BP1 and S6K in brown adipocytes ( B ) , BAT from mice fed asparagine ( C ) or asparaginase ( D ) . ( B ) Asparagine was added to cultured media from day 4 to day 6 . Brown preadipocytes were induced to differentiation with or without 1mM asparagine . Rapamycin ( 75nM ) was added to cells for 48h . Then western blot detected protein expression on day 6 . Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation . Protein expression analysis was performed on day 6 .

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Figure 7Quantification of intracellular amino acid levels in brown fat cells (day 6) treated with or without 1mM asparagine. n=4 independent experiments. Data information: Data represent means±SEM. *P<0.05; **P<0.01; ***P<0.001; unpaired two-tailed Student's t test.B-D. mTORC1 pathway activity was analyzed by immunoblotting for the phosphorylation of 4E-BP1 and S6K in brown adipocytes (B), BAT from mice fed asparagine (C) or asparaginase (D). (B) Asparagine was added to cultured media from day 4 to day 6.Brown preadipocytes were induced to differentiation with or without 1mM asparagine. Rapamycin (75nM) was added to cells for 48h. Then western blot detected protein expression on day 6.Brown preadipocytes were knocked down Raptor following standard induction to mature adipocytes with or without asparagine supplementation. Protein expression analysis was performed on day 6.


Figure 1CSF p3 - Alcβ37 values in subjects who were categorized along the AD continuum ( n = 131 ) . Cut - off values are 359 . 6 pg / mL for Aβ42 ( A + indicates < 359 . 6 pg / mL ) , 30 . 6 pg / mL for p - tau ( T + indicates > 30 . 6 pg / mL ) , and 105 . 3 pg / mL for t - tau ( N + indicates > 105 . 3 pg / mL ) respectively . Statistical significance was determined by a one - way ANOVA followed by Tukey ' s multiple comparisons test ( means ± SEM ; A - : n = 36 , A + T - N - : n = 51 , A + T + N - : n = 30 , A + T + N + : n = 14 ) , and significant p - values ( p < 0 . 01 , p < 0 . 0001 ) are indicated on the graph .

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Figure 1CSF p3-Alcβ37 values in subjects who were categorized along the AD continuum (n = 131). Cut-off values are 359.6 pg/mL for Aβ42 (A+ indicates <359.6 pg/mL), 30.6 pg/mL for p-tau (T+ indicates >30.6 pg/mL), and 105.3 pg/mL for t-tau (N+ indicates >105.3 pg/mL) respectively. Statistical significance was determined by a one-way ANOVA followed by Tukey's multiple comparisons test (means ± SEM; A-: n = 36, A+T-N-: n = 51, A+T+N-: n = 30, A+T+N+: n = 14), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.


Figure 2 ( C ) Effect of p3 - Alcβ37 and its partial peptides on the viability of mouse primary cultured neurons . Wild - type ( WT ) neurons ( div 15 - 20 ) were incubated for 24 h in the presence ( 10 μM ) or absence ( - ) of p3 - Alcβ1 - 11 , p3 - Alcβ9 - 19 , p3 - Alcβ20 - 37 , and p3 - Alcβ1 - 37 . Neuronal viability was evaluated with MTT assays and expressed relative to that of cells cultured in the absence of peptides ( assigned a value of 1 . 0 ) . Statistical analysis was performed using a one - way ANOVA , followed by Dunnett ' s multiple comparisons test ( means ± SEM ; n = 10 - 12 ) , and significant p - values ( p < 0 . 01 , p < 0 . 0001 ) are indicated on the graph . ( D , E , F ) Effect of p3 - Alcβ on the neuronal toxicity of Aβ42 oligomers . WT neurons ( div 15 - 20 ) were incubated for 24 h in the presence ( 10 μM ) or absence ( - ) of p3 - Alcβ9 - 19 and p3 - Alcβ1 − 37 with ( + ) or without ( - ) Aβ42 oligomers ( Aβo , 2 . 5 μM ) . Neuronal viability was evaluated by MTT ( D ) , ATP generation ( E ) , and LDH release ( F ) assays and expressed relative to neurons cultured in the absence of p3 - Alcβ and Aβo ( assigned a value of 1 . 0 ) . Statistical significance was determined by Dunnett ' s multiple comparisons tests ( means ± SEM ; MTT : n = 36 , ATP : n = 40 , LDH : n = 60 ) . The significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 , p < 0 . 0001 ) versus cells incubated in the absence ( - ) of p3 - Alcβ and Aβo are indicated in the graphs . The significant p - values ( # p < 0 . 05 , # p < 0 . 01 ) versus cells incubated in the presence ( + ) of Aβo and in the absence ( - ) of p3 - Alcβ ) are indicated in the graphs . ( G ) Effect of Aβ42 oligomers ( Aβo ) and p3 - Alcβ peptides on the generation of reactive oxygen species ( ROS ) . WT neurons ( div 15 - 17 ) were incubated for 24 h with ( + ) or without ( - ) Aβo ( 2 . 5 μM ) and in the presence ( + ) or absence ( - ) of p3 - Alcβ ( 10 μM ) . ROS generation was assessed and expressed relative to that of neurons incubated in the absence ( - ) of Αβo , which was assigned a value of 1 . 0 . Statistical significance between samples with or without Aβo was evaluated with a Student ' s t - test ( means ± SEM ; n = 10 ) , and the significant p - value ( p < 0 . 05 ) is indicated on the graph .

