D , E , F , G . 3D electron microscopy of thalamus in 12 - month - old Ifnb + / + Ifnb - / - mice . D . Volume rendering of mitochondria ( left panel ) corresponding mitochondria in 2D electron microscopy image ( right panel ) in bodies . Each mitochondrion is colored differently . Scale bars equal 2 microns . Voxel equals 0 . 00878908x0 . 00878908x0 . 03 microns for Ifnb + / + neuron 0 . 00439453x0 . 00438453x0 . 04 microns for Ifnb - / - . Nuclei are in grey marked with ' N . ' E . Reconstituted axonal mitochondria . extracted from Movies EV1 2 . F . of mitochondria in bodies non - myelinated myelinated axons . N > 30 ; error bars are SD . G . Number of branches per mitochondrion . N > 30 ; error bars are SD .

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

D, E, F, and G. 3D electron microscopy of thalamus in 12-month-old Ifnb+/+ and Ifnb-/- mice. D. Volume rendering of mitochondria (left panel) and corresponding mitochondria in 2D electron microscopy image (right panel) in cell bodies. Each individual mitochondrion is colored differently. Scale bars equal 2 microns. Voxel size equals 0.00878908x0.00878908x0.03 microns for the Ifnb+/+ neuron and 0.00439453x0.00438453x0.04 microns for Ifnb-/-. Nuclei are in grey and marked with 'N.' E. Reconstituted axonal mitochondria. Images extracted from Movies EV1 and 2. F. Size of individual mitochondria in cell bodies and non-myelinated and myelinated axons. N>30; error bars are SD. G. Number of branches per mitochondrion. N>30; error bars are SD.


D - E . Respiration of isolated mitochondria from Ifnb + / + Ifnb - / - whole brains . OCR measurements under 1 substrates ( pyruvate / malate ) were obtained at baseline ( B ) on addition of ADP , oligomycin , FCCP rotenone / antimycin A to capture S3 , S4o , S3u , nonmitochondrial respiration . N = 3 - 4 mice per group ; bar graphs show mean + SE .

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

D-E. Respiration of isolated mitochondria from Ifnb+/+ and Ifnb-/- whole brains. OCR measurements under Complex 1 substrates (pyruvate/malate) were obtained at baseline (B) and on addition of ADP, oligomycin, FCCP and rotenone/antimycin A to capture S3, S4o, S3u, and nonmitochondrial respiration. N=3-4 mice per group; bar graphs show mean + SE.


K . Immunofluorescence of 8OHdG in CN cultures . Neurons were labeled with Nissl . Scale bars equal 50 microns . L . of 8OHdG by ELISA in brain tissues in young ( aged 1 . 5 - 3 months ) old ( aged 6 - 12 months ) Ifnb + / + Ifnb - / - mice .

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

K. Immunofluorescence of 8OHdG in CN cultures. Neurons were labeled with Nissl. Scale bars equal 50 microns. L. Quantification of 8OHdG by ELISA in brain tissues in young (aged 1.5-3 months) and old (aged 6-12 months) Ifnb+/+ and Ifnb-/- mice.


M . Immunoblot for optineurin in 6 - week - old midbrains of Ifnb + / + Ifnb - / - mice . N . Immunoblot for optineurin in DIV6 Ifnb + / + Ifnb - / - CNs with or without 30 U / mL rIFN - β .

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

M. Immunoblot for optineurin in 6-week-old midbrains of Ifnb+/+ and Ifnb-/- mice. N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without 30 U/mL rIFN-β.


H . Kinetics of Drp1 dephosphorylation at S643 on rIFN - β treatment in wild - type CN cultures . PD - L1 was used as a positive control of rIFN - β activity .

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

H. Kinetics of Drp1 dephosphorylation at S643 on rIFN-β treatment in wild-type CN cultures. PD-L1 was used as a positive control of rIFN-β activity.


I . MFN2 OPA1 immunoblots in Ifnb + / + Ifnb - / - CN cultures . Vinculin was used as a loading control . Error bars are from 3 independent experiments .

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

I. MFN2 and OPA1 immunoblots and quantification in Ifnb+/+ and Ifnb-/- CN cultures and quantification. Vinculin was used as a loading control. Error bars are SEM from 3 independent experiments.


J . Optineurin Tom20 immunofluorescence in Ifnb + / + Ifnb - / - MEFs transfected with Drp1 or phosphomimetic Drp1 .

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

J. Optineurin and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1.


E . Volume rendering from 3D of Ifnb + / + Ifnb - / - thalami . Mitochondria are blue , ER is yellow , nuclei are grey . Mitochondria undergoing fission are cyan indicated with a arrow . 2D pictures are shown in Fig 2 .

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

E. Volume rendering from 3D TEM images of Ifnb+/+ and Ifnb-/- thalami. Mitochondria are blue, the ER is yellow, and nuclei are grey. Mitochondria undergoing fission are cyan and indicated with a white arrow. 2D pictures are shown in Fig 2.


H . Projections of 3D from immunostaining of Drp1 ( blue ) , Hsp60 ( mitochondria , green ) , INF2 ( yellow ) , calnexin ( ER , magenta ) in Ifnb - / - MEFs with or without rIFN - β treatment for 6 h . Scale bar equal 2 microns . Arrows indicate fission foci . I . Quantifications of triple - positive MT - DR + Drp1 + Cnx + in MEFs . are shown in Fig EV3 .

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

H. Projections of 3D images from immunostaining of Drp1 (blue), Hsp60 (mitochondria, green), INF2 (yellow), and calnexin (ER, magenta) in Ifnb-/- MEFs with or without rIFN-β treatment for 6 h. Scale bar equal 2 microns. Arrows indicate fission foci. I. Quantifications of triple-positive MT-DR+Drp1+Cnx+ in MEFs. Images are shown in Fig EV3.


D . Projections of 3D from immunostaining for phospho - S622 Drp1 ( yellow ) , Hsp60 ( mitochondria , green ) , calnexin ( ER , magenta ) in N2A depleted of STAT5 A B ( δStat5 ) or IFN - β ( δIfnb ) or with nontargeting control ( NTC ) . with or without rIFN - β treatment for 6 h . Scale bar equals 5 microns . Arrows indicate triple - positive foci . E . of D .

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

D. Projections of 3D images from immunostaining for phospho-S622 Drp1 (yellow), Hsp60 (mitochondria, green), and calnexin (ER, magenta) in N2A cells depleted of STAT5 A and B (δStat5) or IFN-β (δIfnb) or with nontargeting control (NTC). Cells with or without rIFN-β treatment for 6 h. Scale bar equals 5 microns. Arrows indicate triple-positive foci. E. Quantification of D.


L . Immunostaining of PGAM5 Hsp60 in NTC δSTAT5 N2As transfected with PGAM5 or empty vector control of mitochondrial section . Scale bars equal 5 microns . Error bars mean from 10 .

