Figure 6Cell death measured using Annexin V ( AnnV ) - Glo assay of THP - 1 ∆ CASP1 cells transfected with non - targeting control ( CTRL ) or GBP1 siRNA and treated as indicated . Cells were primed with IFNγ , transfected with soluble Tg antigen ( STAg ) or both , or left untreated ( UT ) . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . AnnV - Glo assay from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 cells transfected with soluble Tg antigen ( STAg ) that was untreated ( UT ) or treated as indicated . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . Immunoblots from IFNγ - primed THP - 1 ∆ CASP - 1 or THP - 1 ∆ ASC cells transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA and then untreated ( - ) or transfected with STAg for 12 h . Images are representative of n = 2 independent experiments . AnnV - Glo assay from naïve or IFNγ - primed THP - 1 ∆ CASP - 1 cells transfected with non - targeting control ( CTRL ) , GBP1 , AIM2 , CASP - 1 or CASP - 8 siRNA and then untreated ( UT ) or transfected with STAg as indicated for 18 h . Area under the curve ( AUC ) from real - time assay plotted as mean ± SEM from n = 3 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 8 activation . Propidium iodide ( PI ) uptake assay from MDMs transfected with non - targeting control ( CTRL ) or GBP1 siRNA and then left untreated ( UT ) , treated with LPS or transfection reagent ( Lipofectamine ) only or transfected with LPS for 4 h . Area under the curve ( AUC ) from real - time assays plotted as mean ± SEM from n = 4 independent experiments . Schematic on right shows an overview of pathways leading to caspase - 4 activation .

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Figure 6Cell death measured using Annexin V (AnnV)-Glo assay of THP-1 ∆CASP1 cells transfected with non-targeting control (CTRL) or GBP1 siRNA and treated as indicated. Cells were primed with IFNγ, transfected with soluble Tg antigen (STAg) or both, or left untreated (UT). Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with soluble Tg antigen (STAg) that was untreated (UT) or treated as indicated. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments.Immunoblots from IFNγ-primed THP-1∆CASP-1 or THP-1 ∆ASC cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (-) or transfected with STAg for 12 h. Images are representative of n = 2 independent experiments.AnnV-Glo assay from naïve or IFNγ-primed THP-1 ∆CASP-1 cells transfected with non-targeting control (CTRL), GBP1, AIM2, CASP-1 or CASP-8 siRNA and then untreated (UT) or transfected with STAg as indicated for 18 h. Area under the curve (AUC) from real-time assay plotted as mean ± SEM from n = 3 independent experiments. Schematic on right shows an overview of pathways leading to caspase-8 activation.Propidium iodide (PI) uptake assay from MDMs transfected with non-targeting control (CTRL) or GBP1 siRNA and then left untreated (UT), treated with LPS or transfection reagent (Lipofectamine) only or transfected with LPS for 4 h. Area under the curve (AUC) from real-time assays plotted as mean ± SEM from n = 4 independent experiments. Schematic on right shows an overview of pathways leading to caspase-4 activation.


Figure 1 ( A ) EAE severity in 6 - to - 8 - week - old female ( left panel ) and male ( right panel ) wild - type ( WT ) and ERα - / - mice ( n = 7 - 9 mice per group ) . ( B ) EAE sensitivity to IFNβ in 6 - to - 8 - week - old female ( top panel ) and male ( bottom panel ) WT and ERα - / - mice . Vehicle ( Veh ) or IFNβ ( 47 μg / kg ) were i . p . injected . every other day from 0 to 10 dpi ( n = 8 - 12 mice per group ) .

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Figure 1(A) EAE severity in 6-to-8-week-old female (left panel) and male (right panel) wild-type (WT) and ERα-/- mice (n=7-9 mice per group). (B) EAE sensitivity to IFNβ in 6-to-8-week-old female (top panel) and male (bottom panel) WT and ERα-/- mice. Vehicle (Veh) or IFNβ (47 μg/kg) were i.p. injected. every other day from 0 to 10 dpi (n= 8-12 mice per group).


Figure 2 ( D ) Sensitivity of EAE to IFNβ in Cyp19 - / - ovary - transferred female WT mice and WT ovary - transferred Cyp19 - / - mice ( n = 5 - 6 mice ) . ( E ) Estradiol levels in LNs ( A : axillary , B : branchial , C : cervical , and I : inguinal ) in sham - operated and OVX mice ( n = 5 mice per group ) .

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Figure 2(D) Sensitivity of EAE to IFNβ in Cyp19-/- ovary-transferred female WT mice and WT ovary-transferred Cyp19-/- mice (n=5-6 mice).(E) Estradiol levels in LNs (A: axillary, B: branchial, C: cervical, and I: inguinal) in sham-operated and OVX mice (n=5 mice per group).


Figure 3 ( A ) Brightfield micrograph of ventral horns of Golgi - Cox stained lumber ( L ) 5 spinal cord section of male WT and ERα - / - EAE mice at 33 dpi with quantitation of area stained in gray matter . The white dotted line indicates the division between white and gray matter , V . H . represents the ventral horn ( n = 5 mice per group ) . ( C ) Representative confocal reflection maximum intensity projection micrograph of Golgi - Cox stained dendrites in the lumbar ventral horn of male WT and ERα - / - mice at 33 dpi with quantitative analysis of dendrite spine density ( n = 4 - 5 mice per group , 20 dendrites analyzed per animal ) .

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Figure 3(A) Brightfield micrograph of ventral horns of Golgi-Cox stained lumber (L)5 spinal cord section of male WT and ERα-/- EAE mice at 33 dpi with quantitation of area stained in gray matter. The white dotted line indicates the division between white and gray matter, V.H. represents the ventral horn (n=5 mice per group).(C) Representative confocal reflection maximum intensity projection micrograph of Golgi-Cox stained dendrites in the lumbar ventral horn of male WT and ERα-/- mice at 33 dpi with quantitative analysis of dendrite spine density (n=4-5 mice per group, 20 dendrites analyzed per animal).


Figure 4 ( A ) Upregulated and downregulated genes in RNAseq analysis were evaluated by qPCR . Splenic CD4 + T cells from male WT EAE and ERα - / - EAE mice at 9 dpi were used ( n = 5 - 8 mice ) . ( D ) Neurite outgrowth of the neurons in Golgi - stained spinal sections of Tcra - / - recipient mice that received passive intrathecal injections of CD4 + splenic cells from male WT EAE and ERα - / - EAE mice at 14 dpi quantified using the Sholl technique . Sholl rings are separated by 10 μm ( n = 4 mice per group , 20 neurons analyzed per animal ) with an end radius of 100 μm .

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Figure 4(A) Upregulated and downregulated genes in RNAseq analysis were evaluated by qPCR. Splenic CD4+ T cells from male WT EAE and ERα-/- EAE mice at 9 dpi were used (n=5-8 mice).(D) Neurite outgrowth of the neurons in Golgi-stained spinal sections of Tcra-/- recipient mice that received passive intrathecal injections of CD4+ splenic cells from male WT EAE and ERα-/- EAE mice at 14 dpi quantified using the Sholl technique. Sholl rings are separated by 10 μm (n=4 mice per group, 20 neurons analyzed per animal) with an end radius of 100 μm.


Figure 5 ( C ) Neurite outgrowth of the neurons in Golgi - stained spinal sections of Tcra - / - recipient mice that received passive intrathecal injections of CD4 + splenic cells from male WT EAE and cERα - / - EAE mice at 14 dpi quantified using the Sholl analysis method . Sholl rings are separated by 10 μm ( n = 4 mice per group , 20 neurons analyzed per animal ) with an end radius of 100 μm .

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Figure 5(C) Neurite outgrowth of the neurons in Golgi-stained spinal sections of Tcra-/- recipient mice that received passive intrathecal injections of CD4+ splenic cells from male WT EAE and cERα-/- EAE mice at 14 dpi quantified using the Sholl analysis method. Sholl rings are separated by 10 μm (n=4 mice per group, 20 neurons analyzed per animal) with an end radius of 100 μm.