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 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 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

Figure 2(C) Effect of p3-Alcβ37 and its partial peptides on the viability of mouse primary cultured neurons. Wild-type (WT) neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ1-11, p3-Alcβ9-19, p3-Alcβ20-37, and p3-Alcβ1-37. Neuronal viability was evaluated with MTT assays and expressed relative to that of cells cultured in the absence of peptides (assigned a value of 1.0). Statistical analysis was performed using a one-way ANOVA, followed by Dunnett's multiple comparisons test (means ± SEM; n = 10-12), and significant p-values (p < 0.01, p < 0.0001) are indicated on the graph.(D, E, F) Effect of p3-Alcβ on the neuronal toxicity of Aβ42 oligomers. WT neurons (div 15-20) were incubated for 24 h in the presence (10 μM) or absence (-) of p3-Alcβ9-19 and p3-Alcβ1−37 with (+) or without (-) Aβ42 oligomers (Aβo, 2.5 μM). Neuronal viability was evaluated by MTT (D), ATP generation (E), and LDH release (F) assays and expressed relative to neurons cultured in the absence of p3-Alcβ and Aβo (assigned a value of 1.0). Statistical significance was determined by Dunnett's multiple comparisons tests (means ± SEM; MTT: n = 36, ATP: n = 40, LDH: n = 60). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) versus cells incubated in the absence (-) of p3-Alcβ and Aβo are indicated in the graphs. The significant p-values (#p < 0.05, #p < 0.01) versus cells incubated in the presence (+) of Aβo and in the absence (-) of p3-Alcβ) are indicated in the graphs.(G) Effect of Aβ42 oligomers (Aβo) and p3-Alcβ peptides on the generation of reactive oxygen species (ROS). WT neurons (div 15-17) were incubated for 24 h with (+) or without (-) Aβo (2.5 μM) and in the presence (+) or absence (-) of p3-Alcβ (10 μM). ROS generation was assessed and expressed relative to that of neurons incubated in the absence (-) of Αβo, which was assigned a value of 1.0. Statistical significance between samples with or without Aβo was evaluated with a Student's t-test (means ± SEM; n = 10), and the significant p-value (p < 0.05) is indicated on the graph.


Figure 3 ( A , B ) Suppression of Ca2 + influx induced by Aβo in neurons treated with p3 - Alcβ9 - 19 ( A ) and p3 - Alcβ37 ( B ) . Mouse neurons ( div 11 - 13 ) pretreated with Fluo 4 - AM were stimulated at 5 min ( arrow ) with ( + ) or without ( - ) Aβo ( 5 . 2μM ) in the presence ( + ) or absence ( - ) of p3 - Alcβ ( 50 μM ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 18 min ( 5 - 23 min at time points ) is shown ( right ) as the AUC expressed relative to that of cells cultured in the absence ( - ) of p3 - Alcβ and Aβo ( assigned a value of 1 . 0 ) . ( C , D ) Suppression of Ca2 + influx into neurons followed by Ca2 + administration in the presence of p3 - Alcβ9 - 19 ( C ) and p3 - Alcβ37 ( D ) . Mouse neurons ( div 11 - 13 ) pretreated with Fluo 4 - AM in calcium - depleted medium were administered Ca2 + ( 2 mM ) at 5 min ( arrow ) in the presence or absence of p3 - Alcβ9 - 19 ( C ) and p3 - Alcβ37 ( D ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 7 . 5 or 9 min ( 5 to 12 . 5 min in panel C and 5 to 14 min in panel D at the indicated time points ) is shown ( right ) . Statistical significance was determined by one - way ANOVA with Tukey ' s multiple comparison test ( means ± SE ; n = 14 ( A ) , n = 15 ( B ) , n = 10 ( C ) ) . The significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 , p < 0 . 0001 ) are indicated on the graphs . Statistical significance was determined with a Student ' s t - test ( means ± SEM ; n = 11 ( D ) ) , and the significant p - value ( p < 0 . 001 ) is indicated on the graph .

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Figure 3(A, B) Suppression of Ca2+ influx induced by Aβo in neurons treated with p3-Alcβ9-19 (A) and p3-Alcβ37 (B). Mouse neurons (div 11-13) pretreated with Fluo 4-AM were stimulated at 5 min (arrow) with (+) or without (-) Aβo (5.2μM) in the presence (+) or absence (-) of p3-Alcβ (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 18 min (5-23 min at time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence (-) of p3-Alcβ and Aβo (assigned a value of 1.0).(C, D) Suppression of Ca2+ influx into neurons followed by Ca2+ administration in the presence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). Mouse neurons (div 11-13) pretreated with Fluo 4-AM in calcium-depleted medium were administered Ca2+ (2 mM) at 5 min (arrow) in the presence or absence of p3-Alcβ9-19 (C) and p3-Alcβ37 (D). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 7.5 or 9 min (5 to 12.5 min in panel C and 5 to 14 min in panel D at the indicated time points) is shown (right). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparison test (means ± SE; n = 14 (A), n = 15 (B), n = 10 (C)). The significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graphs. Statistical significance was determined with a Student's t-test (means ± SEM; n = 11 (D)), and the significant p-value (p < 0.001) is indicated on the graph.