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

L. Immunostaining of PGAM5 and Hsp60 in NTC and δSTAT5 N2As transfected with PGAM5 or empty vector control and quantification of mitochondrial section area. Scale bars equal 5 microns. Error bars mean SEM from 10 individual cells.


M . Optineurin immunostaining in Ifnb + / + Ifnb - / - MEFs overexpressing PGAM5 . Empty vector was used as control . Additional controls are shown in Fig EV5L . Error bars are SD from N = 30 .

O O B-MEASURED_VAR O O B-CONTROLLED_VAR 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

M. Optineurin immunostaining in Ifnb+/+ and Ifnb-/- MEFs overexpressing PGAM5 and quantification. Empty vector was used as control. Additional controls are shown in Fig EV5L. Error bars are SD from N=30 cells.


E - K . Immunofluorescence in striatal region in mice treated with 6OH - DA rIFN - β for optineurin ( E , quantified in F ) Tom20 ( E ) , with of mitochondrial by mitochondrial section ( G ) . 8OHdG - ( H - I ) , OxDJ1 - ( J - K ) , DAPI - labeled nuclei . Scale bars equal 10 microns .

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

E-K. Immunofluorescence in the striatal region in mice treated with 6OH-DA and rIFN-β for optineurin (E, quantified in F) and Tom20 (E), with quantification of individual mitochondrial size by mitochondrial section area (G). 8OHdG- (H-I), OxDJ1- (J-K), and DAPI-labeled nuclei. Scale bars equal 10 microns.


, Total of Munc18 - 1 . HEK293T transfected with WT or mutant Munc18 - 1 variants either GFP , syntaxin - 11 - 264 or syntaxin - 11 - 180 were lysed lysates were analyzed by quantitative immunoblotting to indicated , normalized to β - actin ( Synt - 1A = syntaxin - 1A ) . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis , followed by Dunn ' s multiple comparison ; n = 11 independent experiments ; exact p values are shown in Appendix Table S1 ) .

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

, Total protein levels of Munc18-1. HEK293T cells transfected with WT or mutant Munc18-1 variants and either GFP, syntaxin-11-264 or syntaxin-11-180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins, normalized to β-actin (Synt-1A = syntaxin-1A). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 by Kruskal-Wallis test, followed by Dunn's multiple comparison test; n = 11 independent experiments; exact p values are shown in Appendix Table S1).


Solubility of Munc18 - 1 . HEK293T transfected as in ( C ) were solubilized in 0 . 1 % Triton X - 100 ( TX ) equal volumes of soluble insoluble fractions were analyzed by quantitative immunoblotting . TX - soluble Munc18 - 1 was measured as of total Munc18 - 1 by quantitative immunoblotting . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis followed by Dunn ' s multiple comparison , or by one - way ANOVA followed by Bonferroni post - hoc ; n = 5 - 9 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).


Same as in ( A , B ) except that compounds 9 , 10 , 11 , 13 , 14 16 were added at 0 . 25 , 1 , 5 , 20 , 100 , or 250 µM . Note , that data for 250 µM were not plotted in ( D ) because of causing neuronal . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 by Kruskal - Wallis with Dunn ' s multiple comparison , or one - way ANOVA with Dunnett ' s post - hoc ; n = 6 - 15 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Same as in (A, B) except that compounds 9, 10, 11, 13, 14 and 16 were added at 0.25, 1, 5, 20, 100, or 250 µM. Note, that the data for 250 µM were not plotted in (D) because of causing neuronal death. Data are means ± SEM (*p < 0.05, **p < 0.01 by Kruskal-Wallis test with Dunn's multiple comparison test, or one-way ANOVA with Dunnett's post-hoc test; n = 6-15 independent experiments; exact n and p values are shown in Appendix Table S1).


Limited proteolysis . Recombinant purified were incubated with increasing concentrations of trypsin . Remaining were analyzed by quantitative immunoblotting . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , by 2 - way ANOVA Dunnett ' s multiple - comparison ; n = 4 independent experiments ; exact p values are shown in Appendix Table S1 ) .

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

Limited proteolysis. Recombinant purified proteins were incubated with increasing concentrations of trypsin. Remaining protein levels were analyzed by quantitative immunoblotting. Data are means ± SEM (* p < 0.05, **p < 0.01, ****p < 0.0001, by 2-way ANOVA and Dunnett's multiple-comparison test; n = 4 independent experiments; exact p values are shown in Appendix Table S1).


Munc18 - 1 knockout neurons expressing G544D Munc18 - 1 were subjected to of mean firing rate before addition of compounds ( 0 h ) or 48 hours after vehicle ( DMSO ) or compound addition . 16 electrodes per well were analyzed for neuronal firing ( A ; purple boxes indicate network activity ) . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA Dunnett ' s multiple comparison , or Kruskal - Wallis followed by Dunn ' s post - hoc ; n = 11 - 16 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Munc18-1 knockout neurons expressing G544D Munc18-1 were subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).


Uptake of synaptotagmin - 1 antibody during high K + stimulation . Neurons expressing cre recombinase / or WT , R406H or G544D Munc18 - 1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake . Endocytosed synaptotagmin - 1 antibody was quantified by immunostaining ( A - C ; scale bar = 30 µm ; 4 - PBA = 4 - phenylbutyrate ) , via counting number of pixels > of 15 ( D ) . Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA Dunnett ' s multiple comparison ; n = 3 - 10 independent experiments ; exact n p values are shown in Appendix Table S1 ) .

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

Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).


Plot of change in from baseline to peak for Munc18 - 1 G544D ( H ) Data are means ± ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way one - way ANOVA Dunnett ' s multiple comparison ; n = 7 - 23 ; exact n p values are shown in Appendix Table S1 ) . Note , that compound 10 is at a similar wavelength than pHluorin , so experiments at concentrations higher than 1 µM were not possible .

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

Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.


Heat - induced paralysis . Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 ° C over a period of 120 min , paralysis was scored at indicated time points . Data are means ± ( n = 3 - 4 independent experiments on ten worms per experiment ; exact n p values are shown in Appendix Table S1 ) . ( B , D , F , H , J , L ) of paralysis at 20 minutes . Data are means ± ( * , # p < 0 . 05 , * * , # # p < 0 . 01 , * * * , # # # p < 0 . 001 , # as compared to WT , * as compared to mutant by two - way ANOVA Dunnett ' s multiple comparison ; n = 3 - 4 independent experiments on ten worms per experiment ; exact n p values are shown in Appendix Table S1 ) .

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

Heat-induced paralysis. Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 °C over a period of 120 min, and paralysis was scored at indicated time points. Data are means ± SEM (n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1). (B, D, F, H, J, L) Quantification of paralysis at 20 minutes. Data are means ± SEM (*,#p < 0.05, **,##p < 0.01, ***,###p < 0.001, # as compared to WT, * as compared to mutant by two-way ANOVA and Dunnett's multiple comparison test; n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1).


