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

O O O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O

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


( d ) IL - 3 was withdrawn from Ba / F3 in presence or absence of BafA1 ( 100 nM ) as indicated .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

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

(d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated.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


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

O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-TISSUE O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O B-TISSUE B-DISEASE O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-TISSUE O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O B-DISEASE I-DISEASE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-TISSUE B-GENEPROD O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O B-TISSUE B-GENEPROD O O O O O B-DISEASE I-DISEASE I-DISEASE O O B-EXP_ASSAY O O O O O B-TISSUE B-GENEPROD O O O O 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-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O

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


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

O O B-TISSUE B-GENEPROD O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O B-DISEASE I-DISEASE O B-EXP_ASSAY O O O B-TISSUE B-GENEPROD O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O B-DISEASE I-DISEASE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O B-DISEASE O O O O B-TISSUE O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O

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


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

O O O B-ORGANISM B-TISSUE B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O

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


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

O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-TISSUE O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

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


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

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-TISSUE O O B-ORGANISM O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O B-ORGANISM O O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O B-DISEASE O O O O B-TISSUE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-DISEASE O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O

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


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

O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

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


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

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


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

O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


F PKG - 3Ty were solubilised in either PBS or 1 % Triton X - 100 ( TX100 ) split into soluble ( S ) pellet ( P ) fractions . Total lysate is also shown .

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F PKG-3Ty parasites were solubilised in either PBS or 1% Triton X-100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown.


G , H signal of Myc - ACβ - iKD followed by IFA ( G ) after 48 hours ± ATc . Data information : Scale bars = 2 μm .

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G, H The signal of Myc-ACβ-iKD followed by IFA (G) after 48 hours ± ATc.Data information: Scale bars = 2 μm.


G , H signal of Myc - ACβ - iKD followed by western blot ( H ) after 48 hours ± ATc .

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G, H The signal of Myc-ACβ-iKD followed by western blot (H) after 48 hours ± ATc.


I Invasion of ACα - KO tachyzoites or Myc - Acβ - iKD after 48 hours ± ATc is not affected . However , addition of ATc for 48 hours reduces invasion efficiency of ACα - KO / Myc - Acβ - iKD ( data is from three independent biological replicates ) .

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I Invasion of ACα-KO tachyzoites or Myc-Acβ-iKD after 48 hours ± ATc is not affected. However, addition of ATc for 48 hours reduces the invasion efficiency of ACα-KO/ Myc-Acβ-iKD (data is from three independent biological replicates).


J As for destabilisation of PKAc1 - iKD or stabilisation of DDmyc - PKArG321E - Ty , ATc - KO / Myc - Acβ - iKD tachyzoites treated with lytic cycle for 33 or 40 hours , exit host invade exit monolayer of HFF leading to lysis while non - treated invade initiate a new lytic cycle . Data information : Scale bars = 2 μm .

O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

J As for destabilisation of PKAc1-iKD or stabilisation of DDmyc-PKArG321E-Ty, ATc -KO/ Myc-Acβ-iKD tachyzoites treated with lytic cycle for 33 or 40 hours, exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non-treated parasites invade and initiate a new lytic cycle. Data information: Scale bars = 2 μm.


A , B Hypocotyl of ChWT , ( A ) RNAi - ChHFR1 transgenic ( B ) chfr1 mutant seedlings grown under different R : FR . Seedlings were grown for 7 days in continuous W ( R : FR > 1 . 5 ) or for 3 days in W then transferred to W supplemented with increasing amounts of FR ( W + FR ) for 4 more days , producing various R : FR . Aspect of representative 7 - day old ChWT , RNAi - HFR1 chfr1 - 1 seedlings grown in W or W + FR ( R : FR , 0 . 02 ) , as indicated , is shown in lower panel . Data information : Values are means ± SE of three independent biological replicates relative to ChWT value at 0 h . Asterisks mark significant differences ( Student t - : * * p - value < 0 . 01 ; * p - value < 0 . 05 ) relative to ChWT value at same time point .

