C ) ELISA binding results of mAbs specific for hRSV F ( left panels ) or hMPV F ( right panels ) with indicated at left . mAbs specific of hRSV F bind to epitopes of antigenic site Ø ( D25 ) , antigenic site II ( Mz 47F ) or antigenic site IV ( 101F 107F ) . mAs specific of hMPV F bind to epitopes of antigenic site II ( MF14 ) , antigenic site IV ( MF11 , MF16 MF29 ) or an epitope shared by hRSV F hMPV F ( MPE8 ) .

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C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind to epitopes of antigenic site Ø (D25), antigenic site II (Mz and 47F) or antigenic site IV (101F and 107F). mAs specific of hMPV F bind to epitopes of antigenic site II (MF14), antigenic site IV (MF11, MF16 and MF29) or an epitope shared by hRSV F and hMPV F (MPE8).


F . Groups of BALB / c mice ( n = 5 ) were inoculated twice with 10 µg of indicated at top of each panel , folded in either prefusion conformation for hRSV F , F - 410 F - 411 or in postfusion conformation for hMPV F . One week after last dose , mice were sacrificed their blood collected . A ) Each mouse serum was tested in ELISA for antibody binding to F indicated at bottom of each panel . * P = 0 . 00046 * * P = 0 . 02459 .

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F. Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum was tested in ELISA for antibody binding to the F proteins indicated at the bottom of each panel. *P = 0.00046 and **P = 0.02459.


Groups of BALB / c mice ( n = 5 ) were inoculated twice with 10 µg of indicated at top of each panel , folded in either prefusion conformation for hRSV F , F - 410 F - 411 or in postfusion conformation for hMPV F . One week after last dose , mice were sacrificed their blood collected . B ) Mouse sera were also tested in microneutralization assays with hRSV hMPV , indicated at bottom . * P = 0 . 04138 , * * P = 0 . 04831 * * * P = 0 . 00339 . results in A B are presented as in Fig . 3 .

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Groups of BALB/c female mice (n = 5) were inoculated twice with 10 µg of the proteins indicated at the top of each panel, folded in either the prefusion conformation for hRSV F, F-410 and F-411 or in the postfusion conformation for hMPV F. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, indicated at the bottom. *P = 0.04138, **P = 0.04831 and ***P = 0.00339. The results in A and B are presented as in Fig. 3.


A Northern blots comparing GlmY GlmZ in wild type Z8 ∆ rapZ mutant Z28 under normal growth GlcN6P starvation conditions . Both strains , which also carried a chromosomal glmS ' - lacZ fusion , were treated with 60 μg / ml Nva - FMDP or H2O ( " mock " ) . Samples were harvested hourly for Northern determination of β - galactosidase activity . Growth curves are shown in Appendix Fig S1 . Blots were re - probed using a 5S rRNA specific probe to provide loading controls .

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A Northern blots comparing GlmY and GlmZ levels in wild type strain Z8 and the ∆rapZ mutant Z28 under normal growth and GlcN6P starvation conditions. Both strains, which also carried a chromosomal glmS'-lacZ fusion, were treated with 60 μg/ml Nva-FMDP or H2O ("mock"). Samples were harvested hourly for Northern analysis and determination of β-galactosidase activity. Growth curves are shown in Appendix Fig S1. Blots were re-probed using a 5S rRNA specific probe to provide loading controls.


B purification profile of Strep - RapZ from ∆ glmS Z904 under GlcN6P replete depletion conditions is shown ( top ) . cleared lysate ( CL ) , flow through ( FT ) , washing steps ( W ) elution fractions ( E1 - 3 ) from StrepTactin affinity chromatography were separated on 12 . 5 % SDS - PAA gels stained with Coomassie blue . Metabolites were extracted from E2 analyzed by HILIC - MS / MS . extracted ion chromatograms of LC - MS targeting GlcN6P ( retention time 16 . 6 minutes ) are shown below . samples derived from purification of Strep - RapZ ( panels i ii ) or Strep - KdpE ( panels iii iv ) were analyzed with SRM transition m / z 258 . 1 to m / z 97 in negative ion mode . identity of metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final of 100 pg / μl ( panel v ) .

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B The purification profile of Strep-RapZ from the ∆glmS strain Z904 under GlcN6P replete and depletion conditions is shown (top). The cleared lysate (CL), flow through (FT), washing steps (W) and the elution fractions (E1-3) from StrepTactin affinity chromatography were separated on 12.5 % SDS-PAA gels and stained with Coomassie blue. Metabolites were extracted from E2 and analyzed by HILIC-MS/MS. The extracted ion chromatograms of the LC-MS analysis targeting GlcN6P (retention time 16.6 minutes) are shown below. The samples derived from purification of Strep-RapZ (panels i and ii) or Strep-KdpE (panels iii and iv) were analyzed with the SRM transition m/z 258.1 to m/z 97 in the negative ion mode. The identity of the metabolite detected in panel i was confirmed by adding chemically pure GlcN6P to a final concentration of 100 pg/μl (panel v).


C SPR addressing interaction of RapZ variants with GlcN6P similar metabolites . Strep - tagged were captured onto a sensor chip various concentrations of respective metabolite ( i . e . 100 , 500 , 1000 , 2500 5000 nM ) were injected using a single cycle kinetics approach .

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C SPR analysis addressing interaction of RapZ variants with GlcN6P and similar metabolites. The Strep-tagged proteins were captured onto a sensor chip and various concentrations of the respective metabolite (i.e. 100, 500, 1000, 2500 and 5000 nM) were injected using a single cycle kinetics approach.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete depletion conditions . strains were grown in 96 - well plates exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . were harvested at indicated times β - galactosidase activities were determined . Strains Z197 Z360 were used , which harbor glmY ' - lacZ glmZ ' - lacZ fusions , respectively .

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Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z197 and Z360 were used, which harbor glmY'-lacZ and glmZ'-lacZ fusions, respectively.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete depletion conditions . strains were grown in 96 - well plates exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . were harvested at indicated times β - galactosidase activities were determined . Expression of glmY ' - lacZ in Z197 ∆ rapZ mutant Z225 is compared .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Expression of glmY'-lacZ in strain Z197 and the ∆rapZ mutant Z225 is compared.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete depletion conditions . strains were grown in 96 - well plates exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . were harvested at indicated times β - galactosidase activities were determined . Strains Z190 Z201 were addressed , which transcribe glmY ' - lacZ either from σ54 - promoter or σ70 promoter , respectively .

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

Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and Z201 were addressed, which transcribe glmY'-lacZ either from the σ54-promoter or the σ70 promoter, respectively.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete depletion conditions . strains were grown in 96 - well plates exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . were harvested at indicated times β - galactosidase activities were determined . Strains Z190 ∆ qseF mutant Z196 are compared , both of which transcribe glmY ' - lacZ fusion solely from σ54 promoter .

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Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Strains Z190 and the ∆qseF mutant Z196 are compared, both of which transcribe the glmY'-lacZ fusion solely from the σ54 promoter.


Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete depletion conditions . strains were grown in 96 - well plates exposed to various degrees of GlcN6P depletion elicited by Nva - FMDP . were harvested at indicated times β - galactosidase activities were determined . Complementation experiment analyzing requirement of rapZ for glmY expression in grown to exponential phase under standard conditions in flask cultures . Strains Z197 ∆ rapZ mutant Z225 were used . Tested plasmids were pFDX4291 ( vector control for pFDX4324 pYG82 = VC1 ) , pFDX4324 ( rapZ ) , pYG82 ( rapZquad ) , pBGG237 ( vector control for pBGG164 = VC2 ) pBGG164 ( strep - rapZ ) .