Figure 4 ( A ) Suppression of Ca2 + influx induced by Aβ42 oligomers ( Aβo ) in neurons treated with the NMDA receptor antagonist D - AP5 . Neurons ( div 14 ) pretreated with Fluo 4 - AM were stimulated with or without Aβo ( 5 . 2 μM ) at 5 min ( arrow ) in the presence or absence of D - AP5 ( 50 μM ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 24 min ( 5 - 29 min at the indicated time points ) is shown ( right ) as the AUC expressed relative to that of cells cultured in the absence of D - AP5 and Aβo ( assigned a value of 1 . 0 ) . Statistical significance was determined by one - way ANOVA with Tukey ' s multiple comparisons test ( means ± SE ; n = 4 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 ) are indicated on the graph . ( D ) Non - synergistic suppression of Ca2 + influx induced by Aβ42 oligomers ( Aβo ) in neurons by p3 - Alcβ and D - AP5 . Neurons ( div 14 ) pretreated with Fluo 4 - AM were stimulated with or without Aβo ( 5 . 2 μM ) at 5 min ( arrow ) in the presence ( + ) or absence ( - ) of p3 - Alcβ9 - 19 ( 50 μM ) and D - AP5 ( 50 μM ) . The fluorescence intensity was recorded at the indicated time ( left ) and the fluorescence area intensity for 23 min ( 5 - 28 min at the indicated time points ) is shown ( right ) as the AUC expressed relative to that of cells cultured in experiment No . 1 ( assigned a value of 1 . 0 ) . Statistical significance was determined with a one - way ANOVA with Tukey ' s multiple comparison test ( means ± SEM ; n = 12 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 , p < 0 . 0001 ) are indicated on the graph .

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Figure 4(A) Suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons treated with the NMDA receptor antagonist D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence or absence of D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 24 min (5-29 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in the absence of D-AP5 and Aβo (assigned a value of 1.0). Statistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test (means ± SE; n = 4), and significant p-values (p < 0.05, p < 0.01) are indicated on the graph.(D) Non-synergistic suppression of Ca2+ influx induced by Aβ42 oligomers (Aβo) in neurons by p3-Alcβ and D-AP5. Neurons (div 14) pretreated with Fluo 4-AM were stimulated with or without Aβo (5.2 μM) at 5 min (arrow) in the presence (+) or absence (-) of p3-Alcβ9-19 (50 μM) and D-AP5 (50 μM). The fluorescence intensity was recorded at the indicated time (left) and the fluorescence area intensity for 23 min (5-28 min at the indicated time points) is shown (right) as the AUC expressed relative to that of cells cultured in experiment No.1 (assigned a value of 1.0). Statistical significance was determined with a one-way ANOVA with Tukey's multiple comparison test (means ± SEM; n = 12), and significant p-values (p < 0.05, p < 0.01, p < 0.001, p < 0.0001) are indicated on the graph.


Figure 5 ( E - G ) The SUVRs of groups administered p3 - Alcβ9 - 19 versus the vehicle - treated group . Statistical analysis was performed using one - way ANOVA , followed by Bonferroni correction for multiple comparisons ( means ± SEM ; n = 6 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 ) are indicated on the graphs . ( I , J ) The SUVRs of [ 18F ] BCPP - EF in the cortex ( I ) and hippocampus ( J ) are compared across the three groups . Statistical analysis was performed using one - way ANOVA , followed by Bonferroni correction for multiple comparisons ( means ± SEM ; n = 6 - 10 ) , and significant p - values ( p < 0 . 05 , p < 0 . 01 , p < 0 . 001 ) are indicated on the graphs . ( K ) Improvement of the inverse SUVR correlation between [ 18F ] BCPP - EF and [ 11C ] DPA713 in AD mice following administration of p3 - Alcβ9 - 19 . AD mice with ( closed circle ) or without ( open circle ) subcutaneous injection of p3 - Alcβ9 - 19 peptide ( 1 mg / kg body weight ) are also analyzed with [ 11C ] DPA713 . A solid linear line shows a significant inverse correlation ( Pearson ' s correlation , r = 0 . 594 , p < 0 . 05 ) in AD mice , whereas a dotted linear line shows a positive correlation tendency in AD mice in response to p3 - Alcβ9 - 19 administration .

