Figure 5A ) Expression of PTGES in normal and tumor tissue . B ) Kaplan - Meier analysis of overall ( left ) and progression - free ( right ) survival in patients with lung adenocarcinoma with high PTGES ( top 10 % ) and low PTGES ( bottom 90 % ) expression . C ) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma . D ) IL15 gene expression analysis in high and low NK gene signature ( NK GS ) . E ) Overall survival in patients with stage I ( n = 269 ) lung adenocarcinoma with high ( top 50 % ) and low ( bottom 50 % ) NK cell gene expression signature expression . F ) Overall survival in patients with high ( top 50 % ) PTGES expression in relation to high ( top 50 % ) and low ( bottom 50 % ) NK cell gene expression signature .

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Figure 5A) Expression of PTGES in normal and tumor tissue.B) Kaplan-Meier analysis of overall (left) and progression-free (right) survival in patients with lung adenocarcinoma with high PTGES (top 10%) and low PTGES (bottom 90%) expression.C) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma.D) IL15 gene expression analysis in high and low NK gene signature (NK GS).E) Overall survival in patients with stage I (n=269) lung adenocarcinoma with high (top 50%) and low (bottom 50%) NK cell gene expression signature expression.F) Overall survival in patients with high (top 50%) PTGES expression in relation to high (top 50%) and low (bottom 50%) NK cell gene expression signature.


Figure 6A ) Real time image - based killing of A549 cells by NK cells isolated from two individual healthy donors at an E : T ratio of 10 : 1 . B ) NK cells were stimulated with IL - 2 or IL - 15 in the presence or absence of PGE2 and then purified as CD25 + / CD54 + or CD25 - / CD54 - cells and tested for cytotoxicity against A549 tumor cell in a 51Cr - release assay . E : T ratio = 5 : 1 ( n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - . C ) Representative image of NK cell infiltration ( red ) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells , scale bar : 400 μm . D , E ) Incucyte quantification of D ) total and E ) tumor - infiltrating CD25 + / CD54 + and CD25 - / CD54 - NK cells . RCU , Red Calibrated Unit , ( n = 4 , biological replicates ) . F , G ) Flow cytometry quantification of ( F ) tumor - infiltrating and ( G ) non - infiltrating NK cells . Results are depicted as total NK cells among CD45 positive live cells . ( n = 4 , biological replicates ) . H ) 48 hours dpi zebrafish injected with A549 , A549 + CD25 + / CD54 + , or A549 + CD25 - / CD54 - NK cells after 24 hours . Fluorescent image was taken by Leica epi - fluorescent stereomicroscope . Scale bar : 500 µm . The tumor injection area ( perivitelline space ) is depicted with a dashed line . I ) 3D view of tumor injection area 24 hours posts tumor inoculation ( left ) . Scale bar = 50 µm . The tumor injection area ( perivitelline space ) is depicted with a dashed line . Magnified view for tumor area ( right ) . The A549 - td tumor cells are annotated with a purple translucent mask and NK cells are color - coded based on the distance to the tumor area ( near - far : blue - red ) . Scale bar = 10 µm . J ) The distance between NK cells and tumor area in CD25 - / CD54 - and CD25 + / CD54 + NK cell treated fish ( n = 5 , biological replicates ) . K ) Frequency of tumor infiltrating NK cells in in CD25 - / CD54 - and CD25 + / CD54 + NK cell treated fish ( n = 5 , biological replicates ) . L , M ) Quantification of ( L ) tumor cell and ( M ) NK cell counts 24 hours after injection of NK cells by Imaris ( n = 5 , biological replicates ) . N ) Frequency of tissue - infiltrating total NK cells ( CD56 + / CD3 - ) ( left ) and CD54 positive NK cells ( right ) among CD45 positive live cells ( n = 10 , biological replicates ) .

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Figure 6A) Real time image-based killing of A549 cells by NK cells isolated from two individual healthy donors at an E:T ratio of 10:1.B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-.C) Representative image of NK cell infiltration (red) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells, scale bar: 400 μm.D, E) Incucyte quantification of D) total and E) tumor-infiltrating CD25+/CD54+ and CD25-/CD54- NK cells. RCU, Red Calibrated Unit, (n=4, biological replicates).F, G) Flow cytometry quantification of (F) tumor-infiltrating and (G) non-infiltrating NK cells. Results are depicted as total NK cells among CD45 positive live cells. (n=4, biological replicates).H) 48 hours dpi zebrafish injected with A549, A549+ CD25+/CD54+, or A549+ CD25-/CD54- NK cells after 24 hours. Fluorescent image was taken by Leica epi-fluorescent stereomicroscope. Scale bar: 500 µm. The tumor injection area (perivitelline space) is depicted with a dashed line.I) 3D view of tumor injection area 24 hours posts tumor inoculation (left). Scale bar=50 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. Magnified view for tumor area (right). The A549-td tumor cells are annotated with a purple translucent mask and NK cells are color-coded based on the distance to the tumor area (near-far: blue-red). Scale bar=10 µm.J) The distance between NK cells and tumor area in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). K) Frequency of tumor infiltrating NK cells in in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). L, M) Quantification of (L) tumor cell and (M) NK cell counts 24 hours after injection of NK cells by Imaris (n=5, biological replicates).N) Frequency of tissue-infiltrating total NK cells (CD56+/CD3-) (left) and CD54 positive NK cells (right) among CD45 positive live cells (n=10, biological replicates).


Figure 2 ( A ) . Cell growth assessment of ΔIndLon ( upper plot ) and ΔIndFtsH ( lower plot ) mutants grown under inducing ( blue ) or depleting conditions ( red ) . Growth was monitored by measuring DNA and protein biomass over time . The average from two independent biological replicates is shown . ( D ) . Role of Lon and FtsH under heat shock stress conditions . ΔIndLon and ΔIndFtsH mutants were grown under inducing ( Lon + or FtsH + ) or depleting conditions ( Lon + or FtsH + , 60h of depletion for both , Lon and FtsH ) , and then exposed at 45 ° C during 0 , 5 , 10 , 15 or 20 min . Then , growth after the heat treatment was monitored over time under inducing conditions by the 430 / 560 absorbance rate index that shows pH changes in the medium . The average from two independent biological replicates is shown for each condition .

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Figure 2(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.(D). Role of Lon and FtsH under heat shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon+ or FtsH+, 60h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15 or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition.


Figure 4 ( C ) In vivo degradation assays of FtsA , FtsZ and DnaB . ΔIndLon mutants expressing the different N - terminal FLAG - tagged derivatives were grown in depleting conditions for 36h . Then , Lon expression was transiently induced for 3h before translation was blocked with gentamicin ( Gm ) . Samples were taken at the indicated time points after gentamicin treatment , and processed for immunoblot analysis using anti - FLAG antibodies . LC , loading control . As controls , non - induced cells were also treated with gentamicin and processed at the same time points . Immunoblots are representative of two independent experiments . ( E and F ) Similar to panel B ( E ) and C ( F ) but for a C - terminal FLAG - tagged derivative of MPN201 as a representative of an unstable HsdS subunit . ( H ) Similar to panel B but for C - terminal FLAG - tagged derivatives of FtsH candidate substrates . Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti - FtsH and anti - FLAG antibodies . LC , loading control . ( I ) Similar to panel C , but for FtsH candidate substrates . In this case , C - terminal FLAG - tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment .

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Figure 4(C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.(E and F) Similar to panel B (E) and C (F) but for a C-terminal FLAG-tagged derivative of MPN201 as a representative of an unstable HsdS subunit.(H) Similar to panel B but for C-terminal FLAG-tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti-FtsH and anti-FLAG antibodies. LC, loading control.(I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.


Figure 5 ( B ) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons . N - terminal FLAG - tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant . Protein levels were then determined by immunoblot using anti - Lon and anti - FLAG antibodies comparing inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

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Figure 5(B) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons. N-terminal FLAG-tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant. Protein levels were then determined by immunoblot using anti-Lon and anti-FLAG antibodies comparing inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


Figure 6 ( B ) Lon surveillance of truncated protein variants . As an example , an N - terminal FLAG - tagged derivative of MPN304 , which encodes a truncated variant of ArcA ( ArcA - Nt ) , was expressed in the ΔIndLon mutant , and its accumulation monitored over time under non - inducing conditions using anti - FLAG antibodies . Anti - Lon antibodies were used to monitor Lon depletion along the time course experiment ( western blot figure on left ) . MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants ( 36h of depletion ) , in which protein expression was blocked with gentamicin ( Gm ) during the indicated time points after transient induction of Lon for 3h . As control , samples after Gm treatment were also taken from non - induced samples . LC , loading control ( western blot figure on right ) . Immunoblots are representative of two independent experiments . A schematic representation of the arginine deiminase pathway , which is inactive in M . pneumoniae , and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing ( + ) or depleting ( - ) conditions . ( E ) Protein quality control of terminal organelle assembly mediated by Lon . Top of panel E , a schematic representation of the genetic organization at the P65 operon after deleting the N - terminal region of the hmw2 gene in the ΔIndLon mutant . The expected PCR products for the deletion are also indicated . Below , an agarose electrophoresis gel showing the PCR validation of the intended deletion , and SDS - PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins , whose stability depend on HMW2 , for ΔIndLon or ΔIndLon _ Δhmw2Nt cell lysates grown in inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