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Reporter gene assays addressing expression of lacZ fusions under GlcN6P replete and depletion conditions. strains were grown in 96-well plates and exposed to various degrees of GlcN6P depletion elicited by Nva-FMDP. Cells were harvested at indicated times and the β-galactosidase activities were determined. Complementation experiment analyzing the requirement of rapZ for glmY expression in cells grown to exponential phase under standard conditions in flask cultures. Strains Z197 and the ∆rapZ mutant Z225 were used. Tested plasmids were pFDX4291 (vector control for pFDX4324 and pYG82 = VC1), pFDX4324 (rapZ), pYG82 (rapZquad), pBGG237 (vector control for pBGG164 = VC2) and pBGG164 (strep-rapZ).


A BACTH addressing interaction of RapZ with QseF QseE . following plasmid combinations were tested using reporter RH785 ( columns 1 - 6 ) : pBGG352 / pBGG349 , pBGG353 / pBGG348 , pYG199 / pBGG349 , pYG246 / pBGG348 , pKT25 / pUT18C ( negative control ) , pKT25 - zip / pUT18C - zip ( positive control ) . , β - galactosidase activities are presented as mean ± SD .

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A BACTH assay addressing interaction of RapZ with QseF and QseE. The following plasmid combinations were tested using reporter strain RH785 (columns 1-6): pBGG352/pBGG349, pBGG353/pBGG348, pYG199/pBGG349, pYG246/pBGG348, pKT25/pUT18C (negative control), pKT25-zip/pUT18C-zip (positive control). , β-galactosidase activities are presented as mean ± SD.


B SPR spectroscopy experiments addressing interaction of RapZ with part ( aa 196 - 475 ) of kinase QseE response regulator QseF . Strep - RapZ was captured onto a sensor chip increasing concentrations of QseE ' - His10 or QseF - His10 were injected .

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B SPR spectroscopy experiments addressing interaction of RapZ with the cytoplasmic part (aa 196-475) of kinase QseE and response regulator QseF. Strep-RapZ was captured onto a sensor chip and increasing concentrations of QseE'-His10 or QseF-His10 were injected.


C To assess role of RapZ for QseF activity , β - galactosidase activities were determined from strains Z196 ( ∆ qseF ) Z1110 ( ∆ qseF ∆ rapZ ) at indicated times during growth . Strains harbored glmY ' - lacZ fusion that is solely expressed from σ54 promoter following plasmids : pKESK23 ( VC = vector control ; black grey ; note that activities are too low for display ) , pYG89 ( qseF , green ) , pYG90 ( qseF - D56E , purple ) , pYG93 ( qseF - D56A , red ) . , β - galactosidase activities are presented as mean ± SD .

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C To assess the role of RapZ for QseF activity, β-galactosidase activities were determined from strains Z196 (∆qseF) and Z1110 (∆qseF ∆rapZ) at indicated times during growth. Strains harbored the glmY'-lacZ fusion that is solely expressed from the σ54 promoter and the following plasmids: pKESK23 (VC = vector control; black and grey; note that activities are too low for display), pYG89 (qseF, green), pYG90 (qseF-D56E, purple), pYG93 (qseF-D56A, red). , β-galactosidase activities are presented as mean ± SD.


D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10 - tagged QseE ' ( aa 196 - 475 ) in presence of various concentrations of RapZ . Samples were removed following [ γ - 32P ] - ATP addition at indicated times separated on 12 . 5 % SDS - PAA gels , which were analyzed by phospho imaging .

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D In vitro phosphorylation assays addressing autophosphorylation of 1 μM His10-tagged QseE' (aa 196-475) in presence of various concentrations of RapZ. Samples were removed following [γ-32P]-ATP addition at indicated times and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.


E of QseE ' autophosphorylation ( lanes 1 - 6 ) phosphoryl group transfer to QseF ( lanes 7 - 12 ) in absence or presence of 5 μM Strep - RapZ . To assess phosphoryl group transfer , 1 μM QseF - His10 was added to .

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E Analysis of QseE' autophosphorylation (lanes 1-6) and phosphoryl group transfer to QseF (lanes 7-12) in absence or presence of 5 μM Strep-RapZ. To assess phosphoryl group transfer, 1 μM QseF-His10 was added to the assay.


F To analyse role of GlcN6P , 5 μM RapZ or equivalent volume of buffer was pre - incubated with indicated GlcN6P for 5 min subsequently 1 μM QseE ' - His10 was added . Following an additional incubation for 5 min , [ γ - 32P ] - ATP was added reactions were stopped after 1 min .

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F To analyse the role of GlcN6P, 5 μM RapZ or the equivalent volume of buffer was pre-incubated with the indicated GlcN6P concentration for 5 min and subsequently 1 μM QseE'-His10 was added. Following an additional incubation for 5 min, [γ-32P]-ATP was added and the reactions were stopped after 1 min.


A β - Galactosidase activities of strains Z197 ( wild type ) Z1118 ( ∆ glmY ∆ glmZ ) , which carry chromosomal glmY ' - lacZ fusion , were determined during growth .

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A β-Galactosidase activities of strains Z197 (wild type) and Z1118 (∆glmY ∆glmZ), which carry the chromosomal glmY'-lacZ fusion, were determined during growth.


B Strains Z197 ( wild type ) Z225 ( ∆ rapZ ) were transformed with following plasmids expressing mentioned sRNAs : pBR - plac ( vector control = VC ) , pYG83 ( glmY ) , pYG84 ( glmZ ) pSD69 ( gcvB ) . sRNA expression was induced with 1 mM IPTG β - galactosidase activities were determined in exponential growth phase .

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B Strains Z197 (wild type) and Z225 (∆rapZ) were transformed with the following plasmids expressing the mentioned sRNAs: pBR-plac (vector control = VC), pYG83 (glmY), pYG84 (glmZ) and pSD69 (gcvB). sRNA expression was induced with 1 mM IPTG and β-galactosidase activities were determined in the exponential growth phase.


C To assess impact of GlmY * GlmZ on stimulation of QseE ' autophosphorylation by RapZ , 1 μM QseE ' - His10 was incubated with [ γ - 32P ] - ATP in absence or presence of 5 μM Strep - RapZ / or sRNAs GlmY * , GlmZ GcvB . In lanes 3 - 11 QseE ' was co - incubated with RapZ as well as indicated sRNAs provided at 0 . 5 μM , 1 . 25 μM 2 . 5 μM . In lanes 14 - 16 , QseE ' was incubated with 2 . 5 μM of each sRNA without RapZ . Samples were removed 1 min after addition of [ γ - 32P ] - ATP separated on 12 . 5 % SDS - PAA gels , which were analyzed by phospho imaging .

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C To assess the impact of GlmY* and GlmZ on stimulation of QseE' autophosphorylation by RapZ, 1 μM QseE'-His10 was incubated with [γ-32P]-ATP in absence or presence of 5 μM Strep-RapZ and/or the sRNAs GlmY*, GlmZ and GcvB. In lanes 3-11 QseE' was co-incubated with RapZ as well as the indicated sRNAs provided at 0.5 μM, 1.25 μM and 2.5 μM. In lanes 14-16, QseE' was incubated with 2.5 μM of each sRNA without RapZ. Samples were removed 1 min after addition of [γ-32P]-ATP and separated on 12.5 % SDS-PAA gels, which were analyzed by phospho imaging.


Northern blot experiments assessing half - lives of GlmY * GlmZ under normal growth GlcN6P starvation conditions . cultures were either treated with 100 μg / ml Nva - FMDP or H2O ( mock ) . Transcription was stopped by rifampicin addition when cultures attained OD600 = 1 . 0 samples were removed at indicated times for Northern . A of GlmY * GlmZ decay in wild type Z8 . B of GlmY * GlmZ decay in ∆ rapZ mutant Z28 . C Semi - logarithmic plots of GlmY * full - GlmZ decay for half - life determination .

