EMBO/SourceData huggingface dataset:
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.
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.
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.
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.
Figure 2A 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 and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control.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 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 the complete block of cell growth indicated by plaque assay (E)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 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 the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).
Figure 3A 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 the absence of plaque formation.Scale bars = 1 mm (B)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 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 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)
Figure 4B 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 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 the parasite.Scale bars = 3 mm (D)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 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 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 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 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).
Figure 5B 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 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 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.
Figure 6A 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 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 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 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.
Figure 7A-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, 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, 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, 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 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 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 The signal of Myc-ACβ-iKD followed by IFA (G) after 48 hours ± ATc.Data information: Scale bars = 2 μm.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 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 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.
Figure 1A, 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 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.
Figure 3B 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 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 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.