Figure 6 ( A ) Number of membrane - bound lymphotoxin ( mLT ) + DCs ( derived from spleen and LNs ) in male cERα - / - EAE mice at 9 dpi ( n = 5 mice per group ) . DC population were gated as Ly6G - and CD11c + ( EV Fig . 1 A ) . ( B ) Tuj1 - stained differentiated N2a cells co - cultured with nothing , non - treated CD4 + T cell , and rLT - treated CD4 + T cell ( 10 ng / ml ) for 24 h . Panels depict representative fields for each condition . Quantification of percentage differentiated neuron and average neurite length . The results are derived from at least six biological samples .

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Figure 6(A) Number of membrane-bound lymphotoxin (mLT)+ DCs (derived from spleen and LNs) in male cERα-/- EAE mice at 9 dpi (n=5 mice per group). DC population were gated as Ly6G- and CD11c+ (EV Fig.1 A).(B) Tuj1-stained differentiated N2a cells co-cultured with nothing, non-treated CD4+ T cell, and rLT-treated CD4+ T cell (10 ng/ml) for 24 h. Panels depict representative fields for each condition. Quantification of percentage differentiated neuron and average neurite length. The results are derived from at least six biological samples.


Figure 7 ( A ) Lta mRNA expression in BMDCs developed from bone marrows of WT and ERα - / - mice 3 h after treatment with LPS ( 100 ng / ml ) with vehicle ( Veh ) or PPT ( 1 µM ) pre - treatment for 18 h ( n = 4 samples per group ) . ( B ) Il1b and Ifnb mRNA expression 3 h after treatment with LPS ( 100 ng / ml ) with vehicle ( Veh ) or PPT ( 1 µM ) pre - treatment for 18 h ( n = 4 - 5 samples per group ) . ( C ) Representative Western blot of TRAF3 and HSC70 in WT BMDCs and ERα - / - BMDCs . TRAF3 expression in BMDCs following vehicle ( Veh ) or PPT ( 1 µM ) pre - treatment for 18 h was evaluated by normalization of HSC70 ( n = 5 - 7 per group ) . ( F ) Nuclear localization of IRF3 in BMDCs treated with vehicle or with LPS for 2 h prior upon a vehicle or PPT ( 1 µM ) pre - treatment for 18 h . The results are presented as a ratio of the nuclear IRF3 signal to the cytosol IRF3 signal ( n = 5 - 9 biological samples )

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Figure 7(A) Lta mRNA expression in BMDCs developed from bone marrows of WT and ERα-/- mice 3 h after treatment with LPS (100 ng/ml) with vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h (n=4 samples per group). (B) Il1b and Ifnb mRNA expression 3 h after treatment with LPS (100 ng/ml) with vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h (n=4-5 samples per group).(C) Representative Western blot of TRAF3 and HSC70 in WT BMDCs and ERα-/- BMDCs. TRAF3 expression in BMDCs following vehicle (Veh) or PPT (1 µM) pre-treatment for 18 h was evaluated by normalization of HSC70 (n=5-7 per group).(F) Nuclear localization of IRF3 in BMDCs treated with vehicle or with LPS for 2 h prior upon a vehicle or PPT (1 µM) pre-treatment for 18 h. The results are presented as a ratio of the nuclear IRF3 signal to the cytosol IRF3 signal (n=5-9 biological samples)


Figure 1E . Validation of individual targets by manually curated in vitro ubiquitylation experiments . HA - tagged ( hemagglutinin ) candidate proteins were purified from Hek293T cells via anti - HA immunoprecipitation ( IP ) followed by HA - peptide elution . Candidates were incubated with 20 µM His6 - Ubiquitin , 170 nM E1 , E2s ( 0 . 5 µM each of UbcH5b and Cdc34 ) and 5 mM ATP in the presence or absence of 0 . 1 µM SCFFbxw5 for 2 hours at 37 ° C . Substrates were detected via SDS - PAGE followed by western blotting using anti - HA antibodies for detection .

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Figure 1E. Validation of individual targets by manually curated in vitro ubiquitylation experiments. HA-tagged (hemagglutinin) candidate proteins were purified from Hek293T cells via anti-HA immunoprecipitation (IP) followed by HA-peptide elution. Candidates were incubated with 20 µM His6-Ubiquitin, 170 nM E1, E2s (0.5 µM each of UbcH5b and Cdc34) and 5 mM ATP in the presence or absence of 0.1 µM SCFFbxw5 for 2 hours at 37°C. Substrates were detected via SDS-PAGE followed by western blotting using anti-HA antibodies for detection.


Figure 2E . Co - IP of HA - Kif2a , HA - Kif2b or HA - MCAK with Flag - Fbxw5F . In vitro pull - down experiment as in B . Asterisk indicates an unspecific protein from E . coli . Note : Kif2b purification from Sf21 cells was much less efficient and it was therefore not included in the experiment .

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Figure 2E. Co-IP of HA-Kif2a, HA-Kif2b or HA-MCAK with Flag-Fbxw5F. In vitro pull-down experiment as in B. Asterisk indicates an unspecific protein from E.coli. Note: Kif2b purification from Sf21 cells was much less efficient and it was therefore not included in the experiment.


Figure 3D . In vitro ubiquitylation experiment as in B . Blots 1 & 2 : Plk1 or RanGAP1 used as substrates instead of MCAK . Blot 3 : Left : Experiment as in B . Asterisks indicate signals from Plk1 antibody ( same species as anti - Fbxw5 ) . Right : Same reaction except that Cul1 ~ Nedd8 / Rbx1 complexes were replaced by Cul4A ~ Nedd8 / DDB1 / Rbx1 complexes Bottom blots : Same blots as above incubated with anti - Fbxw5 antibodies . Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex . G . In vitro ubiquitylation experiment as in B using either wild type , K48R , K63R , KK48 , 63RR or methylated ubiquitin ( Kmethyl , see Fig EV2K ) . Top : Cdc34 used as E2 . Bottom : UbcH5b used as E2 .

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Figure 3D. In vitro ubiquitylation experiment as in B. Blots 1&2: Plk1 or RanGAP1 used as substrates instead of MCAK. Blot 3: Left: Experiment as in B. Asterisks indicate signals from Plk1 antibody (same species as anti-Fbxw5). Right: Same reaction except that Cul1~Nedd8/Rbx1 complexes were replaced by Cul4A~Nedd8/DDB1/Rbx1 complexes Bottom blots: Same blots as above incubated with anti-Fbxw5 antibodies. Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex.G. In vitro ubiquitylation experiment as in B using either wild type, K48R, K63R, KK48,63RR or methylated ubiquitin (Kmethyl, see Fig EV2K). Top: Cdc34 used as E2. Bottom: UbcH5b used as E2.


Figure 4B . Quantification of the MCAK / Uba2 signal ratio normalised to each non - targeting control of 4 independent experiments . Error bars indicate standard deviation , asterisks the p - value of a two - tailed unpaired Student ' s t - test comparing each Fbxw5 - directed siRNA sample with the according non - targeting control ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . C . RPE - 1 cells were transfected with the indicated siRNAs ( 72 hours total time ) , split on coverslips 24 hours later and serum - starved for the last 24 hours . Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining . Left : Maximum intensity projections ( same for all following microscopy images ) of representative images . Arrows indicate ODF2 signal . Scale bar = 5 µm . Note : Middle panel shows an example image of siFw5 - 3 . Right : Quantification of MCAK signals co - localising with ODF2 . Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells . Asterisks indicate p - value of a Mann Whitney test comparing each sample set with the non - targeting control ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Figure 4B. Quantification of the MCAK/Uba2 signal ratio normalised to each non-targeting control of 4 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test comparing each Fbxw5-directed siRNA sample with the according non-targeting control (*P < 0.05, **P < 0.01, ***P < 0.001).C. RPE-1 cells were transfected with the indicated siRNAs (72 hours total time), split on coverslips 24 hours later and serum-starved for the last 24 hours. Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining. Left: Maximum intensity projections (same for all following microscopy images) of representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Note: Middle panel shows an example image of siFw5-3. Right: Quantification of MCAK signals co-localising with ODF2. Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells. Asterisks indicate p-value of a Mann Whitney test comparing each sample set with the non-targeting control (**P < 0.01, ***P < 0.001).