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 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

Figure 5(E-G) The SUVRs of groups administered p3-Alcβ9-19 versus the vehicle-treated group. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons (means ± SEM; n = 6), and significant p-values (p < 0.05, p < 0.01) are indicated on the graphs.(I, J) The SUVRs of [18F]BCPP-EF in the cortex (I) and hippocampus (J) are compared across the three groups. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni correction for multiple comparisons (means ± SEM; n = 6-10), and significant p-values (p < 0.05, p < 0.01, p < 0.001) are indicated on the graphs.(K) Improvement of the inverse SUVR correlation between [18F]BCPP-EF and [11C]DPA713 in AD mice following administration of p3-Alcβ9-19. AD mice with (closed circle) or without (open circle) subcutaneous injection of p3-Alcβ9-19 peptide (1 mg/kg body weight) are also analyzed with [11C]DPA713. A solid linear line shows a significant inverse correlation (Pearson's correlation, r = 0.594, p < 0.05) in AD mice, whereas a dotted linear line shows a positive correlation tendency in AD mice in response to p3-Alcβ9-19 administration.


Figure 1 ( Top ) PCR detection of the recombined BrafLox − V600E allele ( Lox - V600E ) in BVE mouse tissues at 8 weeks of age without tamoxifen induction . Substantial recombination is observed in the lung , while weaker recombination is also detected in the liver . No recombination was detected in hematopoietic tissues ( bone marrow and spleen ) even after 40 cycles of amplification ( right ) . ( Bottom ) H & amp ; amp ; E staining of lung sections from wild - type ( WT , 10 weeks of age ) , BVE ( 1 - 10 weeks of age ) and Braf + / LSL − V600E mouse 8 weeks after nasal delivery of AdCre is indicated below . V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development . Scale bars , 100 μm . Ki67 immunostaining of lung sections from BVE mice at 3 - 10 weeks of age . Scale bars , 25 μm . The right bar graph summarises % Ki67 + cells in tumours at different ages post - partum . Four to six mice were analysed for each age group as indicated , and more than 3 , 500 tumour cells per mouse were evaluated . The data represent mean ± SD . Representative H & amp ; amp ; E staining and Mac2 immunohistochemistry of serial lung sections from 10 - week - old BVE mice at low ( upper panels ) and high ( lower panels ) magnifications . Scale bars , 100 μm ( upper panels ) or 25 μm ( lower panels ) .

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 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

Figure 1(Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&amp;E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1-10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm.Ki67 immunostaining of lung sections from BVE mice at 3-10 weeks of age. Scale bars, 25 μm. The right bar graph summarises %Ki67+ cells in tumours at different ages post-partum. Four to six mice were analysed for each age group as indicated, and more than 3,500 tumour cells per mouse were evaluated. The data represent mean ± SD.Representative H&amp;E staining and Mac2 immunohistochemistry of serial lung sections from 10-week-old BVE mice at low (upper panels) and high (lower panels) magnifications. Scale bars, 100 μm (upper panels) or 25 μm (lower panels).


Figure 2Giemsa staining of isolated stroma IMCs . These cells show round or oval - shaped nuclei that lack indentations , and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes , suggestive of immature myeloid - lineage cells that are morphologically distinct from monocytes / DCs . Scale bar , 25 μm . Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b , which is expressed at low levels ( overlaid with isotype control histograms shown in grey ) . Cell surface marker analysis ( dot plots ) of IMCs demonstrating CD11c + IMCs do not express macrophage ( F4 / 80 ) , DC ( CD24 ) or M1 - polarisation ( CD86 ) markers , but weakly express the M2 marker CD206 . Insets show isotype control staining . Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines . The cells grown on culture plates were imaged by phase - contrast microscopy ( left ) . Then , the cells were trypsinised and smeared on glass slides for Giemsa staining ( right ) . These cells are intermediate to large sized , round or amoeboid cells with cytoplasmic vacuolation , consistent with a macrophage morphology . Scale bars , 50 μm . Cell surface marker analysis of cultured IMCs showing differentiation into F4 / 80 + macrophages with CD11bhighCD11c + CD86 + CD206low surface phenotype ( overlaid with isotype control histograms shown in light grey ) . PCR detection of Braf recombination in purified IMCs , AT2 cells and tumour cell aggregates .

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Figure 2Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm.Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey).Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining.Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm.Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey).PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates.