Rescue of subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP were immobilized , ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger , broken arrowheads to single , smaller ( N , O ) . Scale bar in ( N ) ( O ) = 10 µm . 4 - PBA = 4 - phenylbutyrate .

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

Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta (N, O). Scale bar in (N) and (O) = 10 µm. 4-PBA = 4-phenylbutyrate.


Rescue of subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP at 1 , 5 , 20 or 100 µM compound were immobilized , ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger , broken arrowheads to single , smaller

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

Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP at 1, 5, 20 or 100 µM compound were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta


of soluble aggregated Munc18 - 1 . Data are means ± ( * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA Dunnett ' s multiple comparison ; n = 3 - 4 worms ; exact n p values are shown in Appendix Table S1 ) .

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 O O O O O

Quantification of soluble and aggregated Munc18-1. Data are means ± SEM (**p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-4 worms; exact n and p values are shown in Appendix Table S1).


( a ) Ba / F3 expressing myrPI ( 3 ) K - ER , myrPKB - ER , FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT for 4 h ( myrPI ( 3 ) K - ER , myrPKB - ER ) or 8 h ( FOXO3 ( A3 ) - ER FOXO4 ( A3 ) - ER ) microarray analyses were performed . Shown are fold changes relative to control for glutamine synthetase ( GS ) , Mxi1 Pink1 . Data are represented as mean values of one experiment performed in quadruplicate .

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

(a) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate.


( b ) Ba / F3 expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight stimulated with 4 - OHT . Relative mRNA of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . normalized for B2M ( n = 4 ) . * P0 . 05 , * * P0 . 01 .

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

(b) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4). *P0.05, **P0.01.


( c ) Ba / F3 expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight stimulated with 4 - OHT . lysates were analysed for of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 actin . Shown are representative blots ( n = 4 ) .

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

(c) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( d ) Ba / F3 expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in presence of mIL - 3 . Relative mRNA of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . values normalized for B2M ( n = 3 ) . * * P0 . 01 .

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 O O O O O O B-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

(d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3). **P0.01.


( e ) Ba / F3 expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in presence of mIL - 3 . lysates were analysed for of glutamine synthetase , p27 actin . Shown are representative blots ( n = 3 ) .

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

(e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).


( f ) Wild - type Ba / F3 were cytokine starved overnight stimulated with mIL - 3 . lysates were analysed for of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 actin . Shown are representative blots ( n = 4 ) .

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

(f) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( g ) Wild - type Ba / F3 were incubated with LY294002 in presence of mIL - 3 . lysates were analysed for of glutamine synthetase , p27 actin . Shown are representative blots ( n = 3 ) .

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

(g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).


( h ) FOXO1 , 3 , 4 − / − MEFs wild - type MEFs were incubated with LY294002 ( 10 μM ) for indicated times . was isolated relative mRNA of glutamine synthetase were analysed using quantitative PCR . Data are represented as mean values normalized for B2M ( n = 2 ) . Uncropped of blots are shown in Supplementary Fig . S7 .

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 B-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

(h) FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT actinomycin D ( 1 μg ml − 1 ) or dimethylsulphoxide ( DMSO ) as a control in presence of mIL - 3 for 16 h . lysates were analysed for of glutamine synthetase ( GS ) , p27 actin . Shown are representative blots ( n = 3 ) .

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

(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and actinomycin D (1 μg ml−1) or dimethylsulphoxide (DMSO) as a control in the presence of mIL-3 for 16 h. Cell lysates were analysed for protein levels of glutamine synthetase (GS), p27 and actin. Shown are representative blots (n=3).


( b ) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO - binding sites were transfected in HEK293 together with Renilla FOXO3 ( A3 ) as indicated . Luciferase activity was measured 40 h after transfection . Data are depicted as relative luciferase units ( RLU ) compared with control . Shown are mean ± s . e . m . values ( n = 3 )

O O O B-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O 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) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO-binding sites were transfected in HEK293 cells together with Renilla and FOXO3(A3) as indicated. Luciferase activity was measured 40 h after transfection. Data are depicted as relative luciferase units (RLU) compared with the control. Shown are mean ± s.e.m. values (n=3)


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 stimulated with 4 - OHT MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for LC3 analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm .

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

(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.


( d ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT . lysates were analysed for of glutamine synthetase , p27 actin . Shown are representative blots ( n = 4 ) .

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

(d) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=4).


( e ) osteosarcoma U2OS expressing FOXO ( A3 ) - ER was stimulated with 4 - OHT , were lysed equal amounts of were analysed for of glutamine synthetase actin .

O O O O 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 O O

(e) The osteosarcoma cell line U2OS expressing FOXO(A3)-ER was stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin.


( f ) MSCs expressing FOXO ( A3 ) - ER were stimulated with 4 - OHT , were lysed equal amounts of were analysed for of glutamine synthetase actin . Shown are representative blots ( n = 3 ) .

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

(f) MSCs expressing FOXO(A3)-ER were stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. Shown are representative blots (n=3).


( g ) Wild - type , daf - 2 daf - 16 mutant worms were synchronized to L1 by hypochlorite treatment were placed on nematode growth medium agar plates . After five days worms were lysed in imidazole analysed for glutamine synthetase activity .

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

(g) Wild-type, daf-2 and daf-16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity.


( h ) Wild - type N2 worms or daf - 2 mutants were synchronized to L1 by hypochlorite treatment placed on nematode growth medium plates with or without expressing DAF - 16 double - stranded . After five days worms were lysed in imidazole analysed for glutamine synthetase activity . ( g , h ) Shown are means of five independent plates for each condition . Uncropped of blots are shown in Supplementary Fig . S7 .

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

(h) Wild-type N2 worms or daf-2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF-16 double-stranded RNA. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (g,h) Shown are the means of five independent plates for each condition. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in presence of mIL - 3 . lysates were analysed for glutamine synthetase ( GS ) activity in an enzyme ( left ) . In addition , expression of glutamine synthetase , p27 actin was determined using western blot ( right ) . Shown are mean ± s . e . m . ( n = 3 ) representative blots of these experiments . * * P0 . 01 .

O O O O O O 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 O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for glutamine synthetase (GS) activity in an enzyme assay (left). In addition, the expression of glutamine synthetase, p27 and actin was determined using western blot (right). Shown are the mean ± s.e.m. (n=3) and representative blots of these experiments. **P0.01.


( b ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT for 16 h in presence of mIL - 3 together with MSO , as indicated . lysates were analysed for glutamine synthetase activity in an enzyme ( left ) . Furthermore expression of glutamine synthetase , p27 actin were determined using western blot ( right ) . Shown are mean ± s . e . m . ( n = 3 ) representative blots of these experiments . * P0 . 05 , * * P0 . 01 .

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

(b) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT for 16 h in the presence of mIL-3 together with MSO, as indicated. Cell lysates were analysed for glutamine synthetase activity in an enzyme assay (left). Furthermore the expression levels of glutamine synthetase, p27 and actin were determined using western blot (right). Shown are the mean ±  s.e.m. (n=3) and representative blots of these experiments. *P0.05, **P0.01.


( c ) Ba / F3 expressing myrPI ( 3 ) K - ER were cytokine - starved overnight . next day , were washed in PBS put in a medium without serum , with or without 4 - OHT . At times indicated , medium samples were taken analysed for acid by high - performance liquid chromatography . were lysed equal amounts of were analysed by western blotting for of phospho - PKB ( S473 ) , phospho - FOXO3 ( T32 ) actin ( inset ) . Shown are mean of relative acid , compared with t = 0 ( n = 2 ) , representative blots of these experiments .

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

(c) Ba/F3 cells expressing myrPI(3)K-ER were cytokine-starved overnight. The next day, cells were washed in PBS and put in a medium without serum, with or without 4-OHT. At the times indicated, medium samples were taken and analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho-PKB (S473), phospho-FOXO3 (T32) and actin (inset). Shown are the mean of relative amino acid levels, compared with t=0 (n=2), and representative blots of these experiments.


( d ) Ba / F3 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in serum - free medium containing mIL - 3 . medium was analysed for acid by high - performance liquid chromatography . were lysed analysed for of glutamine synthetase , p27 actin ( inset ) . Shown are mean ± s . e . m . of relative acid , compared with t = 0 ( n = 4 ) , representative blots of these experiments . * * P0 . 01 . Uncropped of blots are shown in Supplementary Fig . S7 .

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

(d) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in serum-free medium containing mIL-3. The medium was analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and analysed for protein levels of glutamine synthetase, p27 and actin (inset). Shown are the mean ±  s.e.m. of relative amino acid levels, compared with t=0 (n=4), and representative blots of these experiments. **P0.01. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT with or without MSO . After 24 h , were starved of serum , glucose in D - PBS containing indicated inhibitors stimulated with for 10 min . lysates were analysed for of phospho - S6K ( Thr 389 ) actin . Shown are representative blots of three independent experiments .

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

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments.


( b , c ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT MSO ( 0 . 25 M ) for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for mTOR LAMP2 analysed by confocal microscopy . ( b ) Shown are representative pictures ( n = 4 ) . Scale bars , 100 μm . ( c ) of co - localization between mTOR LAMP2 as seen in b . Depicted are mean ± s . e . m . of of co - localized pixels from four experiments . * P0 . 05 , * * P0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O 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 O O O O O O O O O O O O O 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,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments. *P0.05, **P0.01.


( d ) DLD1 expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT , MSO BafA1 ( 200 nM ) . After 24 h , lysates were analysed for of LC3 tubulin . Shown are representative blots ( n = 4 ) .

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

(d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4).


( e ) DLD1 expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or a non - targeting siRNA stimulated with 4 - OHT in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , lysates were analysed for of glutamine synthetase , LC3 actin . Shown are representative blots ( n = 2 )

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

(e) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2)


( f ) DLD1 expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetasesiRNA or non - template ( NT ) control siRNA stimulated with 4 - OHT for 16 h . were lysed equal amounts of were analysed for of p62 or glutamine synthetase . Shown are representative blots ( n = 2 ) .

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

(f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetasesiRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2).


( g ) DLD1 were stimulated with LY294002 or PKB inhibitor VIII for 24 h . lysates were analysed for of LC3 actin . Shown are representative blots ( n = 4 ) . Uncropped of blots are shown in Supplementary Figs S7 S8 .

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

(g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for LC3 ER analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm . ( b ) of LC3 - positive spots as seen in a using ImageJ software . Depicted are mean ± s . e . m . of number of LC3 - positive spots divided by number of DAPI - positive ( n = 4 ) . * P0 . 05 .

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

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4). *P0.05.


( c ) MSCs expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT BafA1 ( 100 nM ) . At indicated time points , lysates were analysed for of glutamine synthetase ( GS ) , LC3 , pS6 ( Ser 235 / 236 ) actin . Shown are representative blots ( n = 2 ) .

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

(c) MSCs expressing FOXO3(A3)-ER were treated with or without 4-OHT and BafA1 (100 nM). At the indicated time points, cell lysates were analysed for protein levels of glutamine synthetase (GS), LC3, pS6 (Ser 235/236) and actin. Shown are representative blots (n=2).


( d ) IL - 3 was withdrawn from Ba / F3 in presence or absence of BafA1 ( 100 nM ) as indicated . lysates were analysed for of glutamine synthetase , LC3 hsp90 . Shown are representative blots ( n = 2 ) .

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

(d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated. Cell lysates were analysed for protein levels of glutamine synthetase, LC3 and hsp90. Shown are representative blots (n=2).


( e ) DLD1 were stimulated with l - glutamine ( l - Gln ) in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , lysates were analysed for of LC3 , p62 , p - S6K ( Thr 389 ) actin . Shown are representative blots ( n = 3 ) .

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(e) DLD1 cells were stimulated with l-glutamine (l-Gln) in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of LC3, p62, p-S6K (Thr 389) and actin. Shown are representative blots (n=3).


( f ) DLD1 were starved stimulated with glutamine ( 5 10 mM ) . were stained for mTOR LAMP2 analysed by confocal microscopy , co - localization between mTOR LAMP2 was quantified by ImageJ . Depicted are means of of co - localized pixels ( n = 2 ) .

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(f) DLD1 cells were starved and stimulated with glutamine (5 and 10 mM). Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy, and the co-localization between mTOR and LAMP2 was quantified by ImageJ. Depicted are the means of the percentage of co-localized pixels (n=2).


( g ) DLD1 expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - WIPI - 1 subsequently stimulated with 4 - OHT in presence or absence of MSO in glutamine - free DMEM containing 0 . 1 % FCS or medium without for 24 h . GFP - WIPI - 1 formation was performed by confocal microscopy . Depicted are percentages of positive for GFP - WIPI - 1 . Shown are mean ± s . e . m . ( n = 4 ) . * P0 . 05 * * P0 . 01 . Uncropped of blots are shown in Supplementary Fig . S8 .

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

(g) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently stimulated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS or medium without amino acids for 24 h. GFP-WIPI-1 puncta formation analysis was performed by confocal microscopy. Depicted are the percentages of cells positive for GFP-WIPI-1 puncta. Shown are the mean ±  s.e.m. (n=4). *P0.05 and **P0.01. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) DLD1 expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 stimulated with 4 - OHT MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . were stained for LC3 analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm .

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(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.


( b ) DLD1 expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - WIPI - 1 subsequently treated with 4 - OHT in presence or absence of MSO in glutamine - free DMEM containing 0 . 1 % FCS . After 24 h , were analysed for GFP p62 expression by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bar , 20 μm .

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(b) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently treated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h, cells were analysed for GFP and p62 expression by confocal microscopy. Shown are representative pictures (n=3). Scale bar, 20 μm.


( c ) DLD1 expressing FOXO3 ( A3 ) - ER were treated with 4 - OHT MSO in glutamine - free DMEM containing 0 . 1 % FCS . After 24 h were analysed for endogenous Atg12 formation by confocal microscopy . Shown are mean ± s . d . ( n = 3 ) . * P0 . 05 .

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(c) DLD1 cells expressing FOXO3(A3)-ER were treated with 4-OHT and MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy. Shown are the mean ± s.d. (n=3). *P0.05.


( d ) DLD1 expressing FOXO ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or non - template ( NT ) control siRNA , stimulated with 4 - OHT for 24 h with or without 3 - methyladenine ( 3 - MA ) . Apoptosis was determined by FACS after labelling with annexin V ( AxV ) - phycoerythrin DAPI . graph shows relative fold versus control samples , mean ± s . e . m . ( n = 3 ) . * P0 . 05 .

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(d) DLD1 cells expressing FOXO(A3)-ER were transfected with glutamine synthetase (GS) siRNA or non-template (NT) control siRNA, and stimulated with 4-OHT for 24 h with or without 3-methyladenine (3-MA). Apoptosis was determined by FACS analysis after labelling cells with annexin V (AxV)-phycoerythrin and DAPI. The graph shows relative fold versus control samples, mean ±  s.e.m. (n=3). *P0.05.


A H & E of sections of intestinal tissues obtained from endoscopic biopsy of COVID - 19 who presented with gastrointestinal ( GI ) symptoms . inflammatory infiltrates were indicated by a yellow arrow . edema was indicated by a red star . Scale bars , 100 µm . B bar chart shows degrees of intestinal inflammation in with COVID - 19 . Number of samples for each group as indicated . Data information : data are shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 H&E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID-19 patients who presented with gastrointestinal (GI) symptoms. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bars, 100 µm. B The bar chart shows the degrees of intestinal inflammation in patients with COVID-19. Number of samples for each group as indicated. Data information: All data are shown as mean ± SD.


C Cytokine in plasma of COVID - 19 with ( n = 12 ) or without ( n = 6 ) GI symptoms by cytokine . Data information : data are shown as mean ± SD . P values are determined by Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Cytokine levels in the plasma of COVID-19 patients with (n=12) or without (n=6) GI symptoms by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


D of plasma VEGF in COVID - 19 with ( n = 8 ) or without ( n = 10 ) disease progression by cytokine . Data information : data are shown as mean ± SD . P values are determined by Student ' s t - .

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

D Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=10) disease progression by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


E Temporal course of plasma VEGF at early late stage of COVID - 19 infection by cytokine . Data shown are of plasma VEGF in with ( n = 8 ) without ( n = 10 ) disease progression at early stage ( one to three days after laboratory - confirmed for COVID - 19 ) late stage ( more than three days after laboratory - confirmed for COVID - 19 ) .

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 O O O O O O O O O O O O O O O O O O O O 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 Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by cytokine assay. Data shown are the levels of plasma VEGF in patients with (n=8) and without (n=10) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19).


F of plasma VEGF in COVID - 19 with ( n = 7 ) or without ( n = 6 ) interstitial vasodilation by cytokine . G of plasma VEGF in COVID - 19 with ( n = 8 ) or without ( n = 5 ) interstitial edema by cytokine . Data information : data are shown as mean ± SD . P values are determined by Student ' s t - .

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 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

F Levels of plasma VEGF in COVID-19 patients with (n=7) or without (n=6) interstitial vasodilation by cytokine assay. G Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=5) interstitial edema by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


A of VEGF - A mRNA in intestinal tissues from COVID - 19 ( n = 5 ) or healthy controls ( n = 5 ) by - seq . Data information : data are shown as mean ± SD . For ( A ) , P values are determined by Student ' s t -

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A Levels of VEGF-A mRNA in the intestinal tissues from COVID-19 patients (n=5) or healthy controls (n=5) by RNA-seq. Data information: All data are shown as mean ± SD. For (A), P values are determined by Student's t-test


C , D Representative H & E ( C ) quantitative of inflammation ( D ) of intestinal tissues from treated by protocol shown in ( B ) . inflammatory infiltrates were indicated by a yellow arrow . Scale bar , 100 µm . Control - Fc , n = 5 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) .

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C, D Representative H&E images (C) and the quantitative analysis of inflammation (D) of the intestinal tissues from animals treated by the protocol shown in (B). The inflammatory infiltrates were indicated by a yellow arrow. Scale bar, 100 µm. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).


E qRT - PCR of of inflammatory factors in duodenum tissues of mice treated with Control - Fc or Spike RBD - Fc . Control - Fc , n = 5 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . ; for ( E ) , P values are determined by Mann - Whitney .

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 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

E qRT-PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test.


F ELISA of VEGF in intestinal tissues of mice treated with Control - Fc or Spike RBD - Fc . Control - Fc , n = 4 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . For ( F ) P values are determined by Student ' s t -

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 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

F ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (F) P values are determined by Student's t-test


G ELISA of VEGF in plasma of mice after six hours after treatment with Control - Fc or Spike RBD - Fc , or seven days after treated with Control - Fc or Spike RBD - Fc daily . Control - Fc , n = 4 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) .

O O O B-MEASURED_VAR 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 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

G ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).


H Evaluation of murine peritoneal permeability six hours after treatment with Control - Fc or Spike RBD - Fc , or seven days after treated with Control - Fc or Spike RBD - Fc daily by TRITC - dextran dye extravasation . For each group , n = 8 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . For ( H ) , P values are determined by Student ' s t -

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 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

H Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (H), P values are determined by Student's t-test


A qRT - PCR of of VEGF mRNA in HUVEC treated with Control - Fc or Spike RBD - Fc . For each group , n = 3 . Data are from 3 independent biological experiments . Data information : data are shown as mean ± SD . For ( A ) P values are determined by Paired Student ' s t -

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 O O O O O O O O O O O O O O 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 qRT-PCR analysis of levels of VEGF mRNA in HUVEC treated with Control-Fc or Spike RBD-Fc. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-test


B ELISA of VEGF in supernatant from Caco - 2 treated with Control - Fc or Spike - Fc . For each group , n = 4 . Data are from 4 independent biological experiments . Data information : data are shown as mean ± SD . For ( B ) , P values are determined by Paired Student ' s t - ;

O O O B-MEASURED_VAR 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 O O

B ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc or Spike-Fc. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-test;


C Immunofluorescence of VEGF , cytokeratin ( CK ) CD34 in intestinal tissues of COVID - 19 . CK CD34 in green , VEGF in red in blue . Scale bars , 50 µm .

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C Immunofluorescence analysis of VEGF, cytokeratin (CK) and CD34 protein in the intestinal tissues of COVID-19 patients. CK and CD34 staining in green, VEGF staining in red and nuclear staining in blue. Scale bars, 50 µm.


D of ERK pERK in Caco - 2 treated with Control - Fc or Spike RBD - Fc by Western blot . expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : data are shown as mean ± SD . For ( D ) , P values are determined by Paired Student ' s t -

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D Levels of ERK and pERK in Caco-2 cells treated with Control-Fc or Spike RBD-Fc by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (D), P values are determined by Paired Student's t-test


E ELISA of VEGF in supernatant from Caco - 2 treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 . For each group , n = 4 . Data are from 4 independent biological experiments . Data information : data are shown as mean ± SD . ; for ( E ) , P values are determined by Mann - Whitney ;

O O O B-MEASURED_VAR O 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 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 ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test;


F , G Immunohistochemical shows ERK ( E ) pERK ( F ) expression in duodenum of COVID - 19 healthy controls . Scale bars , 100 μm . For each group , n = 7 . n , biologically independent samples ( human specimen ) . Data information : data are shown as mean ± SD . For ( F ) ( G ) , P values are determined by Student ' s t -

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

F, G Immunohistochemical staining shows ERK (E) and pERK (F) expression in the duodenum of COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. For (F) and (G), P values are determined by Student's t-test


H Immunohistochemical shows ERK pERK expression in intestinal tissues of mice treated with Control - Fc , Spike RBD - Fc or Spike RBD - Fc combined with SCH772984 . Scale bar , 100 µm . For each group , n = 4 . Data information : data are shown as mean ± SD . for ( H ) P values are determined by one - way ANOVA .

O 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 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

H Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. Data information: All data are shown as mean ± SD. for (H) P values are determined by one-way ANOVA.


I of Ras , c - Raf , pMEK , ERK , pERK p - P90RSK in Caco - 2 treated with Control - Fc , Spike RBD - Fc or Spike RBD - Fc combined with SCH772984 by Western blot . expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : data are shown as mean ± SD . for ( I ) , P values are determined by one - way ANOVA .

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

I The levels of Ras, c-Raf, pMEK, ERK, pERK and p-P90RSK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. for (I), P values are determined by one-way ANOVA.


A normalized of TRITC - Dextran dye from coculture systems in which Caco - 2 were incubated with Control - Fc or Spike RBD - Fc after 24 hours . For each group , n = 3 . Data are from 3 independent biological experiments . Data information : data are shown as mean ± SD . For ( A ) P values are determined by Paired Student ' s t -

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A The normalized fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc or Spike RBD-Fc after 24 hours. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-test


B expression of junctional ZO - 1 , VE - cadherin ( VE - cad ) , phosphorylated VE - cad ( pVE - cad ; 658 ) pVE - cad ( 731 ) in HUVECs co - cultured with supernatant of Caco - 2 that were treated with either Control - Fc or Spike RBD - Fc detected by Western blot . expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : data are shown as mean ± SD . For ( B ) , P values are determined by Paired Student ' s t -

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

B The expression of junctional proteins ZO-1, VE-cadherin (VE-cad), phosphorylated VE-cad (pVE-cad; 658) and pVE-cad (731) in HUVECs co-cultured with the supernatant of Caco-2 cells that were treated with either Control-Fc or Spike RBD-Fc detected by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-test


C , D Immunohistochemical shows of VE - cad ( C ) pVE - Cad ( 731 ) ( D ) in duodenum tissue from COVID - 19 healthy controls . Scale bars , 100 μm . For each group , n = 7 . n , biologically independent samples ( human specimen ) . Data information : data are shown as mean ± SD . for ( C ) ( D ) , P values are determined by Student ' s t -

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C, D Immunohistochemical staining shows levels of VE-cad (C) and pVE-Cad (731) (D) in the duodenum tissue from COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. for (C) and (D), P values are determined by Student's t-test


E Immunofluorescence of VE - cad expression subcellular localization in Caco - 2 that were treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab by confocal microscopy . VE - cad in green in blue . Scale bar , 20 µm .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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

E Immunofluorescence analysis of VE-cad expression and subcellular localization in Caco-2 cells that were treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab by confocal microscopy. VE-cad staining in green and nuclear staining in blue. Scale bar, 20 µm.


F of TRITC - Dextran dye from coculture systems in which Caco - 2 were incubated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab . Data are from five technical replicates with similar results from three biological replicates . Data information : data are shown as mean ± SD . ; for ( F ) , P values are determined by one - way ANOVA .

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F Fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Data are from five technical replicates with similar results from three biological replicates. Data information: All data are shown as mean ± SD. ; for (F) , P values are determined by one-way ANOVA.


G Immunohistochemical shows of VE - cad pVE - Cad ( 731 ) in intestinal tissues of mice treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab . Scale bar , 100 µm . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . for ( G ) , P values are determined by one - way ANOVA .

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G Immunohistochemical staining shows levels of VE-cad and pVE-Cad (731) in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. for (G), P values are determined by one-way ANOVA.


A ELISA of VEGF in intestinal tissues of mice treated with ERK inhibitor or anti - VEGF antibody . SCH772984 , ERK inhibitor ; Bevacizumab , anti - VEGF antibody . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . P values are determined by one - way ANOVA .

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A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


B Evaluation of intestinal permeability in treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation . For each group , n = 6 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . P values are determined by one - way ANOVA .

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B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


C , D Representative H & E ( C ) degrees of inflammation ( D ) of intestinal tissues from treated with SCH772984 or Bevacizumab . inflammatory infiltrates were indicated by a yellow arrow . edema was indicated by a red star . Scale bar , 100 µm . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : data are shown as mean ± SD . P values are determined by one - way ANOVA .

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C, D Representative H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


A , B Western blot of ΔKu80 , PKAc1 - 3Ty ( A ) lysates probed with αTy antibodies . Profilin ( αPRF ) served as loading control .

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A, B Western blot analysis of ΔKu80, PKAc1-3Ty (A) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control.


A , B Western blot of ΔKu80 , PKAr - 3Ty ( B ) lysates probed with αTy antibodies . Profilin ( αPRF ) served as loading control .

A, B Western blot analysis of ΔKu80, PKAr-3Ty (B) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control.


C , D Peripheral localization of PKAc1 - 3Ty ( C ) shown by IFA using anti - Ty antibodies . Antibodies against GAP45 IMC1 were used as markers of plasma IMC , respectively .

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C, D Peripheral localization of PKAc1-3Ty (C) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively.


C , D Peripheral localization of PKAr - 3Ty ( D ) shown by IFA using anti - Ty antibodies . Antibodies against GAP45 IMC1 were used as markers of plasma IMC , respectively .

C, D Peripheral localization of PKAr-3Ty (D) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively.


E Plasma stained with an αSAG1 antibody is separated from IMC stained with anti - Ty after aerolysin treatment , indicating that PKAr - 3Ty localises at IMC .

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E Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti-Ty after aerolysin treatment, indicating that PKAr-3Ty localises at the IMC.


F N - terminus putative acylation site of PKAr is essential for its IMC targeting .

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F The N-terminus putative acylation site of PKAr is essential for its IMC targeting.


G A second copy of DDmyc - PKAc1 is stabilised after 1 hour in presence of Shld - 1 .

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G A second copy of DDmyc-PKAc1 is stabilised after 1 hour in presence of Shld-1.


H A second copy of DDmyc - PKAc1 stabilised with Shld - 1 is targeted to IMC in presence of a second copy of PKArWT - Ty . When of a second copy of PKArG2A - Ty is provided , stabilised DDmyc - PKAc1 does not localise at periphery . Data information : Scale bars = 2 μm .

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H A second copy of DDmyc-PKAc1 stabilised with Shld-1 is targeted to the IMC in presence of a second copy of PKArWT-Ty. When of a second copy of PKArG2A-Ty is provided, stabilised DDmyc-PKAc1 does not localise at the cell periphery. Data information: Scale bars = 2 μm.


A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host . Destabilisation of PKAr - TyDD leads to absence of plaque formation .

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A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells. Destabilisation of PKAr-TyDD leads to the absence of plaque formation.


B A drop in PKAr - TyDD expression is detected 5 hours after Shld - 1 removal a significant growth defect is observed as early as 12 hours later . Profilin ( αPRF ) served as loading control .

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B A drop in PKAr-TyDD expression is detected 5 hours after Shld-1 removal and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control.


C IFA showed that down regulation of PKAr - TyDD leads to abnormal segregation of apicoplast - associated thioredoxin family ( ATrx1 ) 24 hours after Shld - 1withdrawal .

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C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal.


D Replication of PKAr - TyDD grown for 24 hours + / - Shld - 1 indicates that no more than four are observed per vacuole upon PKAr destabilisation . ( 100 were counted in three independent replicates ) .

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D Replication assay of PKAr-TyDD parasites grown for 24 hours +/- Shld-1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation. (100 parasites were counted in three independent replicates).


E , F Stabilisation of a second copy of DDmyc - PKAc1 leads to complete block of growth indicated by plaque ( E )

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E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by plaque assay (E)


E , F Stabilisation of a second copy of DDmyc - PKAc1 leads to complete block of growth indicated by western blot ( F ) .

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E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by western blot analysis (F).


G , H Stabilisation of a second copy of DDmyc - PKAc1 leads to observation of mainly one per vacuole in treated with Shld - 1 ( 100 were counted in three independent replicates ) . Data information : In C G , data are presented as mean ± . Scale bars = 2 μm ( C G ) , 3 mm ( A E ) .

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G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).Data information: In C and G, data are presented as mean ± SEM. Scale bars = 2 μm (C and G), 3 mm (A and E).


G , H Stabilisation of a second copy of DDmyc - PKAc1 leads to observation of mainly one per vacuole in treated with Shld - 1 ( 100 were counted in three independent replicates ) .

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G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).


A A drop in PKAc1 - Ty - iKD expression is detected as early as 24 hours upon ATc treatment with almost no detectable after 36 hours .

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A A drop in PKAc1-Ty-iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours.


B Conditional down - regulation of PKAc1 - Ty - iKD leads to absence of plaque formation . Scale bars = 1 mm ( B )

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B Conditional down-regulation of PKAc1-Ty-iKD leads to the absence of plaque formation.Scale bars = 1 mm (B)


C , D Down - regulation of PKAc1 - Ty - iKD upon ATc treatment leads to increased dispersion after 40 hours premature egress ( data is from three independent biological replicates ) . Scale bars = 20 μm ( C )

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C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Scale bars = 20 μm (C)


C , D Down - regulation of PKAc1 - Ty - iKD upon ATc treatment leads to increased dispersion after 40 hours premature egress ( data is from three independent biological replicates ) . Data information : In D , data are presented as mean ± SD .

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C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).Data information: In D, data are presented as mean ± SD.


E Prematurely egressed PKAc1 - Ty - iKD after 24 hours of ATc treatment invade exit fully lysing monolayer of HFF while non - treated invade initiate a new lytic cycle . Scale bars = 2 . 5 mm ( E )

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E Prematurely egressed PKAc1-Ty-iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle. Scale bars = 2.5 mm (E)


B Addition of Shld - 1 leads to rapid stabilisation of DDmyc - PKArWT - Ty DDmyc - PKArG321E - Ty .

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B Addition of Shld-1 leads to the rapid stabilisation of DDmyc-PKArWT-Ty and DDmyc-PKArG321E-Ty.


C PKAc1 is co - immunoprecipitated with DDmyc - PKArWT - Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [ 35S ] - labeled methionine / cysteine metabolic labelling . Conversely , PKAc1 is co - immunoprecipitated with DDmyc - PKArG321E - Ty in presence or absence of cAMP indicating G321E substitution prevents release of active PKAc1 in presence of cAMP . autoradiogram shown is representative of two independent biological replicates . Data information : In C E - H , data are presented as mean ± .

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C PKAc1 is co-immunoprecipitated with DDmyc-PKArWT-Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [35S]-labeled methionine/cysteine metabolic labelling. Conversely, PKAc1 is co-immunoprecipitated with DDmyc-PKArG321E-Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP. The autoradiogram shown is representative of two independent biological replicates.Data information: In C and E-H, data are presented as mean ± SEM.


D Conditional overexpression of a dominant - negative DDmyc - PKArG321E - Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over - expression of DDmyc - PKArWT - Ty is well tolerated by . Scale bars = 3 mm ( D )

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D Conditional overexpression of a dominant-negative DDmyc-PKArG321E-Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over-expression of DDmyc-PKArWT-Ty is well tolerated by the parasite.Scale bars = 3 mm (D)


E Overexpression of dominant negative DDmyc - PKArG321E - Ty triggers premature egress from infected host prevents initiation of a new lytic cycle as revealed by IFA . are representative of ~ 80 % of population . Blue = DAPI ; red = GAP45 ; green = GRA3 . Data information : In C E - H , data are presented as mean ± . Scale bars = 10 μm ( E )

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E Overexpression of the dominant negative DDmyc-PKArG321E-Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA. Images are representative of ~80% of the parasite population. Blue = DAPI; red = GAP45; green = GRA3.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 10 μm (E)


F In heavily infected , addition of Shld - 1 at 32 hours post - inoculation for 4 hours leads to premature egress of DDmyc - PKArG321E - Ty but not of DDmyc - PKAr - Ty control as assessed by IFA . Data information : In C E - H , data are presented as mean ± . Scale bars = 5 µm ( F )

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F In heavily infected cells, addition of Shld-1 at 32 hours post-inoculation for 4 hours leads to premature egress of DDmyc-PKArG321E-Ty parasites but not of the DDmyc-PKAr-Ty control line as assessed by IFA.Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 5 µm (F)


G of extracellular DDmyc - PKArG321E - Ty released from heavily infected after addition of Shld - 1 at 32 hours post - inoculation for 4 hours ( 100 were counted in three independent replicates ) . Data information : In C E - H , data are presented as mean ± .

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G Quantification of extracellular DDmyc-PKArG321E-Ty parasites released from heavily infected cells after addition of Shld-1 at 32 hours post-inoculation for 4 hours (100 parasites were counted in three independent replicates).Data information: In C and E-H, data are presented as mean ± SEM.


H A non - selective PKA inhibitor , KT5270 , significantly enhances premature egress of DDmyc - PKArWT - Ty in presence of Shld - 1 ( data is from three independent biological replicates ; statistical was done by two - t - ) . Data information : In C E - H , data are presented as mean ± .

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H A non-selective PKA inhibitor, KT5270, significantly enhances the premature egress of DDmyc-PKArWT-Ty parasites in presence of Shld-1 (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).Data information: In C and E-H, data are presented as mean ± SEM.


I Prematurely egressed DDmyc - PKArG321E - Ty after 4 hours of Shld - 1 treatment invade exit host , fully lysing monolayer of HFF while non - treated invade initiate a new lytic cycle . Scale bars = 2 . 5 mm ( I ) .

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I Prematurely egressed DDmyc-PKArG321E-Ty parasites after 4 hours of Shld-1 treatment invade and exit host cells, fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle.Scale bars = 2.5 mm (I).


B Upon Shld - 1 addition , a putative cGMP - specific PDE , PDE2 was more phosphorylated at Serine 1317 in DDmyc - PKArG321E - Ty compared with DDmyc - PKArWT - Ty suggesting a crosstalk between cAMP cGMP signaling .

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B Upon Shld-1 addition, a putative cGMP-specific PDE, PDE2 was more phosphorylated at Serine 1317 in DDmyc-PKArG321E-Ty parasites compared with DDmyc-PKArWT-Ty parasites suggesting a crosstalk between cAMP and cGMP signaling.


C Chemical inhibition of cGMP - dependent PKG by Compound 1 Compound 2 blocks egress induced by 5 µM BIPPO , a PDE inhibitor , or by stabilisation of DDmyc - PKArG321E - Ty in presence of Shld - 1 . A PKGT761M substitution that renders PKG resistant to both compounds indicates that block in egress in only mediated by PKG when 0 . 2 µM Compound 1 is used while block in egress associated with C2 is not specific to PKG inhibition only ( data is from three independent biological replicates ) . Data information : In C D , data are presented as mean ± SD .

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C Chemical inhibition of the cGMP-dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO, a PDE inhibitor, or by stabilisation of DDmyc-PKArG321E-Ty in presence of Shld-1. A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0.2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only (data is from three independent biological replicates).Data information: In C and D, data are presented as mean ± SD.


D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer . Anti - GRA1 antibodies were used as a loading control anti - catalase antibodies served as a lysis control ( data is from three independent biological replicates ; statistical was done by two - t - ) Data information : In C D , data are presented as mean ± SD .

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D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer. Anti-GRA1 antibodies were used as a loading control and anti-catalase antibodies served as a lysis control (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test) Data information: In C and D, data are presented as mean ± SD.


A premature egress induced by PKA genetic inhibition is exacerbated when host are heavily infected ( data is from three independent biological replicates ) .

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A The premature egress induced by PKA genetic inhibition is exacerbated when host cells are heavily infected (data is from three independent biological replicates).


B Transferring supernatants of Shld - 1 treated cultures from infected with DDmyc - PKArG321E - Typarasites at a MOI of 8 induces egress of from infected at a MOI of 2 within three hours ( data is from three independent biological replicates ; statistical was done by two - t - ) .

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B Transferring supernatants of Shld-1 treated cultures from cells infected with DDmyc-PKArG321E-Typarasites at a MOI of 8 induces egress of parasites from cells infected at a MOI of 2 within three hours (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).


C Acidification of culture medium induces egress in less than 10 minutes . effect is inhibited in presence of compound 1 ( data is from two independent biological replicates ) .

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C Acidification of the culture medium induces parasite egress in less than 10 minutes. The effect is inhibited in presence of compound 1 (data is from two independent biological replicates).


D Neutralisation of cultures with weak base NH4Cl blocks premature egress induced by DDmyc - PKArG321E - Ty stabilisation . This block is circumvented by addition of Ca2 + ionophore A23187 or PDE inhibitor BIPPO ( data is from three independent biological replicates ) . Data information : Data are presented as mean ± SD .

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D Neutralisation of cultures with the weak base NH4Cl blocks premature egress induced by DDmyc-PKArG321E-Ty stabilisation. This block is circumvented by addition of the Ca2+ ionophore A23187 or the PDE inhibitor BIPPO (data is from three independent biological replicates). Data information: Data are presented as mean ± SD.


A - D Localization of PDE1 - 3Ty ( A ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , myc - GAP70 were used as markers of plasma , IMC , residual , apical pole , respectively . Data information : Scale bars = 2 μm .

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A-D Localization of PDE1-3Ty (A) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


A - D Localization of PDE2 - 3Ty ( B ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , myc - GAP70 were used as markers of plasma , IMC , residual , apical pole , respectively . Data information : Scale bars = 2 μm .

A-D Localization of PDE2-3Ty (B) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


A - D Localization of GCα - 3Ty ( C ) shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , myc - GAP70 were used as markers of plasma , IMC , residual , apical pole , respectively . Data information : Scale bars = 2 μm .

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A-D Localization of GCα-3Ty (C) shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


A - D Localization of PKG - 3Ty shown by IFA using an anti - Ty antibody . Antibodies against GAP45 , IMC1 , cb - GFP , myc - GAP70 were used as markers of plasma , IMC , residual , apical pole , respectively . Data information : Scale bars = 2 μm .

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

A-D Localization of PKG-3Ty shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.Data information: Scale bars = 2 μm.


E Western blot indicates that GCα - 3Ty is present in three distinct isoforms . A PKG - 3Ty western blot also shows three immune reactive isoforms . Catalase or actin were used as loading controls ΔKu80 as parental .

O O O O 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

E Western blot analysis indicates that GCα-3Ty is present in three distinct isoforms. A PKG-3Ty western blot also shows three immune reactive isoforms. Catalase or actin were used as loading controls and ΔKu80 as the parental strain.