Figure 5A . Fluorescence - based pulse - chase experiment using a monoclonal RPE - 1 cell line expressing mNG - MCAK under a doxycycline - inducible promoter . Cells were transfected with the indicated siRNAs ( siFw5 - 1 & 3 together ) and split 24 hours later on Ibidi 8 Well Glass Bottom µ - slides while simultaneously inducing mNG - MCAK expression with 6 ng / ml doxycycline ( pulse ) . 24 hours later , doxycycline was washed out and cells were released into serum - free medium . Cells that have just finished or were about to finish mitosis ( distinguishable for example by the midbody signal of mNG - MCAK ( arrow heads ) ) were selected and imaged over 24 hours , taking an image every 20 min ( chase ) . Left : representative images of selected time points . Scale bar = 10 µm . Arrows indicate centrosomal mNG - MCAK signals , arrowheads mNG - MCAK signals at the midbody . Right : Quantification of centrosomal mNG - MCAK signals . Error bars show standard error of the mean of 3 independent experiments with 25 cells in total . P - value of a two - tailed unpaired Students t - test comparing signal intensity of Fbxw5 knockdown over non - targeting control was mostly below 0 . 01 for time points 0 to 8 hours and below 0 . 05 for time points 9h to 20h . Dark and light blue lines indicate the time points at which mNG - MCAK signals are reduced by 50 % compared to time point 0 for siNT and siFbxw5 samples , respectively . D . Same samples as in C , except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence . Left : Representative images . Arrows indicate ODF2 signal . Scale bar = 5 µm . Right : Quantification . Error bars show standard error of the mean of 3 independent experiments , covering in total more than 150 cells . Asterisks indicate the p - value of a Mann Whitney test ( * * * P < 0 . 001 ) .

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Figure 5A. Fluorescence-based pulse-chase experiment using a monoclonal RPE-1 cell line expressing mNG-MCAK under a doxycycline-inducible promoter. Cells were transfected with the indicated siRNAs (siFw5-1&3 together) and split 24 hours later on Ibidi 8 Well Glass Bottom µ-slides while simultaneously inducing mNG-MCAK expression with 6 ng/ml doxycycline (pulse). 24 hours later, doxycycline was washed out and cells were released into serum-free medium. Cells that have just finished or were about to finish mitosis (distinguishable for example by the midbody signal of mNG-MCAK (arrow heads)) were selected and imaged over 24 hours, taking an image every 20 min (chase). Left: representative images of selected time points. Scale bar = 10 µm. Arrows indicate centrosomal mNG-MCAK signals, arrowheads mNG-MCAK signals at the midbody. Right: Quantification of centrosomal mNG-MCAK signals. Error bars show standard error of the mean of 3 independent experiments with 25 cells in total. P-value of a two-tailed unpaired Students t-test comparing signal intensity of Fbxw5 knockdown over non-targeting control was mostly below 0.01 for time points 0 to 8 hours and below 0.05 for time points 9h to 20h. Dark and light blue lines indicate the time points at which mNG-MCAK signals are reduced by 50% compared to time point 0 for siNT and siFbxw5 samples, respectively.D. Same samples as in C, except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence. Left: Representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Right: Quantification. Error bars show standard error of the mean of 3 independent experiments, covering in total more than 150 cells. Asterisks indicate the p-value of a Mann Whitney test (***P < 0.001).


Figure 6B . CHX chase experiment as in A , except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h , released for 9 hours and again arrested for 16h . Following the 2nd release , 50 µg / ml CHX was added as indicated and samples were analysed as in A . Left : representative blots . Asterisks indicate unspecific bands detected by the Fbxw5 antibody . Right : Quantification of MCAK / Uba2 signal intensity ratios normalised to time point zero . Error bars show standard deviation of 6 independent experiments and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) . C . Extracts of RPE - 1 cells treated with the indicated siRNAs for 72 hours , grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO ( - ) or 10 µM MG - 132 ( + ) for the very last 6 hours . Left : Representative blots . Asterisk indicates an unspecific band detected by the Fbxw5 antibody . Right : Quantification of MCAK / Uba2 signal ratio normalised to non - targeting control with DMSO of 6 independent experiments . Error bars indicate standard deviation , asterisks the p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

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

Figure 6B. CHX chase experiment as in A, except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h, released for 9 hours and again arrested for 16h. Following the 2nd release, 50 µg/ml CHX was added as indicated and samples were analysed as in A. Left: representative blots. Asterisks indicate unspecific bands detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 6 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).C. Extracts of RPE-1 cells treated with the indicated siRNAs for 72 hours, grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO of 6 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


Figure 7A . Polyclonal RPE - 1 cells expressing mNG - MCAK under a doxycycline - inducible promoter were seeded on coverslips , induced with 20 ng / ml doxycycline and grown for 24 hours . Subsequently , cells were washed twice with PBS and incubated for another 24 hours in serum - free medium without doxycycline . Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining . Left : Representative images . Scale bar = 10 µm . Right : Quantification of ciliated cells . Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) . C . RPE - 1 cells were treated with the indicated siRNA for 48 hours followed by serum - starvation for another 24 hours . Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining . Left : Representative images . Note : Left panel shows an example image of siFw5 - 1 . Scale bar = 10 µm . Right : Quantification of ciliated cells . Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Figure 7A. Polyclonal RPE-1 cells expressing mNG-MCAK under a doxycycline-inducible promoter were seeded on coverslips, induced with 20 ng/ml doxycycline and grown for 24 hours. Subsequently, cells were washed twice with PBS and incubated for another 24 hours in serum-free medium without doxycycline. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).C. RPE-1 cells were treated with the indicated siRNA for 48 hours followed by serum-starvation for another 24 hours. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Note: Left panel shows an example image of siFw5-1. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).


Figure 1E . RT - qPCR of Axin2 in bEnd . 3 cells treated with NDP ( 200 ng / ml ) , isotype control or F4L5 . 13 ( 1200 ng / ml ) and transfected with control , Tspan12 or Fzd4 siRNAs . Data are presented as mean ± SD , n = 3 technical replicates . Data is representative of two independent experiments . F . Time course of phosphorylated Disheveled - 3 ( p - DVL3 ) and ßcatenin protein levels in bEnd . 3 cells treated with 30nM of F4L5 . 13 or NDP . Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß - Tubulin measured by densitometry of independent experiments . Data are presented as mean ± SEM , n = 4 - 6 ( * p < 0 . 05 as compared with NT ) . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test ( * p < 0 . 05 as compared with NT ) .

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Figure 1E. RT-qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n=3 technical replicates. Data is representative of two independent experiments. F. Time course of phosphorylated Disheveled-3 (p-DVL3) and ßcatenin protein levels in bEnd.3 cells treated with 30nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n=4-6 (*p<0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared with NT).


Figure 2A . Volcano plot of gene expression changes in bEnd . 3 cells following 24h of treatment with 30nM F4L5 . 13 . Genes with significant changes in expression level are highlighted in blue and genes co - regulated by treatment with 30nM NDP for 24h are highlighted in red . Statistical analysis was performed using the DESeq2 R package and adjusted p - value ( Wald test ) threshold less than 0 . 1 was used . Labelled genes are associated with enriched GO biological processes indicated below .

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Figure 2A. Volcano plot of gene expression changes in bEnd.3 cells following 24h of treatment with 30nM F4L5.13. Genes with significant changes in expression level are highlighted in blue and genes co-regulated by treatment with 30nM NDP for 24h are highlighted in red. Statistical analysis was performed using the DESeq2 R package and adjusted p-value (Wald test) threshold less than 0.1 was used. Labelled genes are associated with enriched GO biological processes indicated below.