Figure 3CMT64 cells ( 1 × 104 / well in 12 - well plates ) were co - cultured with 1 . 5 - 3 × 105 IMCs for 4 days . Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate . Harvested cells were subjected to counting ( left ) . Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells . The bar graph ( left ) shows CMT64 growth ( cell counting ) data pooled from two independent experiments . Phase - contrast images of CMT64 cells cultured without IMCs ( CMT64 only ) or co - cultured with IMCs ( CMT64 / IMC co - culture ) are also indicated in the middle and right . A higher magnification image of co - cultured CMT64 cells ( right ) highlights IMCs migrating into CMT64 cells ( arrow heads ) . Scale bars are 200 μm , except for the high magnification image ( right ) in which the scale bar is 100 μm . ( Left ) Growth and EMT - like morphological changes of CMT64 cells cultured with 25 % IMC - CM . CMT64 cells cultured for 4 days at low - density in DMEM / 10 % FCS without IMC - CM ( - IMC - CM ) or with 25 % IMC - CM ( + IMC - CM ) were counted and normalised to the average of control as 100 % . The data in the bar chart represent mean + SD of five cultures utilising IMC - CM obtained from three independent sources . ( Middle ) Phase - contrast images to indicate representative morphologies of CMT64 cells maintained without IMC - CM ( control ) or cultured with 25 % IMC - CM ( + IMC - CM ) for 2 weeks . ( Right ) E - cadherin immunofluorescence of CMT64 cells cultured as above . Scale bars are 200 μm ( phase contrast ) and 10 μm ( immunofluorescence ) . Co - culture of AT2 cells and autologous IMCs from BVE mice ( 8 - to 10 - week - old ) for 48 h using the Transwell ® culture system . In vitro BrdU incorporation ( flow cytometry , left and middle ) and EMT marker expression ( RT - PCR , right ) in co - cultured AT2 cells are indicated . Representative flow cytometry plots are indicated on the left , and the bar chart in the middle indicates % BrdU + AT2 cell numbers normalised to control cultures without IMCs ( n = 4 , mean + SD ) . Gapdh serves as a loading control for the RT - PCR on the right . Morphological alterations and intracellular signalling in AT2 cells cultured with IMC - CM . On the left and middle , primary AT2 cells from 3 - week - old BVE mice were cultured for 48 h and then incubated in serum - free IMC - CM ( AT2 with IMC - CM ) or DMEM ( AT2 w / o IMC - CM , serving as a control ) for another 48 h , followed by immunostaining for E - cadherin ( red ) and vimentin ( green ) and confocal laser scanning microscopy ( CLSM ) imaging . The 3 × zoomed image in the middle right highlights dividing vimentin + cells that show internalised E - cadherin ( arrows ) . Scale bars , 50 μm . On the right , primary AT2 cells from 10 - week - old BVE mice were cultured for 48 h , serum starved for 5 h and treated with or without serum - free IMC - CM for 30 min . Phosphorylation of AKT , SMAD3 , MEK and ERK were analysed by immunoblotting .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5-3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm.(Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence).Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT-PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT-PCR on the right.Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting.


Figure 4CCR1 immunofluorescence of primary IMCs ( left , co - stained with Mac2 ) and AT2 cells ( right , co - stained with E - cadherin ) detected by CLSM . Scale bars , 10 μm . CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs , but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 ( inset in the left microphotograph ) . CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs . Scale bar , 50 μm . CT imaging of lungs of AdCre - infected Braf + / LSL − V600E mice treated with vehicle ( top ) or CCR1 inhibitor ( bottom ) . H : heart ; * indicates a tumour region accompanied by atelectasis . H & amp ; amp ; E staining of lung sections from vehicle ( left ) or CCR1 inhibitor ( right ) treated AdCre - infected Braf + / LSL − V600E mice . Scale bars , 250 μm ( top ) or 50 μm ( bottom ) . Flow cytometry analysis of CD11c + ( top , green ) and SPC + ( bottom , red ) cells in the lung of AdCre - infected Braf + / LSL − V600Emice treated with vehicle ( left ) or CCR1 inhibitor ( right ) . LungCD11c + ( left ) and SPC + ( right ) cell numbers ( per left lobe ) were quantitated in AdCre - infected Braf + / LSL − V600Emice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) . % LungCD4 + ( left ) and CD8 + ( right ) T lymphocytes were quantitated in AdCre - infected Braf + / LSL − V600E mice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) .

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Figure 4CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph).CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm.CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis.H&amp;E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom).Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600Emice treated with vehicle (left) or CCR1 inhibitor (right).LungCD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600Emice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).% LungCD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).


Figure 5NPC2 immunoblots of concentrated whole lung CM ( top ) and whole lung lysates ( bottom ) generated from Braf + / + ; CreER ™ ( WT ) or BVE ( VE ) mice . Whole lung lysates were obtained from 3 - to 8 - week - old mice . NPC2 immunoblots of un - concentrated CM ( containing 10 % FCS ) from fractionated IMC and AT2 cultures . Total protein staining of the membrane by Amido black ( bottom ) serves as a loading control showing serum albumin at 70 kDa , derived from FCS in the media . Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT - PCR . Mean + SD ( n = 3 ) of Npc2 expression normalised to Gapdh is indicated . NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells . NPC2 immunoblots of cell lysates from freshly isolated or cultured ( 48 h in serum - free DMEM ) IMCs . NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . CLSM imaging of NPC2 - Alexa488 taken up by IMCs . ( Left ) Freshly isolated IMCs were incubated for 2 h with ( + NPC2 - Alexa488 ) or without ( - NPC2 - Alexa488 ) 82 . 5 nM NPC2 - Alexa488 , chased in NPC2 - free media for additional 2 h and live imaged by CLSM . Single - colour ( top ) and DIC - merged ( bottom ) fluorescence images are indicated . Scale bar , 20 μm . ( Right ) Zoomed images ( DIC - merged and single - colour ) from different fields showing vesicular and reticular distribution of incorporated NPC2 - Alexa488 . Scale bar , 10 μm . Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg / ml bNPC2 as determined by qRT - PCR . Mean ± SD ( n = 3 ) of Npc2 expression normalised to Hprt1 is indicated .