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Northern blot experiments assessing the half-lives of GlmY* and GlmZ under normal growth and GlcN6P starvation conditions. Bacterial cultures were either treated with 100 μg/ml Nva-FMDP or H2O (mock). Transcription was stopped by rifampicin addition when cultures attained OD600 = 1.0 and samples were removed at indicated times for Northern analysis. A Analysis of GlmY* and GlmZ decay in the wild type strain Z8. B Analysis of GlmY* and GlmZ decay in the ∆rapZ mutant Z28. C Semi-logarithmic plots of GlmY* and full-length GlmZ decay for half-life determination.


Northern blot experiments ( A - C ) assessing GlmY * half - life under GlcN6P replete ( + GlcN ) depletion conditions ( - GlcN ) . Transcription was stopped by addition of rifampicin samples were harvested at indicated times for Northern . A of ∆ glmS Z1126 . GlcN6P depleted culture was split 8 min after rifampicin addition one of sub - cultures was resupplied with GlcN ( indicated by arrow ) . B ∆ glmS ∆ rapZ double mutant Z1127 was tested . C Semi - logarithmic plots of GlmY * decay for half - life determination .

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Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination.


D EMSA experiments addressing role of GlcN6P for GlmY * / RapZ interaction . Radiolabelled GlmY * was incubated with incremental concentrations of RapZ ( left panel ) or RapZ - CTD ( right panel ) in absence or presence of 7 . 5 mM GlcN6P . Binding reactions were separated on native PAA gels analyzed by phospho - imaging . RapZ / GlmY * is indicated by an arrow .

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D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.


E EMSA following incubation of GlmY * with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 ( lane 2 ) to 8 mM ( lane 10 ) . fraction of GlmY * remaining in gel pocket is marked with an asterisk . RapZ / GlmY * is indicated by an arrow .

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E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.


F EMSA following incubation of GlmY * with 1200 nM RapZ in absence or presence of 7 . 5 mM of indicated metabolite . RapZ / GlmY * is indicated by an arrow .

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F EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.


Western blot for phosphorylated total ERK in HEK293 transfected with native ΔA781 _ N783 ( left ) or P824R ( right ) CSF - 1R treated with CSF - 1 ( D , E ) or IL - 34 ( F , G ) for 10 minutes . Horizontal indicates untreated , with increasing concentrations 10 , 50 100 ng / ml . Corresponding densitometry displays of phospho - ERK to total ERK , normalised to of untreated control for native receptor ( * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 01 , * p < 0 . 05 , one - way ANOVA with Sidak ' s post - for multiple comparison , n = 3 biological replicates , , error bars indicate ) .

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

Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM).


Western blot for CSF - 1R in HEK293 transfected with native ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R treated with rapamycin . Horizontal indicates untreated , with concentrations 0 . 1 , 0 . 5 , 1 , 5 , 10μM . Corresponding densitometry ( below ) displays fold change of CSF - 1R relative to untreated control for each transfection ( * * * * p < 0 . 0001 , one - way ANOVA with Sidak ' s post - for multiple comparison to untreated , n = 3 biological replicates , error bars indicate ) . Western blot for CSF - 1R in HEK293 transfected with native ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R treated with 3 - methyladenosine . Horizontal indicates untreated , with increasing concentrations 0 . 1 , 0 . 2 , 0 . 5 , 1mM . Corresponding densitometry ( below ) displays fold change of CSF - 1R relative to untreated control for each transfection ( * * * * p < 0 . 0001 , * p = 0 . 0114 , one - way ANOVA with Sidak ' s post - for multiple comparison to untreated , n = 3 biological replicates , error bars indicate ) . Western blot for CSF - 1R Actin in HEK293 transfected with native ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R treated with MG132 or PLX3397 . Horizontal indicates untreated , 20μM PLX3397 , with increasing concentrations 0 . 1 , 0 . 5 , 0 . 1 , 5μM . Corresponding densitometry ( below ) displays fold change of CSF - 1R relative to untreated control for each transfection ( * * * p = 0 . 0002 , * * p = 0 . 002 , one - way ANOVA with Sidak ' s post - for multiple comparison to untreated , n = 3 biological replicates , error bars indicate ) .

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

Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).


Immunocytochemistry of microglia isolated from Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + P0 mouse pup brains . Microglia were stimulated with LPS exposed to opsonised latex beads for 1 hour before fixation of bead + via microscopy . were stained using MitoTracker ( red ) . Arrowheads ( ) indicate bead + . of mouse microglial phagocytic activity expressed as bead + ( n = 4 assays , error bars indicate ) .

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Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells. Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).


L ) Immunocytochemistry of macrophages differentiated in vitro from Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + mouse bone marrow . Macrophages were stimulated with LPS exposed to opsonised latex beads for 1 hour before fixation of bead + via microscopy . were stained using MitoTracker ( red ) DAPI ( blue ) . Arrowheads ( ) indicate bead + . M ) of mouse macrophage phagocytic activity expressed as bead + . ( * p = 0 . 024 , Student ' s t - with Welch ' s correction , n = 4 assays , error bars indicate ) .

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L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells. M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).


A ) Immunohistochemistry of Csf - 1rflx / wt ; Cx3cr1 - Cre - ( top ) Csf - 1rflx / wt ; Cx3cr1 - Cre + ( bottom ) mice unilaterally injected with Aβ1 - 42 in hippocampus stained for IB4 ( green ) , F4 / 80 ( ) Aβ ( red ) . Scale bars indicate 50 μm . B ) of F4 / 80 IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . of immunopositivity in both injected uninjected hippocampi of Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + mice . ( Two - way ANOVA with Sidak ' s for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of uninjected hippocampus , obliques ( # ) indicate comparison between Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + mice , # # or * * p < 0 . 003 , # # # # or * * * * p < 0 . 0001 , n = 5 mice per group , error bars indicate ) .

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A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).


C ) Immunohistochemistry of Csf - 1rflx / wt ; Tie2 - Cre - ( top ) Csf - 1rflx / wt ; Tie2 - Cre + mice ( ottom ) mice unilaterally injected with Aβ1 - 42 in hippocampus stained for IB4 ( green ) , F4 / 80 ( ) Aβ ( red ) . D ) of F4 / 80 IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . of immunopositivity in both injected uninjected hippocampi of Csf - 1rflx / wt ; Tie2 - Cre - Csf - 1rflx / wt ; Tie2 - Cre + mice . ( n = 5 mice per group . Two - way ANOVA with Sidak ' s for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of uninjected hippocampus , * p < 0 . 01 , * * * p < 0 . 0005 , error bars indicate ) ) .

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C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).


qPCR of wild type ( blue ) or Csf - 1r + / - ( red ) endothelial treated with control , Csf - 1r + / + MCM or Csf - 1r + / - MCM . Statistical analyses of inter - genotype changes . ( * * * * p < 0 . 0001 , * * p < 0 . 005 , Scatter plots represent technical replicates of n = 2 independent isolations microglia conditionings , Two - way ANOVA with multiple comparisons Sidak post - , error bars indicate ) ) .

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qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).


of ALSP post mortem tissue for amyloid - β ( green ) CLD5 ( red ) , ( B ) human IgG ( green ) PDGFRβ ( red ) , ( C ) GFAP ( green ) CLD5 ( red ) . ΔA781 _ N783 P824R indicate CSF - 1R variant present .

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IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.


( D ) Iba1 ( green ) GFAP ( red ) , ( E ) amyloid - β ( green ) CD68 ( red ) , ( f ) CD163 ( green ) CLD5 ( red ) , imaging of an ALSP with ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 P824R indicate CSF - 1R variant present .

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(D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


( G ) Fibrinogen ( green ) CLD5 ( red ) ( H ) FLAIR imaging of an ALSP with ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 P824R indicate CSF - 1R variant present .

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(G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


B : PK vs . PIPLC treatment to determine prion loads in infected in 384 - well plates . Both treatments discriminate between RML prion infected CAD5 non - infectious brain homogenate ( NBH ) treated CAD5 . * * * * = p - value < 0 . 0001 ( Student ' s t - ) . Shown are mean ± SD , n ≤ 30 individually cultured wells .