Figure 3B . Immunofluorescence of ZO - 1 , CLDN3 and CLDN5 localization at bEnd . 3 cell junctions . bEnd . 3 cells were treated or not with 30 nM F4L5 . 13 or 30 nM Norrin ( NDP ) with or without 100 ng / ml VEGF for 1h . ZO - 1 is shown in green , CLDN3 / 5 in red and DAPI in blue . Scale bars : 15 µm . C . Quantification of ZO - 1 , CLDN3 and CLDN5 fluorescence intensity . Each column represents 40 measurements of fluorescence intensity per condition ( y - axis ) . Data are presented as mean ± SEM . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test ( * p < 0 . 05 as compared to VEGF treatment ) . D

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Figure 3B. Immunofluorescence of ZO-1, CLDN3 and CLDN5 localization at bEnd.3 cell junctions. bEnd.3 cells were treated or not with 30 nM F4L5.13 or 30 nM Norrin (NDP) with or without 100 ng/ml VEGF for 1h. ZO-1 is shown in green, CLDN3/5 in red and DAPI in blue. Scale bars: 15 µm. C. Quantification of ZO-1, CLDN3 and CLDN5 fluorescence intensity. Each column represents 40 measurements of fluorescence intensity per condition (y-axis). Data are presented as mean ± SEM. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (*p<0.05 as compared to VEGF treatment). D


Figure 4C . IB4 - stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5 . 13 treatment . Boxed areas are shown enlarged in the panels on the right . Scale bars : 500 µm . D . Quantification of the vascularized area in the outer plexiform layer . 10 mg / kg F4L5 . 13 or isotype control antibody was injected every 48h from P5 to P20 . Data are presented as mean ± SD , n = 5 retinas ( 2 - 4 fields of view per retina ) from 4 - 5 mice per group . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test . E E . F4L5 . 13 rescues barrier function defects in Tspan12 - / - mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP . IB4 - Alexa647 was used to stain ECs . Scale bars : 100 µm .

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Figure 4C. IB4-stained retinal cross sections show the restored retinal vasculature and the absence of misdirected angiogenesis after F4L5.13 treatment. Boxed areas are shown enlarged in the panels on the right. Scale bars: 500 µm. D. Quantification of the vascularized area in the outer plexiform layer. 10 mg/kg F4L5.13 or isotype control antibody was injected every 48h from P5 to P20. Data are presented as mean± SD, n=5 retinas (2-4 fields of view per retina) from 4-5 mice per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test. E E. F4L5.13 rescues barrier function defects in Tspan12-/- mice as shown by an increased expression of the tight junction component CLDN5 and decreased expression of the EC fenestration component PLVAP. IB4-Alexa647 was used to stain ECs. Scale bars: 100 µm.


Figure 5D . Flat - mounted retinas of adult Tspan12 - / - mice injected systemically with sulfo - NHS - biotin show that F4L5 . 13 partially restored BRB function . PECAM was used to stain endothelial cells . Images are representative of 4 retinas per group . Scale bar : 500 µm . E . Maximum intensity projection of adult Tspan12 - / - retinas after mice were injected with F4L5 . 13 or isotype control . Endothelial cells were stained with IB4 - Alexa 647 . Images are representative of 4 retinas per group . Scale bar : 100 µm .

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Figure 5D. Flat-mounted retinas of adult Tspan12-/- mice injected systemically with sulfo-NHS-biotin show that F4L5.13 partially restored BRB function. PECAM was used to stain endothelial cells. Images are representative of 4 retinas per group. Scale bar: 500 µm.E. Maximum intensity projection of adult Tspan12-/- retinas after mice were injected with F4L5.13 or isotype control. Endothelial cells were stained with IB4-Alexa 647. Images are representative of 4 retinas per group. Scale bar: 100 µm.


Figure 6A . Schematic diagram of the OIR model ( top ) and representative images of P17 OIR retinas are shown ( bottom ) . Neonatal mice were exposed to 75 % oxygen from P7 to P12 to induce vessel loss . Then mice received intravitreal injections of PBS vehicle , mouse - specific anti - VEGF ( as a positive control ) delivered at 0 . 1 μg / μl , or F4L5 . 13v2 ( a second - generation F4L5 . 13 modality , in which the N - terminal FZD4 - specific diabody was replaced with a FZD4 - specific Fab ) at 50nM or 500nM target vitreous concentrations . Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17 . At P17 , retinas from injected mice were collected , dissected as flat ‐ mounts and stained with Isolectin B4 . Scale bar = 500 µm . B . Quantification of the ratio of neovascular and avascular area on total area of vascularization . Data are presented as mean ± SEM , n = 11 - 13 retinas per group . Significance was calculated by one - way ANOVA with Bonferroni ' s multiple comparisons test ( indicated p - values are in comparison to Vehicle ) . E

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

Figure 6A. Schematic diagram of the OIR model (top) and representative images of P17 OIR retinas are shown (bottom). Neonatal mice were exposed to 75% oxygen from P7 to P12 to induce vessel loss. Then mice received intravitreal injections of PBS vehicle, mouse-specific anti-VEGF (as a positive control) delivered at 0.1 μg/μl, or F4L5.13v2 (a second-generation F4L5.13 modality, in which the N-terminal FZD4-specific diabody was replaced with a FZD4-specific Fab) at 50nM or 500nM target vitreous concentrations. Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17. At P17, retinas from injected mice were collected, dissected as flat‐mounts and stained with Isolectin B4. Scale bar = 500 µm. B. Quantification of the ratio of neovascular and avascular area on total area of vascularization. Data are presented as mean ± SEM, n=11-13 retinas per group. Significance was calculated by one-way ANOVA with Bonferroni's multiple comparisons test (indicated p-values are in comparison to Vehicle). E


Figure 2A stable inducible HEK - 293T cell line expressing myc - PP2AC ( panel A were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2AC affinity purified , respectively . The binding of the indicated proteins was analysed by Western blotting A ) representative of 3 independent experimentsA stable inducible HEK - 293T cell line expressing myc - PP2AC ( panel B ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2AC by quantitative mass spectrometry ( B ) . B ) analysis of a biological triplicate . a stable inducible HeLa cell line expressing myc - PP2R1A ( C ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2R1A affinity purified , respectively . The binding of the indicated proteins was analysed by Western blotting C ) representative of 2 technical repeatsD ) HEK - 293T cells were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes . Bound complexes were analysed by mass spectrometry to identify PP2A components . Analysis conducted on a biological triplicate . E ) Doxycyclin - inducible CRISPR - Cas9 HeLa cell lines allow for depletion of specific B56 subunits . Growth assays in the presence of the indicated concentrations of DT - 061 or iHAP1 measured after 12 days of treatment . Mean and SD of three independent experiments are shown .

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Figure 2A stable inducible HEK-293T cell line expressing myc-PP2AC (panel A were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting A) representative of 3 independent experimentsA stable inducible HEK-293T cell line expressing myc-PP2AC (panel B) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC by quantitative mass spectrometry (B). B) analysis of a biological triplicate.a stable inducible HeLa cell line expressing myc- PP2R1A (C) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2R1A affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting C) representative of 2 technical repeatsD) HEK-293T cells were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes. Bound complexes were analysed by mass spectrometry to identify PP2A components. Analysis conducted on a biological triplicate.E) Doxycyclin-inducible CRISPR-Cas9 HeLa cell lines allow for depletion of specific B56 subunits. Growth assays in the presence of the indicated concentrations of DT-061 or iHAP1 measured after 12 days of treatment. Mean and SD of three independent experiments are shown.


Figure 3A ) Schematic of the experimental setup and results of CRISPR screen . Genes were ranked based on their normZ score . The top hits which were further analysed are highlighted . B ) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay . Cells were treated with DMSO ( Ctr ) or DT - 061 5 . 5 µM or iHAP1 0 . 5 µM . C ) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5 . 5 μM DT - 061 or 0 . 5 μM iHAP1 treatment . Data information : B - C ) Mean and SD of three independent experiments is shown .

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Figure 3A) Schematic of the experimental setup and results of CRISPR screen. Genes were ranked based on their normZ score. The top hits which were further analysed are highlighted.B) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay. Cells were treated with DMSO (Ctr) or DT-061 5.5 µM or iHAP1 0.5 µM. C) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5.5 μM DT-061 or 0.5 μM iHAP1 treatment. Data information: B-C) Mean and SD of three independent experiments is shown.