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Figure 5NPC2 immunoblots of concentrated whole lung CM (top) and whole lung lysates (bottom) generated from Braf+/+;CreER™ (WT) or BVE (VE) mice. Whole lung lysates were obtained from 3-to 8-week-old mice.NPC2 immunoblots of un-concentrated CM (containing 10% FCS) from fractionated IMC and AT2 cultures. Total protein staining of the membrane by Amido black (bottom) serves as a loading control showing serum albumin at 70 kDa, derived from FCS in the media.Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT-PCR. Mean + SD (n = 3) of Npc2 expression normalised to Gapdh is indicated.NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells.NPC2 immunoblots of cell lysates from freshly isolated or cultured (48 h in serum-free DMEM) IMCs.NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg/ml bNPC2 for 48 h.CLSM imaging of NPC2-Alexa488 taken up by IMCs. (Left) Freshly isolated IMCs were incubated for 2 h with (+NPC2-Alexa488) or without (-NPC2-Alexa488) 82.5 nM NPC2-Alexa488, chased in NPC2-free media for additional 2 h and live imaged by CLSM. Single-colour (top) and DIC-merged (bottom) fluorescence images are indicated. Scale bar, 20 μm. (Right) Zoomed images (DIC-merged and single-colour) from different fields showing vesicular and reticular distribution of incorporated NPC2-Alexa488. Scale bar, 10 μm.Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg/ml bNPC2 as determined by qRT-PCR. Mean ± SD (n = 3) of Npc2 expression normalised to Hprt1 is indicated.


Figure 6Shortened survival of BVE mice following reduction in Npc2 gene dosage . Genotypes of mice are as follows : + / + = Braf + / LSL − V600E ; CreER + / 0 ; Npc2 + / + , + / hypo = Braf + / LSL − V600E ; CreER + / 0 , Npc2 + / hypo , hypo / hypo = Braf + / LSL − V600E ; CreER + / 0 ; Npc2hypo / hypo . Median survival time ( MST ) for each genotype is indicated . Representative H & amp ; amp ; E staining of lung sections from 5 - week - old BVE mice on the Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) background is shown . Scale bars , 500 μm . Stroma IMC quantification per lung in Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) BVE mice at different ages ( 39 - 109 days p . p . ) . Ki67 / Mac2immunofluorescence analysis of Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVElung sections at 5 weeks of age . Merged images for Ki67 ( green ) , Mac2 ( red ) and DAPI ( blue ) are indicated . Arrows indicate dual positive cells for nuclearKi67 and Mac2 . Scale bars , 50 μm . The bar chart indicates % Ki67 in Mac2 + lung stroma cells ( mean + SD ) , obtained from 4 independent Npc2 + / + and Npc2 + / hypoBVE pairs . In vivo BrdU incorporation of CD11c + IMCs from Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVE mice are shown . Representative flow cytometry plots of three independent experiments are indicated . Ki67 ( green ) / Mac2 ( red ) dual immunofluorescencestaining of lung sections from 5 - week - old Npc2 + / hypoBVEmice with nuclearDAPI ( blue ) staining , focusing on intratumour Mac2 + cell clusters . Scale bar , 50 μm . Enlarged images of the boxed areas 1 and 2 highlight Ki67 + Mac2 − cells associated with Mac2 + cells ( arrows ) . Lung SPC + / CD11c + cell numbers of Braf + / LSL − V600E ; Npc2 + / + ( + / + , n = 3 ) and Braf + / LSL − V600E ; Npc2 + / hypo ( + / hypo , n = 7 ) mice at 10 weeks post - AdCre infection .

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Figure 6Shortened survival of BVE mice following reduction in Npc2 gene dosage. Genotypes of mice are as follows: +/+ = Braf+/LSL−V600E;CreER+/0;Npc2+/+, +/hypo = Braf+/LSL−V600E;CreER+/0,Npc2+/hypo, hypo/hypo = Braf+/LSL−V600E;CreER+/0;Npc2hypo/hypo. Median survival time (MST) for each genotype is indicated.Representative H&amp;E staining of lung sections from 5-week-old BVE mice on the Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) background is shown. Scale bars, 500 μm.Stroma IMC quantification per lung in Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) BVE mice at different ages (39-109 days p.p.).Ki67/Mac2immunofluorescence analysis of Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVElung sections at 5 weeks of age. Merged images for Ki67 (green), Mac2 (red) and DAPI (blue) are indicated. Arrows indicate dual positive cells for nuclearKi67 and Mac2. Scale bars, 50 μm. The bar chart indicates % Ki67 in Mac2+lung stroma cells (mean + SD), obtained from 4 independent Npc2+/+ and Npc2+/hypoBVE pairs.In vivo BrdU incorporation of CD11c+ IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice are shown. Representative flow cytometry plots of three independent experiments are indicated.Ki67 (green)/Mac2 (red) dual immunofluorescencestaining of lung sections from 5-week-old Npc2+/hypoBVEmice with nuclearDAPI (blue) staining, focusing on intratumour Mac2+cell clusters. Scale bar, 50 μm. Enlarged images of the boxed areas 1 and 2 highlight Ki67+Mac2− cells associated with Mac2+ cells (arrows).Lung SPC+/CD11c+cell numbers of Braf+/LSL−V600E;Npc2+/+ (+/+, n = 3) and Braf+/LSL−V600E;Npc2+/hypo (+/hypo, n = 7) mice at 10 weeks post-AdCre infection.