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B: PK vs. PIPLC treatment to determine prion loads in infected cells in 384-well plates. Both treatments discriminate between RML prion infected CAD5 and non-infectious brain homogenate (NBH) treated CAD5 cells. **** = p-value < 0.0001 (Student's t-test). Shown are mean ± SD, n ≤ 30 individually cultured wells.


C : infectivity of PIPLC PK - treated lysates was determined by infecting CAD5 with lysates as indicated . signal of highest dilution was measured compared to untreated RML CAD5 lysate . PIPLC - treated PK - treated lysates showed similar infectivity titers . Prion - infected NBH , as well as RML on CAD5ΔPrnp were used for control .

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C: The infectivity of PIPLC and PK-treated cell lysates was determined by infecting CAD5 cells with lysates as indicated. The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate. PIPLC-treated cells and PK-treated lysates showed similar infectivity titers. Prion-infected and NBH, as well as RML on CAD5ΔPrnp were used for control.


D : RML - infected CAD5 GT - 1 / 7 were transfected with non - targeting ( NT ) or Prnp targeting siRNAs in a 384 - well plate subjected to PK or PIPLC treatment . Z ' - factors were calculated for each condition . Shown are mean ± SD , n = 22 individually cultured wells .

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D: RML-infected CAD5 and GT-1/7 cells were transfected with non-targeting (NT) or Prnp targeting siRNAs in a 384-well plate and subjected to PK or PIPLC treatment. Z'-factors were calculated for each condition. Shown are mean ± SD, n = 22 individually cultured wells.


A : Western blot showing Hnrnpk siRNA transfection ( 96 hrs . ) decreases Hnrnpk while increases PrPSc in RML prion infected GT - 1 / 7 . Prnp siRNAs suppressed both PrPC PrPSc as expected . ⍺ : anti Quantifications are reported as normalized to Actin in comparison , to NT . PK - western blot is quantified relative to NT .

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A: Western blot showing Hnrnpk siRNA transfection (96 hrs.) decreases Hnrnpk protein levels while increases PrPSc in RML prion infected GT-1/7. Prnp siRNAs suppressed both PrPC and PrPSc as expected. ⍺: anti Quantifications are reported as normalized to Actin and in comparison, to NT. PK-western blot is quantified relative to NT.


B : Western Blot of PSA - treated uninfected infected GT - 1 / 7 . Increasing of PSA leads to a more prominent reduction of PrPSc in mouse . PK - western blot is quantified relative to DMSO .

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B: Western Blot of PSA-treated uninfected and infected GT-1/7 cells. Increasing concentration of PSA leads to a more prominent reduction of PrPSc in mouse cells. PK-western blot is quantified relative to DMSO.


C : mRNA of Hnrnpk Prnp following siRNA PSA treatment . FPKM : Fragments per kilobase of transcript per million mapped reads . Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA as well as an increase in Prnp mRNA . No difference is seen between DMSO treated PSA treated for either Hnrnpk of Prnp . n = 2 per treatment group .

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C: mRNA levels of Hnrnpk and Prnp following siRNA and PSA treatment. FPKM: Fragments per kilobase of transcript per million mapped reads. Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA levels as well as an increase in Prnp mRNA levels. No difference is seen between DMSO treated and PSA treated cells for either Hnrnpk levels of Prnp levels. n = 2 per treatment group.


D : of PrPSc in RML GT - 1 / 7 following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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D: Quantification of PrPSc levels in RML GT-1/7 cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


E : Brightfield microscopy of effect of siRNA mediated HNRNPK downregulation on prion - induced vacuolation in PG127 hovS . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA . ovPRNP siRNA transfected , as well as uninfected were used as controls Downregulation of ovPRNP in infected hovS eliminates vacuoles ; Right panel : of vacuoles of NT , HNRNPK ovPRNP siRNA treated PG127 hovS . from pictures at three different positions in well were manually counted amount of vacuolated was normalized to total amount of . Values represent mean ± SD . * p = 0 . 0113 , * * * * p = < 0 . 0001 ( Dunnett ' s multiple comparisons ) . Scale bar = 100 µm . n = 3 technical replicates .

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E: Brightfield microscopy images of the effect of siRNA mediated HNRNPK downregulation on prion-induced vacuolation in PG127 hovS cells. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA. ovPRNP siRNA transfected, as well as uninfected cells were used as controls Downregulation of ovPRNP in the infected hovS eliminates the vacuoles; Right panel: Quantification of vacuoles of NT, HNRNPK and ovPRNP siRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. * p = 0.0113, **** p = <0.0001 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


F : of PrPSc in PG127 hovS following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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F: Quantification of PrPSc levels in PG127 hovS cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


G : Same as in E , using shRNAs instead of siRNA . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA . Uninfected were used as controls Right panel : of vacuoles of NT HNRNPK shRNA treated PG127 hovS . from pictures at three different positions in well were manually counted amount of vacuolated was normalized to total amount of . Values represent mean ± SD . * * * p = < 0 . 0009 ( Dunnett ' s multiple comparisons ) . Scale bar = 100 µm . n = 3 technical replicates .

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G: Same as in E, using shRNAs instead of siRNA. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA. Uninfected cells were used as controls Right panel: Quantification of vacuoles of NT and HNRNPK shRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. *** p = <0.0009 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


H : of PrPSc in PG127 hovS following treatment with shRNA against HNRNPK 1 µM PSA in comparison to DMSO NT . Each dot represents an experiment . * = p - value < 0 . 027 ( Student ' s t - ) . Shown are mean ± SD .

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H: Quantification of PrPSc levels in PG127 hovS cells following treatment with shRNA against HNRNPK and 1 µM PSA in comparison to DMSO and NT. Each dot represents an experiment. *= p-value < 0.027 (Student's t-test). Shown are mean ± SD.


I : Gene set overrepresentation of differentially expressed genes ( log2FC - 0 . 25 ≥ or 0 . 25 ≤ FDR ≤ 0 . 05 ) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT - 1 / 7 analyzed by RNAseq . Differentially regulated genes ( up in siRNA treatment down in PSA or vice versa ) were overlapped used for pathway . No significantly enriched pathways are detected for upregulated genes in Hnrnpk downregulated in PSA treatment . For opposing direction , an enrichment of genes involved in glucose metabolism was detected .

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I: Gene set overrepresentation analysis of differentially expressed genes (log2FC -0.25 ≥ or 0.25 ≤ and FDR ≤ 0.05) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT-1/7 cells analyzed by RNAseq. Differentially regulated genes (up in siRNA treatment and down in PSA or vice versa) were overlapped and used for pathway analysis. No significantly enriched pathways are detected for upregulated genes in Hnrnpk and downregulated in PSA treatment. For the opposing direction, an enrichment of genes involved in glucose metabolism was detected.


A : Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO . PSA treatment was started two after infection continued until lysis . PSA reduced amount of PrPSc . Right panel : of Western Blot ; n = 5 biological replicates . Values : mean ± SD . * p = 0 . 0211 ( Student ' s t - ) .

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A: Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO. PSA treatment was started two weeks after infection and continued until lysis. PSA reduced the amount of PrPSc. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values: mean ± SD. * p = 0.0211 (Student's t-test).


B : Western Blot of NBH - treated COCS . PSA treatment was identical to samples of A . PSA did not affect PrPC expression . Right panel : of Western Blot ; n = 5 biological replicates . Values represent mean ± SD . n . s . = not significant ( Student ' s t - ) . ⍺ :

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B: Western Blot analysis of NBH-treated COCS. PSA treatment was identical to the samples of A. PSA did not affect PrPC expression. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values represent mean ± SD. n.s. = not significant (Student's t-test). ⍺: anti


C : Negative geotaxis climbing in prion infected Drosophila . Flies were treated with DMSO , 0 . 5 mM PSA , 0 . 75 mM PSA , or 1 mM PSA at larval stage during adulthood for duration of . Climbing ability was assessed on groups of flies ( n = 3 x 15 ) three times a week expressed as a performance index . Statistical on difference between PG127 prion infected versus control prion - free treatment group data in each graph was performed by an unpaired t : DMSO : p = 0 . 0002 ; PSA : 0 . 5 mM : p = 0 . 0186 ; 0 . 75 mM : p = n . s . ; 1 mM : p = n . s . n . s . = not significant . Shown are mean ±

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C: Negative geotaxis climbing assay in prion infected Drosophila. Flies were treated with DMSO, 0.5 mM PSA, 0.75 mM PSA, or 1 mM PSA at the larval stage and during adulthood for the duration of the assay. Climbing ability was assessed on groups of flies (n=3 x 15) three times a week and expressed as a performance index. Statistical analysis on the difference between PG127 prion infected versus control prion-free treatment group data in each graph was performed by an unpaired t test: DMSO: p = 0.0002; PSA: 0.5 mM: p = 0.0186; 0.75 mM: p = n.s.; 1 mM: p = n.s. n.s. = not significant. Shown are mean ± SD


D : RT - QuIC of whole - head homogenates of prion infected Drosophila . For each sample , 10 10 heads from same treatment group were homogenized , 1 : 20 diluted applied to RT - QuIC . Shown are RT - QuIC reactions of three independent homogenates per treatment group , each assessed in quadruplicates . For , lag - time of each reaction was calculated plotted in a graph . Shown are mean ± SD . assays were performed for 100 hours , samples not yielding a positive reaction are considered negative . * * * = p ≤ 0 . 0006 , n . s . = not significant , flies fed with NBH treated with DMSO were used as a negative control , a standard prion - free a prion - containing sample were used as controls . To control for potential interference of DMSO or PSA with RT - QuIC reaction , prion positive sample was spiked with 1 µM PSA .

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D: RT-QuIC analysis of whole-head homogenates of prion infected Drosophila. For each sample, 10 male and 10 female heads from the same treatment group were homogenized, 1:20 diluted and applied to the RT-QuIC. Shown are the RT-QuIC reactions of three independent homogenates per treatment group, each assessed in quadruplicates. For quantification, the lag-time of each reaction was calculated and plotted in a graph. Shown are mean ± SD. The assays were performed for 100 hours, samples not yielding a positive reaction are considered negative. *** = p ≤ 0.0006, n.s. = not significant, flies fed with NBH and treated with DMSO were used as a negative control, a standard prion-free and a prion-containing sample were used as assay controls. To control for potential interference of DMSO or PSA with the RT-QuIC reaction, prion positive sample was spiked with 1 µM PSA.


SHAPE of complete sfRNAs of ZIKV ( D ) Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , moderately reactive site ( 0 . 4 - 0 . 85 ) are labeled in cyan . Unlabeled sites have low or no SHAPE reactivity ( < 0 . 4 ) . SHAPE reactivity were also annotated onto corresponding secondary structures in ( B ) Nucleotides which SHAPE reactivity was not determined are labeled in gray in ( B )

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SHAPE analysis of the complete sfRNAs of ZIKV (D) Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (B) Nucleotides which SHAPE reactivity was not determined are labeled in gray in (B)


SHAPE of complete sfRNAs of WNV ( E ) . Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , moderately reactive site ( 0 . 4 - 0 . 85 ) are labeled in cyan . Unlabeled sites have low or no SHAPE reactivity ( < 0 . 4 ) . SHAPE reactivity were also annotated onto corresponding secondary structures in ( C ) . Nucleotides which SHAPE reactivity was not determined are labeled in gray in ( C ) .

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SHAPE analysis of the complete sfRNAs of WNV (E). Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (C). Nucleotides which SHAPE reactivity was not determined are labeled in gray in (C).


( A ) - ( D ) scattering profiles ( A ) , pair distribution functions ( PDDFs ) ( B ) , dimensionless Kratky plots ( C ) Porod - Debye plots ( D ) of complete sfRNAs of DENV2 ( cyan ) , ZIKV ( red ) WNV ( blue ) . fine features of P1 in scattering profiles in ( A ) arise from helical interstrand pair - correlation . PDDF profiles in ( B ) were calculated using GNOM ( qmax = 0 . 3 ) . dimensionless Kratky plot Porod - Debye plot for ZIKV xrRNA1 ( red dashed ) were included in ( C ) ( D ) for comparison , indicating that structures of sfRNAs are more extended open than that of ZIKV xrRNA1 .

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(A)-(D) The scattering profiles (A), pair distance distribution functions (PDDFs) (B), dimensionless Kratky plots (C) and Porod-Debye plots (D) of the complete sfRNAs of DENV2 (cyan line), ZIKV (red line) and WNV (blue line). The fine features of P1 in the scattering profiles in (A) arise from helical interstrand pair-distance correlation. The PDDF profiles in (B) were calculated using GNOM (qmax=0.3). The dimensionless Kratky plot and Porod-Debye plot for ZIKV xrRNA1 (red dashed line) were included in (C) and (D) for comparison, indicating that the structures of sfRNAs are more extended and open than that of ZIKV xrRNA1.


( C , D ) scattering profiles ( left ) , PDDFs ( middle ) dimensionless Kratky plots ( right ) of xrRNA1s ( C ) xrRNA2s ( D ) of DENV2 ( cyan ) , ZIKV ( red ) , WNV ( blue ) MVEV ( green ) . insets in ( C ) ( D ) show Guinier regions of respective scattering profiles with a linear fit . back - calculated scattering profile of ZIKV - xrRNA1 crystal structure ( black , open circle ) can be nicely fitted onto its experimental SAXS data ( red ) in ( C ) , while back - calculated scattering profile of MVEV - xrRNA2 crystal structure ( black , open circle ) fits poorly to its experimental SAXS data ( green ) in ( D ) . asymmetric PDDFs of xrRNA1 ( C ) xrRNA2 ( D ) , indicate slightly elongated molecules with asymmetric shapes . dimensionless Kratky plots of xrRNA1 ( C ) xrRNA2 ( D ) of DENV2 , ZIKV , WNV MVEV suggest xrRNA1s xrRNA2s are well - folded of reduced flexibility in solution .

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(C, D) The scattering profiles (left), the PDDFs (middle) and the dimensionless Kratky plots (right) of individual xrRNA1s (C) and xrRNA2s (D) of DENV2 (cyan), ZIKV (red), WNV (blue) and MVEV (green). The insets in (C) and (D) show the Guinier regions of the respective scattering profiles with a linear fit line. The back-calculated scattering profile of ZIKV- xrRNA1 crystal structure (black, open circle) can be nicely fitted onto its experimental SAXS data (red line) in (C), while the back-calculated scattering profile of MVEV- xrRNA2 crystal structure (black, open circle) fits poorly to its experimental SAXS data (green line) in (D). The asymmetric PDDFs of xrRNA1 (C) and xrRNA2 (D), indicate slightly elongated molecules with asymmetric shapes. The dimensionless Kratky plots of xrRNA1 (C) and xrRNA2 (D) of DENV2, ZIKV, WNV and MVEV suggest all individual xrRNA1s and xrRNA2s are well-folded and of reduced flexibility in solution.


( A ) - ( C ) scattering profiles ( A ) , PDDFs ( B ) dimensionless Kratky plots ( C ) of xrRNA1 - 2s from DENV2 ( cyan ) , ZIKV ( red ) WNV ( blue ) . inset in ( A ) shows guinier regions of respective scattering profiles with linear fit lines . dimensionless Kratky plot of ZIKV xrRNA1 ( red dashed ) was included in ( C ) for comparison , indicating that structures of xrRNA1 - 2s are well folded .

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(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of xrRNA1-2s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of xrRNA1-2s are all well folded.


( A ) - ( C ) scattering profiles ( A ) , PDDFs ( B ) dimensionless Kratky plots ( C ) of DB12s from DENV2 ( cyan ) , ZIKV ( red ) WNV ( blue ) . inset in ( A ) shows Guinier regions of respective scattering profiles with linear fit lines . dimensionless Kratky plot of ZIKV xrRNA1 ( red dashed ) was included in ( C ) for comparison , indicating that structures of DB12s of ZIKV WNV are well folded , but that of DB12 of DENV2 is partially folded . Kratky plots for two PK3 mutants of DB12s , ZIKV - DB12 - PK3 ∆ ( green ) WNV - DB12 - PK3 ∆ ( black ) , respectively , are also included for comparison , suggesting reduced compactness upon PK3 mutations .

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(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of DB12s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the Guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of DB12s of ZIKV and WNV are well folded, but that of DB12 of DENV2 is partially folded. The Kratky plots for the two PK3 mutants of DB12s, the ZIKV- DB12-PK3∆ (green) and WNV- DB12-PK3∆ (black), respectively, are also included for comparison, suggesting reduced compactness upon PK3 mutations.


( C ) E expression of ZIKV mutants showed by IFA .

(C) E protein expression of ZIKV and the mutants showed by IFA.


( D ) progeny in cultured supernatants of ZIKV mutants were detected by qRT - PCR in three technical replicates . Two - way ANOVA Dunnett ' s multiple comparison was used for statistical . error bars represent standard deviation . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(D) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT-PCR in three technical replicates. Two-way ANOVA and Dunnett's multiple comparison test was used for statistical analysis. The error bars represent the standard deviation. *** p<0.001, **** p<0.0001.


( E ) luciferase activity of ZIKV - Rep mutants were measured at different time points after transfection in three technical repelicates . error bars represent standard deviation .

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(E) The luciferase activity of the ZIKV-Rep and the mutants were measured at different time points after transfection in three technical repelicates. The error bars represent the standard deviation.


( A ) - ( C ) scattering profiles ( A ) , PDDFs ( B ) dimensionless Kratky plots ( C ) of 3 ' SLs from DENV2 ( cyan ) , ZIKV ( red ) , WNV ( blue ) WNV - 3 ' SLM ( 438UAG441A → 438GUC441U ) mutant ( green ) . inset in ( A ) shows guinier regions of respective scattering profiles with linear fit lines .

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(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of 3'SLs from DENV2 (cyan), ZIKV (red), WNV (blue) and WNV-3'SLM (438UAG441A→438GUC441U) mutant (green). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines.


( A ) - ( C ) fitting chi2 are plotted against ensemble for complete sfRNAs of DENV2 ( A ) , ZIKV ( B ) WNV ( C ) , suggesting optimized minimal structural ensembles with 3 , 3 , 3 conformers , respectively . respective insets show distribution of minimal ensembles initial pools of 10000 random conformers as a function of Rg .

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(A)-(C) The fitting chi2 are plotted against the ensemble size for complete sfRNAs of DENV2 (A), ZIKV (B) and WNV (C), suggesting optimized minimal structural ensembles with 3, 3 ,3 conformers, respectively. The respective insets show the distribution of the minimal ensembles and the initial pools of 10000 random conformers as a function of Rg.


( a ) Hiw - associated was purified by TAP ( see Online Methods ) analyzed by one - dimensional SDS - PAGE gel followed by Sypro Ruby . Mass spectrometry identified full - Hiw , β - tubulin , Rae1 Fsn , indicated by arrows .

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(a) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. Mass spectrometry identified the full-length Hiw, β-tubulin, Rae1 and Fsn, indicated by arrows.


( b ) Rae1 - associated purified from fly brains by TAP was analyzed by one - dimensional SDS - PAGE gel followed by Coomassie blue G - 250 . Mass spectrometry identified Hiw , Nup98 Rae1 , indicated by arrows .

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(b) The Rae1-associated complex purified from fly brains by TAP was analyzed by one-dimensional SDS-PAGE gel followed by Coomassie blue G-250 staining. Mass spectrometry identified Hiw, Nup98 and Rae1, indicated by arrows.


( c ) Larval brain lysate of wild - type ( WT ) , hiw null mutant ( hiwΔN ) Fsn mutant ( Fsnf06595 / Df ( 2R ) 7872 ) flies was subject to co - immunoprecipitation with antibody to Hiw ( Hiw2b ) . Both input immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1 . was also blotted with antibody to β - tubulin to ensure equal amount of total in input samples . Full - blots are presented in Supplementary Figure 8 .

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(c) Larval brain lysate of wild-type (WT), hiw null mutant (hiwΔN) and Fsn mutant (Fsnf06595/Df(2R)7872) flies was subject to co-immunoprecipitation with antibody to Hiw (Hiw2b). Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1. The membrane was also blotted with antibody to β-tubulin to ensure equal amount of total proteins in the input samples. Full-length blots are presented in Supplementary Figure 8.


( a ) gene structure of Rae1 . black - arrowed boxes indicate exons . A transposable element , P { GawB } NP3499 , located ∼ 400 bp upstream of first exon of Rae1 was used to generate imprecise excision mutants of Rae1 . PCR indicates that ∼ 900 bp a ∼ 1 . 5 - k bp fragment in Rae1 promoter region are missing in Rae1EX28 Rae1EXB12 , respectively . ( b ) Western blot on Rae1 in brain lysate of wild - type , Df ( 2R ) 5764 / + , Rae1EX28 / + , or late second instar Rae1EX28 , Rae1EX28 / Df ( 2R ) 5764 ( / Df ) Rae1EXB12 / Df ( 2R ) 5764 larvae . β - tubulin was used as loading control . Full - blots are presented in Supplementary Figure 9 . ( c ) of Rae1 in Rae1 heterozygous homozygous mutant brains . Rae1 were first normalized to β - tubulin are presented as of wild - type level . * P 0 . 05 , * * P 0 . 001 . Error bars denote s . e . m .

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

(a) The gene structure of Rae1. The black-arrowed boxes indicate exons. A transposable element, P{GawB}NP3499, located ∼400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1. PCR analysis indicates that ∼900 bp and a ∼1.5-k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12, respectively. (b) Western blot analysis on Rae1 protein in the brain lysate of wild-type, Df(2R)5764/+, Rae1EX28/+, or late second instar Rae1EX28, Rae1EX28/Df(2R)5764(/Df) and Rae1EXB12 /Df(2R)5764 larvae. β-tubulin was used as the loading control. Full-length blots are presented in Supplementary Figure 9. (c) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains. Rae1 protein levels were first normalized to β-tubulin protein levels and are presented as percentage of wild-type level. *P 0.05, **P 0.001. Error bars denote s.e.m.


( a ) Representative confocal of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) FasII ( red ) , in late 2nd / early 3rd instar wild - type , Rae1EX28 , Rae1EX28 presynaptic rescue ( Rae1EX28 ; elav - Gal4 / UAS - NTAP - Rae1 ) Rae1EX28 wallenda suppression ( Rae1EX28 ; wnd3 ) larvae . Scale bar represents 10 μm . ( b , c ) of bouton number ( b ) ( c ) in wild - type , Rae1EX28 / Df , Rae1EX28 , Rae1EX28 rescue , Rae1EX28 ; wnd3 suppression hiwΔN larval NMJs ( segment A3 muscle 6 / 7 ; n = 20 , 21 , 18 , 20 , 20 11 , respectively , for bouton number ; n = 271 , 465 , 548 , 726 , 486 826 , respectively , for bouton ) . There was no significant difference in either bouton number or bouton between Rae1EX28 Rae1EX28 / Df ( P > 0 . 1 for both comparisons ) . * P 0 . 001 . Error bars denote s . e . m .

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

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae. Scale bar represents 10 μm. (b,c) Quantification of bouton number (b) and size (c) in wild-type, Rae1EX28/Df, Rae1EX28, Rae1EX28 rescue, Rae1EX28; wnd3 suppression and hiwΔN larval NMJs (segment A3 muscle 6/7; n = 20, 21, 18, 20, 20 and 11, respectively, for bouton number; n = 271, 465, 548, 726, 486 and 826, respectively, for bouton size). There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28/Df (P > 0.1 for both comparisons). *P 0.001. Error bars denote s.e.m.


( a ) Representative confocal of muscle 4 synapses stained for both DVGLUT ( green ) FasII ( red ) in wild - type , Rae1EX28 / + , hiwND51 , hiwND51 ; Rae1EX28 / + , hiwΔN hiwΔN ; Rae1EX28 / + larvae . Scale bar represents 10 μm . ( b ) of bouton number at muscle 4 NMJs in wild - type , Rae1EX28 / + , hiwND51 , hiwND51 ; Rae1EX28 / + , hiwΔN hiwΔN ; Rae1EX28 / + larvae ( n = 27 , 23 , 42 , 46 , 23 25 , respectively ) . There was a significant increase in number of boutons formed in hiwND51 ; Rae1EX28 / + compared with hiwND51 3rd instar larval NMJs ( * P 0 . 001 ) . number of boutons in hiwΔN ; Rae1EX28 / + double mutant was not significantly different from that in hiwΔN ( * * P > 0 . 5 ) , demonstrating a lack of enhancement of hiwΔN phenotype by Rae1EX28 / + . Error bars denote s . e . m .

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

(a) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae. Scale bar represents 10 μm. (b) Quantification of bouton number at muscle 4 NMJs in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae (n = 27, 23, 42, 46, 23 and 25 cells, respectively). There was a significant increase in the number of boutons formed in hiwND51; Rae1EX28/+ compared with hiwND51 3rd instar larval NMJs (*P 0.001). The number of boutons in the hiwΔN; Rae1EX28/+ double mutant was not significantly different from that in hiwΔN (**P > 0.5), demonstrating a lack of enhancement of the hiwΔN phenotype by Rae1EX28/+. Error bars denote s.e.m.


( a ) Schematic presentation of NTAP - tagged ( NT - ) HM - tagged ( HM - ) hiw transgenes encoding full - , mutated or truncated Hiw . Hiw functional domains are marked with colored boxes . positions of - acid substitution in NT - HiwΔRING added acid residue necessary to maintain reading frame in NT - Hiw - RCC1 NT - Hiw - PHR are indicated . right column summarizes ability of given hiw transgene to rescue synaptic overgrowth phenotype in hiw mutants , to cause dominant negative overgrowth phenotype when expressed in wild - type background to bind to Rae1 . ( b ) Western blot of fly brain lysate using antibody to TAP ( PAP , Sigma ) or Myc revealed expression of wild - type mutant hiw transgenes in predicted .

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

(a) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins. Hiw functional domains are marked with colored boxes. The positions of amino-acid substitution in NT-HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT-Hiw-RCC1 and NT-Hiw-PHR are indicated. The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants, to cause dominant negative overgrowth phenotype when expressed in wild-type background and to bind to Rae1. (b) Western blot analysis of fly brain lysate using antibody to TAP (PAP, Sigma) or Myc revealed the expression of wild-type and mutant hiw transgenes in predicted size.


( c ) Representative confocal of muscle 4 synapses stained for DVGLUT ( green ) FasII ( red ) in wild - type ( + / hiwND8 ; + / + ; GS - elav - Gal4 / + ) , NT - Hiw overexpression ( + / hiwND8 ; UAS - NT - hiw / + ; GS - elav - Gal4 / + ) , NT - Hiw - NT overexpression ( + / hiwND8 ; UAS - NT - hiw - NT / + ; GS - elav - Gal4 / + ) , NT - Hiw - CT1000 overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - CT1000 ) , NT - Hiw - PHR overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - PHR ) , HM - Hiw - HindIII overexpression ( + / hiwND8 ; UAS - HW - hiw - HindIII / + ; GS - elav - Gal4 / + ) , NT - Hiw - RCC1 overexpression ( NT - hiw - RCC1 / hiwND8 ; + / + ; GS - elav - Gal4 / + ) , or NT - Hiw - CT overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - CT ) 3rd larval NMJs . Overexpression is indicated by an up arrow . Scale bar represents 10 μm . ( d ) of number of boutons at muscle 4 NMJs in wild - type larvae or larvae overexpressing wild - type or mutant hiw transgenes ( n = 23 , 24 , 26 , 27 , 26 , 21 , 25 27 , respectively ; * P 0 . 001 ) . Error bars denote s . e . m .

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

(c) Representative confocal images of muscle 4 synapses stained for DVGLUT (green) and FasII (red) in wild-type (+/hiwND8; +/+; GS-elav-Gal4/+), NT-Hiw overexpression (+/hiwND8; UAS-NT-hiw/+; GS-elav-Gal4/+), NT-Hiw-NT overexpression (+/hiwND8; UAS-NT-hiw-NT/+; GS-elav-Gal4/+), NT-Hiw-CT1000 overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT1000), NT-Hiw-PHR overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-PHR), HM-Hiw-HindIII overexpression (+/hiwND8; UAS-HW-hiw-HindIII/+; GS-elav-Gal4/+), NT-Hiw-RCC1 overexpression (NT-hiw-RCC1/hiwND8; +/+; GS-elav-Gal4/+), or NT-Hiw-CT overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT) 3rd larval NMJs. Overexpression is indicated by an up arrow. Scale bar represents 10 μm. (d) Quantification of the number of boutons at muscle 4 NMJs in wild-type larvae or larvae overexpressing wild-type or mutant hiw transgenes (n = 23, 24, 26, 27, 26, 21, 25 and 27, respectively; *P 0.001). Error bars denote s.e.m.


( a ) Indicated NTAP - tagged hiw transgenes ( described in Fig . 5 ) a TAP only transgene were expressed in neurons under control of BG380 - Gal4 driver . Larval brain lysates from each sample were subject to IgG pulldown . Both inputs IgG pulldown complexes were analyzed by western blot using antibody to Rae1 . Rae1 was present in of pulldown complexes except for those from TAP only , NT - Hiw - CT1000 NT - Hiw - NT . Full - blots are presented in Supplementary Figure 10 . ( b ) of interaction between various Hiw transgenic Rae1 . Rae1 intensities in TAP pulldown blots were normalized to both intensities molecular weights of corresponding TAP pulldown .

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

(a) Indicated NTAP-tagged hiw transgenes (described in Fig. 5) and a TAP only transgene were expressed in neurons under the control of the BG380-Gal4 driver. Larval brain lysates from each sample were subject to IgG pulldown. Both the inputs and the IgG pulldown complexes were analyzed by western blot using antibody to Rae1. Rae1 was present in all of the pulldown complexes except for those from TAP only, NT-Hiw-CT1000 and NT-Hiw-NT. Full-length blots are presented in Supplementary Figure 10. (b) Quantification of the interaction between various Hiw transgenic proteins and Rae1. Rae1 intensities in TAP pulldown blots were normalized to both the intensities and molecular weights of the corresponding TAP pulldown proteins.


( c ) Representative confocal of muscle 4 synapses stained for both DVGLUT ( green ) FasII ( red ) in wild - type ( BG380 / Y ; + / + ; + / + ) , NT - Hiw - CT GAP - GFP ( BG380 / Y ; UAS - GAP - GFP / + ; UAS - NT - hiw - CT / + ) , NT - Hiw - CT Rae1 ( BG380 / Y ; + / + ; UAS - NT - hiw - CT / UAS - GFP - Rae1 ) larvae . Scale bar represents 10 μm . ( d ) of number of boutons at muscle 4 NMJs in wild - type , NT - Hiw - CT overexpression ( BG380 / Y ; + / + ; UAS - NT - hiw - CT / + ) , NT - Hiw - CT GAP - GFP , NT - Hiw - CT Rae1 ( n = 24 , 27 , 32 26 , respectively ) larvae . Error bars denote s . e . m . ; * P 0 . 001 .