Figure 4A ) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics . Taxol and Nocodazole served as controls for the experimental setup . The assay was performed in the presence of the indicated drug concentrations . Mean and SEM are shown from three to six independent experiments . B ) Representative colour - coded temporal projection of U2OS EB1 - GFP cells following indicated treatment . Scale bar 10µm . C ) Quantification of microtubule dynamics acquired from manual tracking of EB1 - GFP comets as indicators for MT growth after drug treatment . The mean and SD are plotted from three independent experiments ( n = 10 cells per experiment ) . Data information : P - values were calculated using Student ' s t - test or Mann - Whitney U - test ( unpaired , two - tailed ) . ns , not significant , * * * * p < 0 . 0001 . D ) Representative temporal projection of mitotic spindles of U2OS EB1 - GFP cells following specified treatment . DNA counterstained with SiR - DNA shown in cyan in merged images . Scale bar 10µm . E ) Analysis of number of EB1 - GFP comets per mitotic spindle . The mean and SD are plotted from three independent experiments ( Total number of cells = 26 ( DMSO ) , 20 ( 30µM DT - 061 ) , 27 ( DMSO + APC / C in . ) and , 26 ( 2µM iHAP1 + APC / C in ) . Data information : P - values were calculated using Student ' s t - test or Mann - Whitney U - test ( unpaired , two - tailed ) . ns , not significant , * * * * p < 0 . 0001 .

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Figure 4A) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics. Taxol and Nocodazole served as controls for the experimental setup. The assay was performed in the presence of the indicated drug concentrations. Mean and SEM are shown from three to six independent experiments.B) Representative colour-coded temporal projection of U2OS EB1-GFP cells following indicated treatment. Scale bar 10µm.C) Quantification of microtubule dynamics acquired from manual tracking of EB1-GFP comets as indicators for MT growth after drug treatment. The mean and SD are plotted from three independent experiments (n = 10 cells per experiment). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.D) Representative temporal projection of mitotic spindles of U2OS EB1-GFP cells following specified treatment. DNA counterstained with SiR-DNA shown in cyan in merged images. Scale bar 10µm.E) Analysis of number of EB1-GFP comets per mitotic spindle. The mean and SD are plotted from three independent experiments (Total number of cells = 26 (DMSO), 20 (30µM DT-061), 27 (DMSO+APC/C in.) and, 26 (2µM iHAP1+APC/C in). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.


Figure 5A ) Measurement of time spent in mitosis by time - lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis . n indicates the number of cells analyzed from at least 2 independent experiments . Schematic of experimental protocol on the top . Cells were treated with iHAP1 when indicated at the concentration stated in the figure . B ) Representative stills from time - lapse imaging experiments depicting the two phenotypes . Scale bar represents 20 µm .

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Figure 5A) Measurement of time spent in mitosis by time-lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis. n indicates the number of cells analyzed from at least 2 independent experiments. Schematic of experimental protocol on the top. Cells were treated with iHAP1 when indicated at the concentration stated in the figure. B) Representative stills from time-lapse imaging experiments depicting the two phenotypes. Scale bar represents 20 µm.


Figure 6A ) Time - lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus ( green ) ( n = 87 of cells from 2 experiments analysed all showing dispersal of marker ) . At time 0 , DT - 061 was added . On the right - hand side , the co - localisation of DT - 061 ( red ) and the Golgi marker is shown . B ) As in A ) but with a marker for ER and either with the addition of DT - 061 alone or DT - 061 + okadaic acid ( 50 nM ) Data information : Experiments were repeated at least twice and stills show a representative cell for each treatment condition . Cells were treated with DT - 061 at the concentration stated in the figure . A - B ) Scale bar indicates 20 µm . C ) Time required for ER signal translocation into the nucleus after drug treatment . Number of cells analysed per condition shown on top and median indicated by red line . Data information : Experiments were repeated at least twiceD ) Percentage of cells with ER signal translocation into the nucleus after drug treatment . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

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

Figure 6A) Time-lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus (green) (n= 87 of cells from 2 experiments analysed all showing dispersal of marker). At time 0, DT-061 was added. On the right-hand side, the co-localisation of DT-061 (red) and the Golgi marker is shown. B) As in A) but with a marker for ER and either with the addition of DT-061 alone or DT-061 + okadaic acid (50 nM) Data information: Experiments were repeated at least twice and stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.C) Time required for ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top and median indicated by red line. Data information: Experiments were repeated at least twiceD) Percentage of cells with ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


Figure 7A - B ) Time - lapse imaging of H358 cells expressing a marker for the Golgi ( A ) or the ER ( B ) . Cells were treated with DT - 061 at time 0 . ( B , low panel ) . For A ) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker . Arrow indicates addition of compound . Data information : Experiments were repeated at least twice except filming of the Golgi marker which was done once . Stills show a representative cell for each treatment condition . Cells were treated with DT - 061 at the concentration stated in the figure . A - B ) Scale bar indicates 20 µm . C ) Time needed for ER translocation into the nucleus after drug treatment . Median ( red line ) : 160 min . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least twiceD ) Percentage of cells with ER nuclear translocation after drug treatment . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

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

Figure 7A-B) Time-lapse imaging of H358 cells expressing a marker for the Golgi (A) or the ER (B). Cells were treated with DT-061 at time 0. (B, low panel). For A) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker. Arrow indicates addition of compound. Data information: Experiments were repeated at least twice except filming of the Golgi marker which was done once. Stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.C) Time needed for ER translocation into the nucleus after drug treatment. Median (red line): 160 min. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twiceD) Percentage of cells with ER nuclear translocation after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


Figure 8B ) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine . MCF7 cells were co - treated with 15 μM DT - 061 , 1 . 5 μM iHAP1 , 3 µg / mL Brefeldin A , or vehicle ( DMSO ) . The determined molar quantities of sphingolipid classes were normalized to that of PC . The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Cer : ceramide , HexCer : hexosylceramide , PC : phosphatidylcholine , and SM : sphingomyelin .

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

Figure 8B) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine. MCF7 cells were co-treated with 15 μM DT-061, 1.5 μM iHAP1, 3 µg/mL Brefeldin A, or vehicle (DMSO). The determined molar quantities of sphingolipid classes were normalized to that of PC. The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t-test. *p<0.05, **p<0.01, ***p<0.001. Cer: ceramide, HexCer: hexosylceramide, PC: phosphatidylcholine, and SM: sphingomyelin.


Figure 1Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton - S control . 24 - hour - sleep curve ( y - axis was average sleep time in every 30 minutes ) , n = 16 . Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118 . 24 - hour - sleep curve , n = 16 .

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Figure 1Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. 24-hour-sleep curve (y-axis was average sleep time in every 30 minutes), n=16. Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. 24-hour-sleep curve, n=16.


O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Rescue experiment of stx mutant. The stx mutant phenotype can be rescued by stx over expression driven by stx-Gal4 (in female flies, P) or EB1-Gal4 (Q). For P, from left to right females mean±SEM: 226.0±11.76, n=49; 336.6±14.10, n=86; 382.7±16.43, n=50; 357.8±16.55, n=72; 296.2±16.04, n=68; 484.9±12.97, n=49; 507.6±13.83, n=86; 539.5±12.99, n=50. 577.0±11.34, n=72; 520.7±14.96, n=68; For Q, from left to right mean±SEM; 398.3±9.535, n=63; 378.4±23.78, n=32; 324.4±12.33, n=45; 495.8±16.43, n=28; 478.5±14.65, n=53; 328.7±16.99, n=34; 540.7±7.690, n=63; 489.9±21.39, n=32; 490.9±13.65, n=45; 494.7±11.99, n=28; 537.8±12.07, n=53; 450.3±15.47, n=34.


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

Figure 3Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. Quantitative RT-PCR of Octβ2R in adult head of stxd77 and w1118 control. (Data represent mean±SEM: w1118, 0.8853±0.1177; N=3; stxd77/Y, 0.3263±0.1575；N=3).Rescue of stxd77 by over expressing Octβ2R in ellipsoid body. From left to right mean±SEM: 399.6±11.49, n=50; 400.3±10.48, n=60; 349.9±14.77, n=48; 495.8±16.43, n=28; 470.1±13.04, n=44; 423.6±14.32, n=47; 548.6±8.707, n=50; 499.0±8.225, n=60; 490.7±16.26, n=48; 494.7±11.99, n=28; 480.2±14.64, n=44; 437.4±14.85, n=47.Rescue of stxd77 by Pc RNAi in ellipsoid body. From left to right mean±SEM: 399.9±11.88, n=48; 409.8±11.50, n=49; 348.7±20.19, n=32; 495.8±16.43, n=28; 459.3±14.78, n=61; 407.5±15.66, n=40; 550.7±8.881, n=48; 516.0±11.76, n=49; 502.3±15.08, n=32; 494.7±11.99, n=28; 546.3±11.35, n=61; 514.2±18.94, n=40. Note that the same data is used for genotype stxd77;; EB1-Gal4 in panel F and G.


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

Figure 4Sleep phenotype of OctβR double and triple mutants. (From left to right mean±SEM: 392.0±12.91, n=60; 395.1±8.56, n=79; 433.7±13.03, n=58; 422.8±10.34; n=62; 474.4±8.36, n=73; 517.4±6.30, n=119; 513.2±7.10, n=79; 536.6±9.05 n=78; 499.1±11.95, n=60; 546.0±8.51, n=79; 519.6±10.31, n=58; 518.9±13.13; n=62; 584.3±8.59, n=73; 579.2±8.35, n=119; 560.9±6.80, n=79; 606.4±6.38, n=78).


Figure 5Quantitative RT - PCR of stx after OA treatment in w1118 control and Octβ1R , Octβ2R mutants , Octβ3R mutants . ( From left to right mean ± SEM : 1 . 000 ± 0 . 0288 , 0 . 781 ± 0 . 0176 , 1 . 477 ± 0 . 043 ， 1 . 403 ± 0 . 0578 , 1 . 157 ± 0 . 0617 , 0 . 881 ± 0 . 0689 , 1 . 149 ± 0 . 0398 , 0 . 953 ± 0 . 0543 , 1 . 017 ± 0 . 0600 , 1 . 090 ± 0 . 0378 , N = 3 ) . Ellipsoid body knock down of Octβ2R results in sleep rebound increase . In response to OA , ellipsoid knock down of Octβ2R rescues control phenotype . Data represent mean ± SEM : 13 . 80 ± 2 . 439 , n = 31 ; 5 . 465 ± 2 . 036 , n = 25 ; 19 . 55 ± 2 . 275 , n = 27 ; 12 . 47 ± 2 . 684 , n = 24 ; 30 . 03 ± 3 . 428 , n = 25 ; 43 . 67 ± 4 . 616 , n = 25 . Sleep profile of PcXT109 mutant with or without OA treatment . In both control and PcXT109 mutant flies , the treatment of OA results in sleep decrease , n = 16 ( G ) . Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies ( From left to right mean ± SEM : 13 . 90 ± 1 . 397 ; 21 . 25 ± 1 . 493 , N = 4 ) ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Quantitative RT-PCR of stx after OA treatment in w1118 control and Octβ1R, Octβ2R mutants, Octβ3R mutants. (From left to right mean±SEM: 1.000±0.0288, 0.781±0.0176, 1.477±0.043，1.403±0.0578, 1.157±0.0617, 0.881±0.0689, 1.149±0.0398, 0.953±0.0543, 1.017±0.0600, 1.090±0.0378, N=3).Ellipsoid body knock down of Octβ2R results in sleep rebound increase. In response to OA, ellipsoid knock down of Octβ2R rescues control phenotype. Data represent mean±SEM: 13.80±2.439, n=31; 5.465±2.036, n=25; 19.55±2.275, n=27; 12.47±2.684, n=24; 30.03±3.428, n=25; 43.67±4.616, n=25.Sleep profile of PcXT109 mutant with or without OA treatment. In both control and PcXT109 mutant flies, the treatment of OA results in sleep decrease, n=16 (G). Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies (From left to right mean±SEM: 13.90±1.397; 21.25±1.493, N=4) (H).


Figure 6Total sleep for Tβh mutant and control flies in video - based method ( From left to right mean ± SEM : 688 . 2 ± 32 . 71 , n = 48 ; 123 . 2 ± 11 . 68 , n = 46 ) .

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

Figure 6Total sleep for Tβh mutant and control flies in video-based method (From left to right mean±SEM: 688.2±32.71, n=48; 123.2±11.68, n=46).


Figure 1Expression of indicated miRNAs in sorted naïve ( CD62L + CD44 - ) CD4 + T cells and following in vitro stimulation with anti - CD3 / anti - CD28 ( 1 - 3 days ) , relative to levels in cells prior to stimulation . Data from 3 independent experiments each using T cells pooled from 4 WT mice . Significance determined by one - way ANOVA . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Expression of indicated miRNAs in purified spleen lymphocytes ( B cells , CD4 + T cells and CD8 + T cells ) from d0 naive ( white ) and day 28 L . donovani - infected ( grey ) mice . Expression of each miRNA normalized to levels in whole naïve spleen ( dotted line ) . Data is mean + SEM of 2 experiments with cells purified from 3 - 5 pooled spleens . Volcano plot of RNAseq gene expression in splenic WT and miR - 132 - / - CD4 + T cells from d28 L . donovani infected mice . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 5 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - ( p < 0 . 05 ) are shown in red . Dotted box indicates transcripts significantly up - regulated in miR - 132 - / - CD4 + T cells by more than 50 % . STRING network analysis of significantly up - regulated transcripts in CD4 + T cells from spleen of d28 L . donovani infected miR - 132 - / - mice compared to WT cells . Cluster of ribosomal proteins shown in green circle , with coding RP transcripts ( black ) and pseudogenes ( red ) indicated . Secondary clusters are shown in grey . Volcano plot of all RP genes in splenic WT and miR - 132 - / - CD4 + T cells from d28 L . donovani infected mice . RPL genes are shown as circles , RPS genes as triangles , and pseudogenes as squares . Red symbols indicate significant difference between WT and miR - 132 - / - cells ( p < 0 . 05 ) whereas black non - significant . Expression of RP transcripts determined by qPCR from L . donovani infected d28 WT ( blue ) and miR - 132 - / - mice ( red ) . N = 9 for each WT and miR - 132 - / - from 2 independent infection experiments . Box extends from 25 - 75th percentile , whiskers are minimum and maximum values , and horizontal lines indicate median . Significance determined by unpaired t - test . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Fold change of all RP transcripts ( grey ) in Th1 cells compared to naïve CD4 + T cells . Data taken from RNA sequencing experiments described in reference 24 . Fold changes in IL - 10 ( red ) and IFNγ ( blue ) indicated for comparison . The statistical significance of the observed up - regulation of RP transcripts in Th1 cells is determined by Chi - squared test .

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

Figure 1Expression of indicated miRNAs in sorted naïve (CD62L+ CD44-) CD4+ T cells and following in vitro stimulation with anti-CD3 / anti-CD28 (1-3 days), relative to levels in cells prior to stimulation. Data from 3 independent experiments each using T cells pooled from 4 WT mice. Significance determined by one-way ANOVA. Data information: * p<0.05, ** p<0.01, *** p<0.001.Expression of indicated miRNAs in purified spleen lymphocytes (B cells, CD4+ T cells and CD8+ T cells) from d0 naive (white) and day 28 L. donovani-infected (grey) mice. Expression of each miRNA normalized to levels in whole naïve spleen (dotted line). Data is mean + SEM of 2 experiments with cells purified from 3-5 pooled spleens.Volcano plot of RNAseq gene expression in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 5 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- (p<0.05) are shown in red. Dotted box indicates transcripts significantly up-regulated in miR-132-/- CD4+ T cells by more than 50%.STRING network analysis of significantly up-regulated transcripts in CD4+ T cells from spleen of d28 L. donovani infected miR-132-/- mice compared to WT cells. Cluster of ribosomal proteins shown in green circle, with coding RP transcripts (black) and pseudogenes (red) indicated. Secondary clusters are shown in grey.Volcano plot of all RP genes in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. RPL genes are shown as circles, RPS genes as triangles, and pseudogenes as squares. Red symbols indicate significant difference between WT and miR-132-/- cells (p<0.05) whereas black non-significant.Expression of RP transcripts determined by qPCR from L. donovani infected d28 WT (blue) and miR-132-/- mice (red). N=9 for each WT and miR-132-/- from 2 independent infection experiments. Box extends from 25-75th percentile, whiskers are minimum and maximum values, and horizontal lines indicate median. Significance determined by unpaired t-test. Data information: * p<0.05, ** p<0.01, *** p<0.001.Fold change of all RP transcripts (grey) in Th1 cells compared to naïve CD4+ T cells. Data taken from RNA sequencing experiments described in reference 24. Fold changes in IL-10 (red) and IFNγ (blue) indicated for comparison. The statistical significance of the observed up-regulation of RP transcripts in Th1 cells is determined by Chi-squared test.


Figure 2A . Volcano plot ( Log2 ( Fold Change ) vs - Log ( P value ) ) of RNA gene expression in purified naïve CD62L + CD44 - WT and miR - 132 - / - CD4 + T cells . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 4 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - cells ( p < 0 . 05 ) shown in red . B . Volcano plot of RNA gene expression in purified naïve CD62L + CD44 - WT and miR - 132 - / - CD4 + T cells following 18hr in vitro stimulation with anti - CD3 / anti - CD28 under Th1 conditions . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 4 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - cells ( p < 0 . 05 ) shown in red . C . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in naïve CD4 + T cells from WT or miR - 132 - / - mice . D . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in in vitro polarised ( Th1 condtions , 18h post stimulation ) CD4 + T cells from WT or miR - 132 - / - mice . E . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in spleen CD4 + T cells from d28 L . donovani infected WT or miR - 132 - / - mice . F . BTAF1 transcript levels determined by qRTPCR in WT ( blue ) or miR - 132 - / - ( red ) in naïve ( d0 ) and Th1 polarised for 18h ( d1 ) CD4 + T cells , and CD4 + T cells from d28 L . donovani infected WT or miR - 132 - / - mice . N = 8 - 9 for each WT and miR - 132 - / Data information : Significance in ( F ) determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 . G . Expression of BTAF1 protein in d0 naïve and d1 ( 18hr ) Th1 - polarised WT and miR - 132 - / - CD4 + T cells , as determined by Western blot . Each lane from individual mouse , and representative of two independent experiments . H . Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3 ' UTR ( white ) or BTAF1 3 ' UTR on which the miR - 132 binding site is mutated ( grey ) downstream of renilla luciferase , in the presence of miR - 132 - 3p or miR - 212 - 3p mimics . Error bars indicate SEM from eight replicate treatments . Data information : Significance in ( H ) determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 .

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Figure 2A. Volcano plot (Log2(Fold Change) vs -Log(P value)) of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.B. Volcano plot of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells following 18hr in vitro stimulation with anti-CD3/anti-CD28 under Th1 conditions. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.C. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in naïve CD4+ T cells from WT or miR-132-/- mice.D. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in in vitro polarised (Th1 condtions, 18h post stimulation) CD4+ T cells from WT or miR-132-/- mice.E. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in spleen CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice.F. BTAF1 transcript levels determined by qRTPCR in WT (blue) or miR-132-/- (red) in naïve (d0) and Th1 polarised for 18h (d1) CD4+ T cells, and CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice. N=8-9 for each WT and miR-132-/ Data information: Significance in (F) determined by unpaired t-test. * p<0.05, ** p<0.01.G. Expression of BTAF1 protein in d0 naïve and d1 (18hr) Th1-polarised WT and miR-132-/- CD4+ T cells, as determined by Western blot. Each lane from individual mouse, and representative of two independent experiments.H. Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3'UTR (white) or BTAF1 3'UTR on which the miR-132 binding site is mutated (grey) downstream of renilla luciferase, in the presence of miR-132-3p or miR-212-3p mimics. Error bars indicate SEM from eight replicate treatments. Data information: Significance in (H) determined by unpaired t-test. * p<0.05, ** p<0.01.


Figure 3A . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non - targeting control ( NTC ) mimics ( white ) or miR - 132 - 3p mimics ( grey ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . B . p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR - 132 - 3p mimics determined by Western blot . GAPDH was used as a loading control . Right panel indicates mean + SEM of 4 experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . C . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA ( white ) or p300 siRNA ( grey ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . D . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA ( white ) or BTAF1 siRNA ( grey ) . Data information : Statistical significance is determined by unpaired t - testE . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR - 132 - 3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h . Levels are normalised to cells transfected with NTC siRNA and NTC mimic . Data information : Statistical significance is determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . F . Puromycin incorporation ( following 10 - minute pulse and 50 - minute chase ) determined by western blot in MEFs transfected with NTC or miR - 132 - 3p or miR - 212 - 3p mimics . Data information : Statistical significance is determined by unpaired t -

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Figure 3A. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non-targeting control (NTC) mimics (white) or miR-132-3p mimics (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.B. p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR-132-3p mimics determined by Western blot. GAPDH was used as a loading control. Right panel indicates mean + SEM of 4 experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.C. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or p300 siRNA (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.D. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or BTAF1 siRNA (grey). Data information: Statistical significance is determined by unpaired t-testE. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR-132-3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h. Levels are normalised to cells transfected with NTC siRNA and NTC mimic. Data information: Statistical significance is determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.F. Puromycin incorporation (following 10-minute pulse and 50-minute chase) determined by western blot in MEFs transfected with NTC or miR-132-3p or miR-212-3p mimics. Data information: Statistical significance is determined by unpaired t-test


Figure 4A . Percentage of IFNγ + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant . B . Percentage of IFNγ + / IL - 10 + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant . C . IL - 10 mRNA levels , determined by RNA - sequencing , in TCRβ + CD4 + cells purified from spleens of L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice ( n = 5 per group ) . D . Percentage of IFNγ + WT ( blue ) or miR - 132 - / - ( red ) in vitro polarised Th1 cells ( 6 days ) in the presence or absence of phenylephrine ( PE ) , determined by intracellular cytokine staining . E . Percentage of IL10 + WT ( blue ) or miR - 132 - / - ( red ) in vitro polarised Th1 cells ( 6 days ) in the presence or absence of phenylephrine ( PE ) , determined by intracellular cytokine staining . F . Total cell counts following in vitro Th1 polarisation ( 6 days ) in the presence or absence of phenylephrine ( PE ) . For ( D - E ) , cells were purified from 3 mice per group and 6 replicates performed . Data information : statistical significance was determined with 1 - way ANOVA followed by Bonferroni ' s multiple comparison test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant .

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Figure 4A. Percentage of IFNγ+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.B. Percentage of IFNγ+/IL-10+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.C. IL-10 mRNA levels, determined by RNA-sequencing, in TCRβ+ CD4+ cells purified from spleens of L. donovani infected WT (blue) or miR-132-/- (red) mice (n=5 per group).D. Percentage of IFNγ+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. E. Percentage of IL10+ WT (blue) or miR-132-/- (red) in vitro polarised Th1 cells (6 days) in the presence or absence of phenylephrine (PE), determined by intracellular cytokine staining. F. Total cell counts following in vitro Th1 polarisation (6 days) in the presence or absence of phenylephrine (PE). For (D-E), cells were purified from 3 mice per group and 6 replicates performed. Data information: statistical significance was determined with 1-way ANOVA followed by Bonferroni's multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.


Figure 5Liver LDU ( Leishman Donovan units ) at day 28 in infected WT mice treated with anti - IL - 10R antibody or isotype control antibody ( left panel , n = 5 mice per group ) , or at day 21 and day 28 from WT ( blue ) , IL - 10 + / - ( open green circles ) and IL - 10 - / - ( filled green circles ) mice ( right panel n = 3 - 6 mice per group ) Data information : Significance determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT ( blue ) and miR - 132 - / - ( miR - 132 - / - ; red ) mice . Data from 4 independent infection experiments . Mean WT and miR - 132 - / - spleen parasite burdens from the 4 independent experiments shown in ( B ) . Lines link individual experiments . Splenic parasite burdens relative to WT group ( WT mean = 1 ) for each of the 4 experiments shown in ( B ) , with each data point representing individual mouse . Data information : Significance determined by unpaired t - test , and in ( C ) by paired t - test of mean values . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Spleen size expressed as % body weight for d0 ( naïve ) or day 28 L . donovani infected WT ( blue ) and miR - 132 - / - ( miR - 132 - / - ; red ) mice . Liver size expressed as % body weight for d0 ( naïve ) or day 28 L . donovani infected WT ( blue ) and miR - 132 - / - ( red ) mice . Data information : Significance determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Figure 5Liver LDU (Leishman Donovan units) at day 28 in infected WT mice treated with anti-IL-10R antibody or isotype control antibody (left panel, n=5 mice per group), or at day 21 and day 28 from WT (blue), IL-10+/- (open green circles) and IL-10-/- (filled green circles) mice (right panel n= 3-6 mice per group) Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.Day 28 splenic parasite burdens expressed as LDU with each data point representing an individual mouse in WT (blue) and miR-132-/- (miR-132-/-; red) mice. Data from 4 independent infection experiments.   Mean WT and miR-132-/- spleen parasite burdens from the 4 independent experiments shown in (B). Lines link individual experiments.   Splenic parasite burdens relative to WT group (WT mean = 1) for each of the 4 experiments shown in (B), with each data point representing individual mouse.   Data information: Significance determined by unpaired t-test, and in (C) by paired t-test of mean values. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.Spleen size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (miR-132-/-; red) mice.   Liver size expressed as % body weight for d0 (naïve) or day 28 L. donovani infected WT (blue) and miR-132-/- (red) mice.   Data information: Significance determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


Figure 1C PICT assay for the interactions between indicated pairs of proteins tagged to FRB and GFP . Representative images before and after adding rapamycin ( RAP ) of RFP - tagged anchor and GFP - tagged prey are shown in the upper and middle row , respectively . Bottom row shows the merged images . White zoom in boxes , 0 . 9µm . Scale bar , 5µm .

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Figure 1C PICT assay for the interactions between indicated pairs of proteins tagged to FRB and GFP. Representative images before and after adding rapamycin (RAP) of RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. Scale bar, 5µm.


Figure 2A Ape1 processing was analyzed by western blot in the indicated strains and temperatures and normalized to the processing achieved in wild - type cells ± SD , n = 3 . D Representative EM images of pep4Δ and pep4Δdrs2Δ strains . Black squares show a zoom - in in the vacuole . White arrowheads point to Cvt bodies . E Representative CLEM images of drs2Δ cells . Ape1 - GFP ( top ) correlates with a membrane - free ribosome exclusion area ( middle ) . Black square ( bottom ) shows a zoom - in at the position correlating with Ape1 - GFP ( white arrowhead ) .

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Figure 2A Ape1 processing was analyzed by western blot in the indicated strains and temperatures and normalized to the processing achieved in wild-type cells ± SD, n = 3.D Representative EM images of pep4Δ and pep4Δdrs2Δ strains. Black squares show a zoom-in in the vacuole. White arrowheads point to Cvt bodies.E Representative CLEM images of drs2Δ cells. Ape1-GFP (top) correlates with a membrane-free ribosome exclusion area (middle). Black square (bottom) shows a zoom-in at the position correlating with Ape1-GFP (white arrowhead).


Figure 3Ape1 processing was analyzed by western blot in the indicated strains , and normalized to the processing achieved in cells expressing wild - type Drs2 ± SD , n ≥ 3 biological replicates .

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Figure 3Ape1 processing was analyzed by western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild-type Drs2 ± SD, n ≥ 3 biological replicates.


Figure 4D Ape1 processing was analyzed by western blot in the indicated strains , and normalized to the processing achieved in cells expressing wild - type Drs2 ± SD , n = 3 biological replicates .

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Figure 4D Ape1 processing was analyzed by western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild-type Drs2 ± SD, n = 3 biological replicates.


Figure 5A Ape1 processing was analyzed by western blot in cells overexpressing N - terminal Drs2 ( 1 - 212 aa ) and normalized to the processing achieved in cells with an empty vector ± SD , n = 3 . C Left , PICT assay for the interaction of Trs85 - FRB ( TRAPPIII ) and Drs2 - GFP or drs2 - 5A - GFP after adding rapamycin ( RAP ) . The Interaction score was normalized to the measurement of Drs2 - GFP . Error bars : mean ± SD , n = 3 . Asterisk indicates significant difference as determined by a two - tailed Student ' s t - test ( * * P < 0 . 01 ) . Right , representative images of the PICT assay . RFP - tagged anchor and GFP - tagged prey are shown in the upper and middle row , respectively . Bottom row shows the merged images . White zoom in boxes , 0 . 9µm . Scale bar , 5µm .

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Figure 5A Ape1 processing was analyzed by western blot in cells overexpressing N-terminal Drs2 (1-212 aa) and normalized to the processing achieved in cells with an empty vector ± SD, n = 3.C Left, PICT assay for the interaction of Trs85-FRB (TRAPPIII) and Drs2-GFP or drs2-5A-GFP after adding rapamycin (RAP). The Interaction score was normalized to the measurement of Drs2-GFP. Error bars: mean ± SD, n = 3. Asterisk indicates significant difference as determined by a two-tailed Student's t-test (**P<0.01). Right, representative images of the PICT assay. RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. Scale bar, 5µm.


Figure 6A Histograms representing the mean speed of Atg9 puncta observed in wild - type , drs2Δ and drs2 - 5A cells . The histograms were fitted to a mixture of Gaussian distributions with two components ; the percentage of each population and the means of the fitting curves are indicated on top of the plot . B Representative images ( left ) and quantification ( right ) of the co - localization between Atg9 - GFP and Ape1 - mCherry ( upper and middle row , respectively ) in cells expressing wild - type Drs2 or drs2 - 5A . White arrowheads point to Ape1 - mCherry clusters co - localizing with Atg9 - GFP . C Values were normalized to the measurements in cells expressing wild - type Drs2 . Error bars : mean ± SD , n = 3 biological replicates . Asterisks indicate significant difference as determined by a two - tailed Student ' s t - test ( * * P < 0 . 01 ) . Scale bar , 5µm . C Representative images ( left ) and quantification ( right ) of the PICT assay for the interaction of Trs85 - FRB ( TRAPPIII ) and Atg9 - GFP in cells expressing wild - type Drs2 or drs2 - 5A after adding rapamycin ( RAP ) . RFP - tagged anchor and GFP - tagged prey are shown in the upper and middle row , respectively . Bottom row shows the merged images . White zoom in boxes , 0 . 9µm . , C Values were normalized to the measurements in cells expressing wild - type Drs2 . Error bars : mean ± SD , n = 3 biological replicates . Asterisks indicate significant difference as determined by a two - tailed Student ' s t - test ( * * P < 0 . 01 ) . Scale bar , 5µm .

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Figure 6A Histograms representing the mean speed of Atg9 puncta observed in wild-type, drs2Δ and drs2-5A cells. The histograms were fitted to a mixture of Gaussian distributions with two components; the percentage of each population and the means of the fitting curves are indicated on top of the plot.B Representative images (left) and quantification (right) of the co-localization between Atg9-GFP and Ape1-mCherry (upper and middle row, respectively) in cells expressing wild-type Drs2 or drs2-5A. White arrowheads point to Ape1-mCherry clusters co-localizing with Atg9-GFP. C Values were normalized to the measurements in cells expressing wild-type Drs2. Error bars: mean ± SD, n = 3 biological replicates. Asterisks indicate significant difference as determined by a two-tailed Student's t-test (**P<0.01). Scale bar, 5µm.C Representative images (left) and quantification (right) of the PICT assay for the interaction of Trs85-FRB (TRAPPIII) and Atg9-GFP in cells expressing wild-type Drs2 or drs2-5A after adding rapamycin (RAP). RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. , C Values were normalized to the measurements in cells expressing wild-type Drs2. Error bars: mean ± SD, n = 3 biological replicates. Asterisks indicate significant difference as determined by a two-tailed Student's t-test (**P<0.01). Scale bar, 5µm.


Figure 1 ( B ) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX . Cut - off for what is regarded as expressed was set to 1 . 0 NX .

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Figure 1(B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.