Figure 7Intracellular un - esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2 + / + and Npc2 + / hypo IMCs . Scale bars , 25 μm . Filipin - stained cells were graded from 0 to 3 + as indicated in the microphotographs ( see Materials and Methods for criteria ) , and % of each grade in IMCs from Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVE mice is shown in the bar chart . The data were obtained by analysing 176 Npc2 + / + and 242 Npc2 + / hypo IMCs from three independent Npc2 + / + and Npc2 + / hypo pairs . CLSM imaging of fresh Npc2 + / hypo IMCs stained with filipin ( cyan ) and LAMP2 immunofluorescence ( purple ) . Scale bars , 5 μm . Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2 . Single - colour greyscale images are also demonstrated in the middle ( filipin ) and right ( LAMP2 ) . Intracellular un - esterified cholesterol distribution as detected by filipin staining in Npc2 + / hypo IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 25 μm . Quantitative data in the bar graph were obtained by analysing 265 bNPC2 - untreated ( - bNPC2 ) and 252 bNPC2 - treated ( + bNPC2 ) cells from three independent cultures using the method described in ( A ) .

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Figure 7Intracellular un-esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2+/+ and Npc2+/hypo IMCs. Scale bars, 25 μm. Filipin-stained cells were graded from 0 to 3+ as indicated in the microphotographs (see Materials and Methods for criteria), and % of each grade in IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice is shown in the bar chart. The data were obtained by analysing 176 Npc2+/+ and 242 Npc2+/hypo IMCs from three independent Npc2+/+ and Npc2+/hypo pairs.CLSM imaging of fresh Npc2+/hypo IMCs stained with filipin (cyan) and LAMP2 immunofluorescence (purple). Scale bars, 5 μm. Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2. Single-colour greyscale images are also demonstrated in the middle (filipin) and right (LAMP2).Intracellular un-esterified cholesterol distribution as detected by filipin staining in Npc2+/hypo IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 25 μm. Quantitative data in the bar graph were obtained by analysing 265 bNPC2-untreated (-bNPC2) and 252 bNPC2-treated (+bNPC2) cells from three independent cultures using the method described in (A).


Figure 8Immunoblot ( left ) and ELISA ( right , n = 3 ) analyses of CCL6 in IMC - CM from primary Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h ( immunoblots ) or 72 h ( ELISA ) . The data in the bar graph ( ELISA ) represent mean + SD . Quantification of secreted CCL6 into IMC - CM during the initial 24 h ( 0 - 24 h ) and subsequent 48 h ( 24 - 72 h ) of IMC culture by ELISA ( n = 3 , mean + SD ) . Immunoblot and qRT - PCR analyses of CCL6 expression in Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . The data in the bar graph ( qRT - PCR ) represent mean + SD ; n = 3 . CLSM imaging of CCL6 ( green ) / LAMP1 ( red ) immunofluorescence of Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 10 μm . The right bottom image highlights the boxed area of the merged image of bNPC2 - treated IMCs showing partial co - localisation of LAMP1 and CCL6 . Immunoblot analysis of intracellular and secreted ( CM ) CCL6 in bafilomycin - treated Npc2 + / + IMC culture . IMCs without ( left ) or with ( right ) 50 μg / ml bNPC2 pre - loading for 30 min were chased in serum - free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h .

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 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 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

Figure 8Immunoblot (left) and ELISA (right, n = 3) analyses of CCL6 in IMC-CM from primary Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h (immunoblots) or 72 h (ELISA). The data in the bar graph (ELISA) represent mean + SD.Quantification of secreted CCL6 into IMC-CM during the initial 24 h (0-24 h) and subsequent 48 h (24-72 h) of IMC culture by ELISA (n = 3, mean + SD).Immunoblot and qRT-PCR analyses of CCL6 expression in Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. The data in the bar graph (qRT-PCR) represent mean + SD; n = 3.CLSM imaging of CCL6 (green)/LAMP1 (red) immunofluorescence of Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 10 μm. The right bottom image highlights the boxed area of the merged image of bNPC2-treated IMCs showing partial co-localisation of LAMP1 and CCL6.Immunoblot analysis of intracellular and secreted (CM) CCL6 in bafilomycin-treated Npc2+/+ IMC culture. IMCs without (left) or with (right) 50 μg/ml bNPC2 pre-loading for 30 min were chased in serum-free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h.