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

(c) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type (BG380/Y;+/+; +/+), NT-Hiw-CT and GAP-GFP (BG380/Y; UAS-GAP-GFP/+; UAS-NT-hiw-CT/+), and NT-Hiw-CT and Rae1 (BG380/Y; +/+; UAS-NT-hiw-CT/UAS-GFP-Rae1) larvae. Scale bar represents 10 μm. (d) Quantification of number of boutons at muscle 4 NMJs in wild-type, NT-Hiw-CT overexpression (BG380/Y; +/+; UAS-NT-hiw-CT/+), NT-Hiw-CT and GAP-GFP, and NT-Hiw-CT and Rae1 (n = 24, 27, 32 and 26, respectively) larvae. Error bars denote s.e.m.; *P 0.001.


( a ) Neuronal expression of neither Hiw nor Rae1 rescues synaptic terminal overgrowth phenotype caused by loss of function of other gene . of number of boutons in wild - type , Rae1EX28 , Rae1EX28 ; Hiw rescue ( Res ) ( Rae1EX28 ; elav - Gal4 / UAS - GFP - hiw ) , hiwΔN hiwΔN ; Rae1 rescue ( hiwΔN ; elav - Gal4 / UAS - NTAP - Rae1 ) larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 7 11 , respectively ) . ( b ) Representative confocal of wild - type ( BG380 - Gal4 / + ; + / + ; UAS - GFP - hiw / + ) or Rae1 mutant ( BG380 - Gal4 / + ; Rae1EX28 ; UAS - GFP - hiw / + ) ventral ganglions with UAS - GFP - hiw trangene expressed in neurons . larvae were stained with antibodies to horseradish peroxidase ( HRP , red ) GFP ( green ) . Scale bar represents 10 μm .

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

(a) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene. Quantification of the number of boutons in wild-type, Rae1EX28, Rae1EX28; Hiw rescue (Res) (Rae1EX28; elav-Gal4/UAS-GFP-hiw), hiwΔN and hiwΔN; Rae1 rescue (hiwΔN; elav-Gal4/UAS-NTAP-Rae1) larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 7 and 11, respectively). (b) Representative confocal images of wild-type (BG380-Gal4/+; +/+; UAS-GFP-hiw/+) or Rae1 mutant (BG380-Gal4/+; Rae1EX28; UAS-GFP-hiw/+) ventral ganglions with UAS-GFP-hiw trangene expressed in neurons. The larvae were stained with antibodies to horseradish peroxidase (HRP, red) and GFP (green). Scale bar represents 10 μm.


( c ) Western blots of total extracted from wild - type , hiwΔN , Rae1EX28 Rae1EX28 rescue ( Rae1EX28 ; elav - Gal4 / UAS - NTAP - Rae1 ) larval brains probed with indicated antibodies . Full - blots are presented in Supplementary Figure 11 .

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

(c) Western blots of total proteins extracted from wild-type, hiwΔN, Rae1EX28 and Rae1EX28 rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) larval brains probed with indicated antibodies. Full-length blots are presented in Supplementary Figure 11.


( d ) Western blot on total extracted from hiwΔN , wild - type ( C155 - Gal4 / + ) Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) larval brains . Full - blots are presented in Supplementary Figure 12 . ( e ) of Hiw in wild - type Rae1 expression larval brains ( * P 0 . 001 , on seven independent experiments ) . Arrows indicate a 38 - kDa endogenous Rae1 arrowheads indicate 58 - kDa transgenic NTAP - Rae1 . Error bars denote s . e . m .

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

(d) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.


( a ) Representative confocal of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) FasII ( red ) , in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + wandering larvae . Scale bar represents 10 μm . ( b ) of number of boutons in wild - type , atg1 / + , atg2 / + , atg18 / + , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 12 , 12 , 16 , 12 21 , respectively ) .

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

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ wandering larvae. Scale bar represents 10 μm. (b) Quantification of the number of boutons in wild-type, atg1/+, atg2/+, atg18/+, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 12, 12, 16, 12 and 21, respectively).


( c ) Western blot on total extracted from wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval brains . neural form of β - catenin ( 82 kDa , see Online Methods ) was used as an internal control of neuronal . Full - blots are presented in Supplementary Figure 13 . ( d ) of Hiw in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval brains ( on six independent experiments ) .

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

(c) Western blot analysis on total proteins extracted from wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains. The neural form of β-catenin (82 kDa, see Online Methods) was used as an internal control of neuronal proteins. Full-length blots are presented in Supplementary Figure 13. (d) Quantification of Hiw protein levels in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains (based on six independent experiments).


( e ) Representative confocal of muscle 4synapses stained for both DVGLUT ( green ) FasII ( red ) in wandering larvae of wild - type ( C155 - Gal4 / + ) , Atg1Gap - GFP ( C155 - Gal4 / + ; UAS - Gap - GFP / + ; UAS - Atg16B / + ) , Atg1NTAP - Rae1 ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ; UAS - Atg1 / + ) . Scale bar represents 10 μm . ( f ) of number of boutons at muscle 4synapses in wild - type , Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) , Atg1Gap - GFP Atg1NTAP - Rae1 wandering larvae ( segment A2 - 4 , n = 28 , 27 , 33 34 , respectively ) . Error bars denote s . e . m . * P 0 . 001 , * * P 0 . 05 .

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

(e) Representative confocal images of muscle 4synapses stained for both DVGLUT (green) and FasII (red) in wandering larvae of wild-type (C155-Gal4/+), Atg1Gap-GFP (C155-Gal4/+; UAS-Gap-GFP/+; UAS-Atg16B/+), and Atg1NTAP-Rae1 (C155-Gal4/+; UAS-NTAP-Rae1/+; UAS-Atg1/+). Scale bar represents 10 μm. (f) Quantification of the number of boutons at muscle 4synapses in wild-type, Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+), Atg1Gap-GFP and Atg1NTAP-Rae1 wandering larvae (segment A2-4, n = 28, 27, 33 and 34, respectively). Error bars denote s.e.m. *P 0.001, **P 0.05.


a , TG in hepatocytes untreated ( None ) or treated with 3 - methyladenine ( 3MA ) cultured in regular medium ( RM ) or oleate ( OL ) ( * P 0 . 02 , * * P 0 . 002 , n = 3 ) .

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a, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).


b , TG in vector - infected ( VEC ) siAtg5 in RM , OL or in MCDM ( * P 0 . 001 , n = 5 ) . OL values are in mM .

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

b, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.


c , TG in wild - type ( WT ) or Atg5 knockout mice embryonic fibroblasts ( Atg5 - / - ) ( * P 0 . 01 or * * P 0 . 0001 , n = 5 ) .

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

c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).


d f , from a b stained with BODIPY 493 / 503 ( d ) . Nuclei are highlighted with 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; d ) . Error bars , s . e . m .

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

df, Cells from a and b stained with BODIPY 493/503 (d). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; d). Error bars, s.e.m.


d f , from a b stained with oil red O ( e ) or visualized by electron microscopy ( f ) . Right : quantifications of LD number ( * P 0 . 01 , * * P 0 . 001 with untreated wild - type ; # P 0 . 01 , # # P 0 . 001 with in RM ; n = 4 6 ) .

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

df, Cells from a and b stained with oil red O (e) or visualized by electron microscopy (f). Right: quantifications of LD number and size (*P  0.01, **P  0.001 with untreated wild-type cells; #P  0.01, # #P  0.001 with cells in RM; n = 46).


a , b , VEC siAtg5 cultured with oleate ( OL ) or in MCDM were examined for their rates of TG synthesis ( a ) β - oxidation ( b ) as compared to in regular medium alone ( * P 0 . 03 , * * P 0 . 004 with VEC in same medium , n = 3 4 ) .

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

a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34).