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Figure 6(B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions.(E) Protein quality control of terminal organelle assembly mediated by Lon. Top of panel E, a schematic representation of the genetic organization at the P65 operon after deleting the N-terminal region of the hmw2 gene in the ΔIndLon mutant. The expected PCR products for the deletion are also indicated. Below, an agarose electrophoresis gel showing the PCR validation of the intended deletion, and SDS-PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins, whose stability depend on HMW2, for ΔIndLon or ΔIndLon_Δhmw2Nt cell lysates grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


Figure 1A , B , and C . Immunostaining of Tom20 ( mitochondria ) in cells positive for β3 - tubulin in DIV6 Ifnb + / + and Ifnb - / - CNs with or without rIFN - β for 24 h . A . Projection of 3D image of Tom20 . Scale bars equal 20 microns . B . Quantification of total mitochondrial mass per neuron . C . Mean individual mitochondria volume in Ifnb + / + and Ifnb - / - CNs . 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 .

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Figure 1A, B, and C. Immunostaining of Tom20 (mitochondria) in cells positive for β3-tubulin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without rIFN-β for 24 h. A. Projection of 3D image of Tom20. Scale bars equal 20 microns. B. Quantification of total mitochondrial mass per neuron. C. Mean individual mitochondria volume in Ifnb+/+ and Ifnb-/- CNs. 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.


Figure 2D - 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 . 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 + / + and Ifnb - / - mice . N . Immunoblot for optineurin in DIV6 Ifnb + / + and Ifnb - / - CNs with or without 30 U / mL rIFN - β .

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Figure 2D-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. 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+/+ and Ifnb-/- mice. N. Immunoblot for optineurin in DIV6 Ifnb+/+ and Ifnb-/- CNs with or without 30 U/mL rIFN-β.


Figure 3H . 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 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 and Tom20 immunofluorescence in Ifnb + / + and Ifnb - / - MEFs transfected with Drp1 or phosphomimetic Drp1 .

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Figure 3H. 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 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 and Tom20 immunofluorescence in Ifnb+/+ and Ifnb-/- MEFs transfected with Drp1 or phosphomimetic Drp1.


Figure 4E . 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 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 .

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Figure 4E. 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 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.


Figure 5D . 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 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 + / + 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 .

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Figure 5D. 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 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+/+ 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.


Figure 6E - 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 .

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Figure 6E-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.


Figure 1 , 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 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 ) .

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

Figure 1, 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 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).


Figure 3Same 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 ) .

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

Figure 3Same 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).


Figure 4Limited 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 ) .

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

Figure 4Limited 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).


Figure 5Munc18 - 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 ) .

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

Figure 5Munc18-1 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).


Figure 6Uptake 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 ) .

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

Figure 6Uptake 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).


Figure 7Plot 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 .

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

Figure 7Plot 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.


Figure 8Heat - 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 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 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 punctaQuantification 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 ) .

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

Figure 8Heat-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 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 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 punctaQuantification 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).


figf1 ( 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 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 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 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 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 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 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 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 .

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

figf1(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 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 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 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 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 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 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 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.


figf2 ( 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 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 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 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 ) 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 , 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 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 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 .

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

figf2(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 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 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 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) 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, 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 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 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.


figf3 ( 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 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 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 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 .

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

figf3(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 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 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 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.


figf4 ( 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 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 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 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 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 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 .

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

figf4(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 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 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 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 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 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.


figf5 ( 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 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 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 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 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 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 .

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

figf5(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 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 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 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 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 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.


figf6 ( 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 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 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 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 .

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

figf6(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 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 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 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.


Figure 1A 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 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 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 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 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 .

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

Figure 1A 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 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 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 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 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.


Figure 2A 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 - testC , 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 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 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 - testG 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 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 -

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

Figure 2A 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-testC, 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 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 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-testG 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 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


Figure 3A 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 - testB 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 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 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 - testE 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 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 - testH 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 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 .

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

Figure 3A 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-testB 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 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 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-testE 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 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-testH 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 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.


Figure 4A 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 - testB 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 - testC , 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 - testE 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 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 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 .

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

Figure 4A 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-testB 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-testC, 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-testE 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 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 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.


Figure 5A 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 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 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 .

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

Figure 5A 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 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 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.


Figure 1A , 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 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 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 and IMC1 were used as markers of plasma membrane and IMC , respectively . 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 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 . 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 .

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

Figure 1A, 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 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 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 and IMC1 were used as markers of plasma membrane and IMC, respectively.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 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. 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.