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A, B Hypocotyl length of ChWT, (A) RNAi-ChHFR1 transgenic and (B) chfr1 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days in continuous W (R:FR>1.5) or for 3 days in W then transferred to W supplemented with increasing amounts of FR (W+FR) for 4 more days, producing various R:FR. Aspect of representative 7-day old ChWT, RNAi-HFR1 and chfr1-1 seedlings grown in W or W+FR (R:FR, 0.02), as indicated, is shown in lower panel. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.


C , D Effect of W + FR exposure on expression of PIL1 , YUC8 XTR7 genes in seedlings of ChWT , ( C ) RNAi - HFR1 ( D ) chfr1 mutant lines . Expression was analyzed in 7 - day old W - grown seedlings transferred to W + FR ( R : FR , 0 . 02 ) for 0 , 1 , 4 , 8 12 h . Transcript abundance is normalized to EF1α . Data information : Values are means ± SE of three independent biological replicates relative to ChWT value at 0 h . Asterisks mark significant differences ( Student t - : * * p - value < 0 . 01 ; * p - value < 0 . 05 ) relative to ChWT value at same time point .

O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-ORGANISM O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O 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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O 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-ORGANISM O O O O O O

C, D Effect of W+FR exposure on the expression of PIL1, YUC8 and XTR7 genes in seedlings of ChWT, (C) RNAi-HFR1 and (D) chfr1 mutant lines. Expression was analyzed in 7-day old W-grown seedlings transferred to W+FR (R:FR, 0.02) for 0, 1, 4, 8 and 12 h. Transcript abundance is normalized to EF1α levels. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.


Seedlings of ChWT AtWT were grown for 3 days in W then either kept under same conditions or transferred to W + FR ( R : FR , 0 . 02 ) for indicated times . material was harvested every 24 h . Transcript abundance of HFR1 PIF7 was normalized to three reference genes ( EF1α , SPC25 , YLS8 ) . Expression values are means ± SE of three independent biological replicates relative to data of AtWT grown in continuous W at day 3 . Asterisks mark significant differences ( 2 - way ANOVA : * * p - value < 0 . 01 , * * * p - value < 0 . 001 ) between ChWT AtWT when grown under W ( black asterisks ) or W + FR ( red asterisks ) .

O O B-ORGANISM I-ORGANISM O O O O O O 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-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O

Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W+FR (R:FR, 0.02) for the indicated times. Plant material was harvested every 24 h. Transcript abundance of HFR1 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: ** p-value <0.01, *** p-value <0.001) between ChWT and AtWT when grown under W (black asterisks) or W+FR (red asterisks).


B Relative expression of HFR1 in seedlings of AtWT , At hfr1 - 5 , hfr1 > pAt : ChHFR1 ( in blue ) hfr1 > pAt : AtHFR1 ( in red ) lines grown under W + FR ( R : FR , 0 . 02 ) . Expression values are means ± SE of three independent biological replicates relative to data of 7 days old AtWT . Transcript abundance is normalized to UBQ10 . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey , p - value < 0 . 05 ) among means .

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B Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, hfr1>pAt:ChHFR1 (in blue) and hfr1>pAt:AtHFR1 (in red) lines grown under W+FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT. Transcript abundance is normalized to UBQ10 levels. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.


C Elongation response of seedlings of indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W + FR ( R : FR , 0 . 02 ) . mean hypocotyl in W ( HypW ) W + FR ( HypW + FR ) of at least four biological replicates was used to calculate HypW + FR - HypW . Error bars represent SE . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey , p - value < 0 . 05 ) among means .

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C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W+FR (R:FR, 0.02). The mean hypocotyl length in W (HypW) and W+FR (HypW+FR) of at least four biological replicates was used to calculate HypW+FR-HypW. Error bars represent SE. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.


D Relative HFR1 in seedlings of indicated lines , normalized to actin , are means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 that is taken as 1 . Seedlings were grown for 7 days in continuous W ( ~ 20 µmol m - 2 s - 1 ) after which they were incubated for 3 h in high W ( ~ 100 µmol m - 2 s - 1 ) transferred to W + FR ( R : FR , 0 . 06 ) for 3 h . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey , p - value < 0 . 05 ) among means .