Figure 1Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( B ) Dose - response assay using increasing numbers of Nrg1 type I β1a - expressing PC12 cells . For each 96 - well , 40 , 000 split TEV assay cells were co - plated with increasing numbers of Nrg1 - expressing cells , and incubated for 24h . Half - maximal activation is reached at 5 , 000 Nrg1 - expressing cells . The Z ' factor is 0 . 5 indicating a large separation band for this assay . Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( C ) Adding 10ng / ml EGFld resulted in a two - fold activation . Per 96 - well , 40 , 000 split TEV assay cells were co - plated with empty PC12 cells ( no Nrg1 expression ) , 10 , 000 Nrg1 - expressing cells , and 10 , 000 Nrg1 - expressing cells plus 10ng / ml EGFld . Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( D ) Lapatinib antagonizes ERBB4 - PIK3R1 signaling in a dose - dependent manner . Per 96 - well , 40 , 000 split TEV assay cells were incubated with increasing amounts of lapatinib , followed by co - plating 10 , 000 Nrg1 type I β1a expressing cells . The inset depicts the IC 50 value in µM . After compound / stimulus addition , each assay was incubated for 24h . RLU , relative luciferase units ; Fluc , firefly luciferase activity ( black line ) ; Rluc , Renilla luciferase activity ( grey line ) ; n = 6 ; data are shown as mean , error bars represent SEM .

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

Figure 1Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(B) Dose-response assay using increasing numbers of Nrg1 type I β1a-expressing PC12 cells. For each 96-well, 40,000 split TEV assay cells were co-plated with increasing numbers of Nrg1-expressing cells, and incubated for 24h. Half-maximal activation is reached at 5,000 Nrg1-expressing cells. The Z' factor is 0.5 indicating a large separation band for this assay.Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(C) Adding 10ng/ml EGFld resulted in a two-fold activation. Per 96-well, 40,000 split TEV assay cells were co-plated with empty PC12 cells (no Nrg1 expression), 10,000 Nrg1-expressing cells, and 10,000 Nrg1-expressing cells plus 10ng/ml EGFld.Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.


Figure 2Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( B ) Graphic visualisation of the primary screen data of the NIH - NCC library set 1 . All counts ( 320 compounds and 64 controls ) from the Nrg1 - ERBB4 - PIK3R1 split TEV compound screen were plotted against the Z - score using the Mondrian programme , with pathway activators displaying high values and inhibitors low values . For the secondary analysis , we selected all candidates that were at least three standard deviations away from the mean . EGFld positive and Lapatinib / CI - 1033 negative controls are shown in red . Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( C and D ) Spironolactone antagonizes Nrg1 - ERBB4 - PIK3R1 signaling . In dose - response assays using ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids transfected into PC12 cells , spironolactone was administered at increasing concentrations before seeding Nrg1 type I ( C ) expressing PC12 cells . Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( C and D ) Spironolactone antagonizes Nrg1 - ERBB4 - PIK3R1 signaling . In dose - response assays using ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids transfected into PC12 cells , spironolactone was administered at increasing concentrations before seeding Nrg1 type III ( D ) expressing PC12 cells . Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( E ) Spironolactone inhibits ERBB4 receptor dimerization . Dose - dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4 - NTEV - tevS - GV and ERBB4 - CTEV plasmids transfected into PC12 cells . 10 ng / ml EGFld was applied as Nrg1 stimulus . Fluc , firefly luciferase activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines , indicating toxicity levels ) ; n = 6 ; data are shown as mean , error bars represent SEM . The insets depict IC 50 values in µM .

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

Figure 2Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(B) Graphic visualisation of the primary screen data of the NIH-NCC library set 1. All counts (320 compounds and 64 controls) from the Nrg1-ERBB4-PIK3R1 split TEV compound screen were plotted against the Z-score using the Mondrian programme, with pathway activators displaying high values and inhibitors low values. For the secondary analysis, we selected all candidates that were at least three standard deviations away from the mean. EGFld positive and Lapatinib/CI-1033 negative controls are shown in red.Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type I (C) expressing PC12 cells.Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen. (C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type III (D) expressing PC12 cells.Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(E) Spironolactone inhibits ERBB4 receptor dimerization. Dose-dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4-NTEV-tevS-GV and ERBB4-CTEV plasmids transfected into PC12 cells. 10 ng/ml EGFld was applied as Nrg1 stimulus. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.


Figure 3Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( A ) Spironolactone , molecular structure shown left , inhibits ERBB4 / PIK3R1 split TEV assay activity ( black line ) , with an IC 50 value of 2 . 76 µM . Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( B ) canrenone . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 . Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( C ) 7α - thiomethyl - spironolactone . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 . Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( D ) eplerenone do not attenuate the ERBB4 / PIK3R1 assay activity ( black lines ) . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 . Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( F ) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays . All single dose - response assays can be found in Fig EV4 ; the combination ERBB4 - ERBB4 is shown in Fig 2E . Fluc , firefly luciferase activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines , indicating toxicity levels ) ; n = 6 ; data are shown as mean , error bars represent SEM . The insets depict IC 50 values in µM .

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

Figure 3Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(A) Spironolactone, molecular structure shown left, inhibits ERBB4/PIK3R1 split TEV assay activity (black line), with an IC 50 value of 2.76 µM.Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (B) canrenone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment. (B-D) The spironolactone metabolites(C) 7α-thiomethyl-spironolactone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (D) eplerenone do not attenuate the ERBB4/PIK3R1 assay activity (black lines). Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(F) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays. All single dose-response assays can be found in Fig EV4; the combination ERBB4-ERBB4 is shown in Fig 2E. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.