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D Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and transferred to W+FR (R:FR, 0.06) for 3 h. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.


A Expression of HFR1 of HFR1 - 3xHA in seedlings of hfr1 > pAt : ChHFR1 ( # 22 ) hfr1 > pAt : AtHFR1 ( # 13 ) . Seedlings were grown for 7 days in continuous W ( ~ 20 µmol m - 2 s - 1 ) after which they were incubated for 3 h in high W ( ~ 100 µmol m - 2 s - 1 ) then either kept at high W or transferred to W + FR ( R : FR , 0 . 06 ) for 3 or 6 h , as indicated in cartoon at top . Relative HFR1 transcript , normalized to UBQ10 , are means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 grown for 3 h under W + FR . Relative , normalized to actin , are means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 . Samples were collected at data points marked in cartoon with asterisks .

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A Expression of HFR1 and protein levels of HFR1-3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and then either kept at high W or transferred to W+FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W+FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks.


C Relative HFR1 transcript transiently expressed in tobacco leaves , normalized to GFP , are means ± SE of three independent biological replicates ( left ) . Relative HFR1 , normalized to GFP , are means ± SE of four independent biological replicates ( right ) . In A C , asterisks mark significant differences ( Student t - : * p - value < 0 . 05 , * * p - value < 0 . 01 ) between indicated pairs .

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C Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In A and C, asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.


D Degradation of ChHFR1 ( 35S : ChHFR1 ) AtHFR1 ( 35S : AtHFR1 ) in tobacco leaf discs treated with cycloheximide ( CHX , 100 µM ) for indicated times . Tobacco were kept under high W ( ~ 200 µmol m - 2 s - 1 ) for 3 days after agroinfiltration then leaf circles were treated with W + FR ( R : FR , 0 . 2 ) CHX . Relative HFR1 ( ChHFR1 , blue bars ; AtHFR1 , red bars ) , normalized to GFP , are means ± SE of four biological replicates relative to data point 0 , taken as 1 for each . Asterisks mark significant differences ( 2 - way ANOVA : * p - value < 0 . 05 ) between ChHFR1 AtHFR1 at same time point .

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D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at the same time point.


B Table summaries of microscale thermophoresis binding of respective synthetic peptides is indicated .

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B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated.


D Relative HFR1 , normalized to GFP , are means ± SE of four independent biological replicates . Asterisks mark significant differences ( Student t - : * p - value < 0 . 05 , * * p - value < 0 . 01 ) between indicated pairs .

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D Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.


A Y2H growth showing interaction between AtHFR1 AtPIF7 . BD - AD - derivative constructs used in are shown on left side of panel . SD - LW or SD - HLW refer to selective medium ( plated as drops in dilutions of 1 , 1 : 10 1 : 100 ) indicative of transformed or interaction between hybrid , respectively . Truncated forms of murine p53 ( BD - fused ) SV40 large T - antigen ( AD - fused ) , known to interact , were used as a positive control . Empty vectors ( / ) were used as negative controls .

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A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7. The BD- and the AD- derivative constructs used in the assay are shown on the left side of the panel. SD-LW or SD-HLW refer to the selective medium (plated as drops in dilutions of 1, 1:10 and 1:100) indicative of transformed cells or interaction between the hybrid proteins, respectively. Truncated forms of murine p53 (BD-fused) and SV40 large T-antigen (AD-fused), known to interact, were used as a positive control. Empty vectors (/) were used as negative controls.


Hypocotyl of seedlings of AtWT , ( B ) pif7 - 1 , hfr1 - 5 , pif7 - 1 hfr1 - 5 ( top graph ) , pif7 - 2 , hfr1 - 5 pif7 - 2 hfr1 - 5 ( bottom graph ) mutants grown under different R : FR . Seedlings were grown in W ( R : FR > 1 . 5 ) for 7 days or for 2 days in W then transferred to two W + FR treatments ( R : FR 0 . 06 or 0 . 02 ) for 5 additional days . Values of hypocotyl are means ± SE of three independent biological replicates ( at least 10 seedlings per replica ) .

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Hypocotyl length of seedlings of AtWT, (B) pif7-1, hfr1-5, pif7-1 hfr1-5 (top graph), pif7-2, hfr1-5 and pif7-2 hfr1-5 (bottom graph) mutants grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica).


C Hypocotyl of seedlings of AtWT , ( C ) transgenic 35S : GFP - ΔNt - HFR1 ( 35S : ΔNt - HFR1 ) , two lines of 35S : PIF7 - CFP ( 35S : PIF7 # 1 # 2 ) , 35S : GFP - ΔNt - HFR1 35S : PIF7 - CFP double transgenic ( 35S : ΔNt - HFR1 x 35S : PIF7 # 1 # 2 ) seedlings grown under different R : FR . Seedlings were grown in W ( R : FR > 1 . 5 ) for 7 days or for 2 days in W then transferred to two W + FR treatments ( R : FR 0 . 06 or 0 . 02 ) for 5 additional days . Values of hypocotyl are means ± SE of three independent biological replicates ( at least 10 seedlings per replica ) . D Aspect of representative 7 - day - old W - grown seedlings shown in C . Scale bar is 1 cm .

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C Hypocotyl length of seedlings of AtWT, (C) transgenic 35S:GFP-ΔNt-HFR1 (35S:ΔNt-HFR1), two lines of 35S:PIF7-CFP (35S:PIF7 #1 and #2), and 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double transgenic (35S:ΔNt-HFR1 x 35S:PIF7 #1 and #2) seedlings grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica). D Aspect of representative 7-day-old W-grown seedlings shown in C. Scale bar is 1 cm.


C Hypocotyl of seedlings of ( left ) AtWT , pifq , pif7 - 2 , ChWT , ( middle ) 35S : GFP - ΔNt - HFR1 ( ΔNtHFR1 ) , hfr1 - 5 ( right ) chfr1 - 1 chfr1 - 2 lines grown at warm temperatures . Hypocotyl lengths are means ± SE of three biological replicates . Asterisks mark significant differences ( Student t - : * p - value < 0 . 05 , * * p - value < 0 . 01 ) relative to same genotype grown at 22ºC ( left right graphs , black asterisks ) , between indicated pairs ( middle graph , red asterisks ) .

O B-TISSUE O B-ORGANISM O O O O B-ORGANISM O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Hypocotyl length of seedlings of (left) AtWT, pifq, pif7-2, ChWT, (middle) 35S:GFP-ΔNt-HFR1 (ΔNtHFR1), hfr1-5 and (right) chfr1-1 and chfr1-2 lines grown at warm temperatures. Hypocotyl lengths are the means ± SE of three biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) relative to the same genotype grown at 22ºC (left and right graphs, black asterisks), and between the indicated pairs (middle graph, red asterisks).


F Relative chlorophylls of ( left ) AtWT , pifq , ChWT , ( middle ) ΔNtHFR1 , hfr1 - 5 ( right ) chfr1 - 1 chfr1 - 2 lines after DIS was promoted for indicated time . For each genotype , values are relative to pigment at time 0 ( 7 days in W ) . Data are means ± SE of four independent biological replicates .

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F Relative chlorophylls levels of (left) AtWT, pifq, ChWT, (middle) ΔNtHFR1, hfr1-5 and (right) chfr1-1 and chfr1-2 lines after DIS was promoted for the indicated time. For each genotype, values are relative to pigment levels at time 0 (7 days in W). Data are the means ± SE of four independent biological replicates.


G Aspect of 4 - day old - grown seedlings of AtWT , pifq , pif7 - 2 , hfr1 - 5 ΔNt - HFR1 ( left panel ) , AtWT , ChWT chfr1 - 1 ( right panel ) .

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G Aspect of 4-day old dark-grown seedlings of AtWT, pifq, pif7-2, hfr1-5 and ΔNt-HFR1 (left panel), and AtWT, ChWT and chfr1-1 (right panel).


( A ) Upper panel : immunoblot of COS7 treated for 12 h with MG132 ( 25 μM ) vinblastine ( 25 μM ) . Lower panel : real ‐ time PCR showing gene expression changes in COS7 following MG132 exposure ( 25 μM , 12 h ) .

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(A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h).


( B , C ) Immunofluorescence of indicated in COS7 treated as in ( A ) . Scale bar , 20 μm .

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(B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm.


( D ) COS7 were transfected for 24 h with nonsense or bag3 siRNA treated with MG132 ( 25 μM , 12 h ) . Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online ) . Diagram shows of aggresome ‐ positive .

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(D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells.


( E ) Aggresome as in supplementary Fig S1D online from analysed in ( H ) . are shown in supplementary Fig S1G online . Diagram shows of aggresome ‐ positive at 4 , 24 48 h after MG132 wash out .

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(E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out.


( F ) were treated as in ( H ) ; however , 4 h after MG132 wash out , BafA1 ( 5 μM ) was added for 20 h to inhibit aggresome clearance . Diagrams show of aggresome ‐ positive mean of aggresomes ( supplementary Fig S1H online ) . are shown in supplementary Fig S1G online .

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(F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes ( supplementary Fig S1H online). Images are shown in supplementary Fig S1G online.


( G ) Dot ‐ blot ( upper panel , in supplementary Fig S1B online ) western blot ( lower panel ) analyses of 0 . 5 % NP40 ‐ soluble ‐ insoluble from shown in Fig 1D ; supplementary Fig S1D online .

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(G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online.


( H ) COS7 transfected with BAG3 or empty vector were treated with either MG132 ( 25 μM , 18 h ) or DMSO as a control . After MG132 wash out , were allowed to recover for 4 , 24 48 h 0 . 5 % NP40 ‐ soluble ‐ insoluble were analysed by immunoblotting .

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(H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting.


( I ) COS7 were transfected for 24 h with GST or GST fused to DIC ( GST ‐ DIC ) treated with MG132 ( 25 μM , 12 h ) . Indicated in whole ‐ lysates in precipitates obtained by GST pull ‐ down with glutathione beads were analysed by immunoblotting . BafA1 , bafilomycin A1 ; ctrl , control ; DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; Hsp70 , heat ‐ shock 70 ; nons , nonsense ; siRNA , short ‐ interfering ; vinblast , vinblastine .

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(I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.


( A ) GST pull ‐ down as in Fig 1I , but performed in transfected for 36 h with full ‐ FLAG ‐ BAG3 ( I ) or BAG3 deletion mutants FLAG - ( Δ421 - 498 ) - BAG3 ( II ) , FLAG - BAG ‐ domain ( III ) , FLAG - PxxP - BAG ‐ domain ( IV ) . Asterisk , nonspecific band .

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(A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG-(Δ421-498)-BAG3 (II), FLAG-BAG‐domain (III), and FLAG-PxxP-BAG‐domain (IV). Asterisk, nonspecific band.


( B ) HEK were transfected for 18 h with SODG85R ‐ GFP , along with indicated FLAG ‐ tagged BAG3 constructs ( shown in A ) HA ‐ tagged Hsp70 ( HA ‐ Hsp70 ) . SODG85R ‐ GFP showed three distinct localization patterns : , pre ‐ aggresomal aggresomal ( see representative pictures on right supplementary Fig S2F online ) . percentages of bearing pre ‐ aggresomal ( upper panel ) aggresomal ( lower panel ) SODG85R ‐ GFP are shown . Scale bar , 10 μm .

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(B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm.


( C ) HEK transfected with SODG85R ‐ GFP , BAG3 Hsp70 were additionally transfected with FLAG ‐ p50 , as indicated . Diagrams show percentages of with pre ‐ aggresomal aggresomal SODG85R ‐ GFP . Right panel : representative image showing pre ‐ aggresomal localization of FLAG ‐ p50 SODG85R ‐ GFP . Scale bar , 5 μm .

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(C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm.


( D ) GST pull ‐ down as in ( A ) , but in HEK transfected for 18 h with SODWT ‐ GFP or mutant SODG85R ‐ GFP , along with FLAG ‐ BAG3 .

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(D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3.


( E ) HEK were transfected with indicated plasmids with nonsense or bag3 siRNA . At 6 h after transfection , lactacystin ( 10 μM ) or GST as vehicle control was added for GST GST pull ‐ down was performed as in ( D ) .

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(E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D).


( F ) Upper panel : sucrose ‐ density gradient of extracts from HEK transfected with SODG85R ‐ GFP GST ‐ DIC , along with FLAG ‐ BAG3 or vector control . Note immunoreactive bands in high ‐ density fraction 14 on BAG3 overexpression ( arrow ) . Migration of a 20S marker is indicated . Lower panel : GST ‐ DIC pull ‐ down analyses of dynein complexes present in sucrose fraction 6 14 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O

(F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14.


( G ) GST pull ‐ down of SODG85R ‐ GFP as in ( D ) but in transfected with FLAG ‐ tagged BAG3 constructs ( I - IV ; indicated as in A ) .

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(G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I-IV; indicated as in A).


( H ) GST pull ‐ down of SODG85R ‐ GFP as in ( D ) but in treated with PYR41 ( 10 μM ; 12 h ) , as indicated . DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; HA , haemagglutinin ; Hsp70 , heat ‐ shock 70 ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short ‐ interfering ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O

(H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( A ) of SODG85R ‐ GFP ( green ) localization with aggresome marker vimentin , autophagic receptors p62 NBR1 , autophagosome marker LC3 lysosomal marker LAMP2 ( in red ) in BAG3 / Hsp70 ‐ transfected HEK . Representative merged of with , pre ‐ aggresomal aggresomal SOD distribution are shown . Arrows indicate LC3 ‐ positive autophagosomes . Scale bar , 5 μm .

O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O

(A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm.


( B ) Transmission electron microscope of SODG85R ‐ GFP aggresome in transfected as in ( A ) . Marked in ( a ) is shown magnified in ( b ) . Arrows , autophagosomes . Arrowheads , autolysosomes . Scale bar , 1 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD 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-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O

(B) Transmission electron microscope analysis of SODG85R‐GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm.


( C ) HEK transfected for 8 h with SODG85R ‐ GFP along with FLAG ‐ BAG3 HA ‐ Hsp70 , as indicated , were treated for 24 h with BafA1 ( 0 . 1 μM ) . Indicated were analysed by immunoblotting . Diagram shows of SODG85R ‐ GFP in BafA1 ‐ treated to those in untreated .

O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O

(C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells.


( D ) Same as in ( C ) but with SODWT ‐ GFP .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O

(D) Same analysis as in (C) but with SODWT‐GFP.


( E ) Similar as in ( C ) but in transfected 24 h before with nonsense or atg7 siRNA .

O O O O O O O O O O O O O O O O O O B-GENEPROD O O

(E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA.


( F ) HEK were transfected with SODG85R ‐ GFP indicated FLAG ‐ tagged BAG3 constructs together with HA ‐ Hsp70 , lysosomal degradation of SODG85R ‐ GFP was examined as in ( C ) . Corresponding immunoblots are shown in supplementary Fig S3F online . Ag , aggresome ; BafA1 , bafilomycin A1 ; GFP , green ; HA , haemagglutinin ; Hsp70 , heat ‐ shock 70 ; N , nucleus ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short interfering ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O

(F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( A ) Spinal cord sections of mice overexpressing SODWT SODG85R ( disease end ‐ stage ) were immunostained for BAG3 SOD1 . Arrows indicate SOD1 ‐ BAG3 ‐ positive in neuropil . Scale bar , 20 μm .

O O O B-TISSUE I-TISSUE O O B-ORGANISM O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O O

(A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm.