EMBO/SourceData huggingface dataset:
Figure 3A PA promotes the phosphorylation of SCaBP8 by SOS2. The phosphorylation activity of His-SOS2 on His-SCaBP8 was detected with or without 1 or 10 μM PA. The solvent of methanol was used as mock control. Coomassie brilliant blue-stained (CBB) gel is shown in the upper panel, and autoradiograph (autorad) results are shown in the middle and lower panels. The numbers indicate the relative ratios of the signal intensity of autoradiograph. Statistical analysis of the relative ratios is shown B PA promotes the phosphorylation of SOS1 C300 by SOS2. CBB gel is shown in the upper panel, and autorad result is shown in the lower panel. The numbers indicate the relative ratios of the signal intensity of autoradiograph. Statistical analysis of the relative ratios is shownC PA promotes the activity of SOS2 under salt stress. Ten-d-old transgenic plants expressing 35S:6×Myc-SOS2 in Col-0 or pldα1 pldδ-1 background were treated with or without 100 mM NaCl or 50 μM PA for 12 h. The total protein was extracted and immunoprecipitated with anti-Myc antibody-conjugated agarose, and then the purified Myc-SOS2 proteins at similar protein levels were incubated with His-SCaBP8 in the kinase assays. His-SCaBP8 was used as a specific substrate of SOS2. The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc-SOS2 bands (Autorad/Myc). Statistical analysis of the relative ratios is shown D PA promotes the PM localization of SOS2 under salt stress. Ten-d-old seedlings were treated as described in Fig 3C. GHR1 was used as a plasma membrane marker. PEPC was used as a cytoplasm marker. The numbers represent the relative ratios of the signal intensity between Myc-SOS2 and GHR1 bands (Myc/GHR1). Statistical analysis of the relative ratios is shown
Figure 4B MST assays show that the site K57 is critical for SOS2 binding to PA. His-SOS2 with indicated point mutations were labeled and kept at a constant concentration (0.5 μM). PA was hydrated in TBST buffer and diluted 15 times in a 1:1 ratio.D SOS2K57G has weak activity and cannot be activated by PA under salt stress. Ten-d-old seedlings expressing 35S:6×Myc-SOS2 or 35S:6×Myc-SOS2K57G in Col-0 or pldα1 pldδ-1 background were treated with or without 100 mM NaCl for 12 h. The brightness and contrast (B&C) of the autoradiograph was adjusted to clearly show the activity of SOS2K57G. The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc-SOS2 bands (Autorad/Myc). Statistical analysis of the relative ratios is shownE The site K57 is important for the PM localization of SOS2 under salt stress. Ten-d-old seedlings were treated with or without 100 mM NaCl for 12 h. The numbers represent the relative ratios of the signal intensity between Myc-SOS2 and GHR1 bands (Myc/GHR1). The asterisk points to nonspecific band. Statistical analysis of the relative ratios is shown
Figure 5A The loss-of-function mutants akt1-1 and akt1-2 are salt-sensitive. Six-d-old seedlings of Col-0 (WT) and akt1 mutants were treated with or without 100 mM NaCl for 7 d. B Analysis for primary root length, related to Fig 5A. Data was shown in a box and whiskers (min to max) form (n ≥ 10). Statistical analysis was done by two-way ANOVA with Holm-Sidak's multiple comparisons test (P ≤ 0.05). CC, D Analysis of the average values of the net Na+ (C) and K+ (D) fluxes of Col-0 and akt1 mutants with or without 100 mM NaCl treatment for 24 h. Error bars represent SD (n ≥ 4).K Time- and voltage-dependent AKT1-mediated K+ current recordings from oocytes. Whole-cell K+ currents were recorded in oocytes that were co-injected with indicated mixture of cRNAs for AKT1, SCaBP8 and SOS2 (SOS2WT or SOS2K57G), in the presence of CIPK23 and CBL1. 30% SOS2 means that the cRNA injection amount of SOS2 was 30% of SCaBP8. The oocytes were treated with or without 50 μM PA for 1.5 h.
Figure 6D SOS2K40N and SOS2T168D∆FISL attenuate the SCaBP8-mediated inhibition of AKT1 in oocytes. The recorded oocytes were co-injected with indicated mixture of cRNAs for AKT1, SCaBP8 and SOS2 (SOS2WT, SOS2T168D∆FISL or SOS2K40N) in the presence of CIPK23 and CBL1.E I-V relationship of AKT1 steady-state currents in oocytes. Error bars represent SD (n ≥ 7).F Comparison of steady-state currents elicited by -150 mV, related to Fig 6E.
Figure 1B. Interaction between PIF7 and ASF1A/ASF1B was detected using a pull-down assay. SUMO-His-fused PIF7 and GST-ASF1A/ASF1B were purified from E. coli.C. Interaction between PIF7 and ASF1A/ASF1B was detected using a semi-in vivo pull-down assay. GST-ASF1A/ASF1B were purified from E. coli. PIF7-Flash protein from overexpressing PIF7-Flash seedlings grown under white light or 1 h of shade treatment was detected using an anti-MYC antibody.E. Interaction between HIRA and ASF1A was detected using a pull-down assay. TF-His-HIRA and GST-ASF1A were purified from E. coli.F. Interactions between ASF1A and PIF7/HIRA were detected in tobacco leaf cells. The PIF7-Flash, HIRA-HA, and GFP-ASF1A (GFP as control) proteins were co-expressed in tobacco leaf cells. Anti-GFP Sepharose beads were used for Co-IP assay.
Figure 2B, Growth analysis of Col-0, pif7-1, asf1ab, and hira-1 seedlings treated with shade. New hypocotyl growth and real-time growth rates were recorded at 15-min intervals after shade treatment.D. Hypocotyl lengths of Col-0, phyB-9, asf1ab, phyB-9 * asf1ab, hira-1, and phyB-9 * hira-1 grown under white light. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one-way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype. E. Hypocotyl lengths of Col-0, pif7-1, asf1ab, pif7-1 * asf1ab, hira-1, and pif7-1 * hira-1 under white light and shade conditions. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one-way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.
Figure 3B. Boxplot displays the fold changes in shade-induced genes in Col-0, pif7-1, asf1ab, and hira-1 by comparing the transcript levels between white light and shade conditions. Different letters indicate significant differences (P < 0.05) as determined by one-way ANOVA.
Figure 4D. Integrative genomics viewer (IGV) screenshots showing the distribution of GFP-ASF1A enrichment at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci.G. ChIP-PCR analysis of ASF1 enrichment using anti-ASF1A antibodies at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci. Col-0 and pif7-1 seedlings were grown under continuous white light or transferred to the shade for 1 h. Top panels show a schematic representation of the gene structures. The bottom panels represent the effects of shade on ASF1A enrichment. Shade-increased enrichment of ASF1A was calculated as input% SH minus input% WL. The difference at the P2 locus in Col-0 was normalized to one for each gene. Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, where n refers to technical replicates).
Figure 5G. IGV screenshots show the distribution of H3.3 and H3.1 enrichment levels at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci.H. Hypocotyl lengths of Col-0, pif7-1, and h3.3kd under white light and shade conditions. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one-way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.I. Relative expression levels of ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 in Col-0, pif7-1, and h3.3kd seedlings grown under white light or shade conditions. The seedlings were grown under white light for 6 d, and then maintained under white light, or transferred to shade for 1 h. Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).
Figure 6E. ChIP-PCR analysis of HTR5 (H3.3) levels at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci. Top panels show a schematic representation of the gene structures. The bottom panels represent the effects of shade on H3.3 enrichment. Shade-increased enrichment of H3.3 was calculated as the input% SH minus input% WL. The difference at the P2 locus in Col-0 was normalized to one for each gene. Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the means ± SDs (n = 3, where n refers to biological replicates).
Figure 1(D, E) Kaplan-Meier curve analysis was conducted to evaluate the relapse-free survival or overall survival probabilities of colon cancer (n=1342) or lung cancer (n=2166) patients based on low or high levels of FBL expression. Data were sourced from an online tool website (https://kmplot.com/analysis/). The lower quartile-based partitioning method was used to select upper and lower thresholds to subset tumor samples into low and high groups. A cut-off value of the 25th percentile was defined as the low level of FBL in colon or lung cancer patients.(F) Representative images of immunohistochemical analysis of FBL expression levels in human lung or colon tumors and their respective normal tissues (n=40). Scale bar, 25 μm.(H, I) Enzymatic activity of FBL is not required for cell proliferation. Colony formation assays were conducted to investigate the role of enzymatic activity in cell proliferation. FBL knockdown HCT116 or A549 cells with ectopic expression of FBL or the catalytically inactive mutant (FBL-E191A/ D236A) were used, with a bar graph showing the relative number of colonies per well (mean ± SD). All cells were allowed to form colonies for 10 days and representative images of colonies in plates stained with Giemsa were displayed. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using one-way ANOVA. ns denotes no statistical significance, * indicates P < 0.05, *** indicates P < 0.001.
Figure 2(A, B) Knockdown of FBL results in increased cellular sensitivity to DNA damage treatment. Cell viability analysis was performed on control (shNC) and shFBL-HCT116/shFBL-A549 cells treated with indicated concentrations of 5 μM MMC using CCK-8 assays. Each data point represents the mean ± SD from three replicates. The statistical significance was determined using Student's t-test. ***p < 0.001.(D-F) Immunofluorescence staining showed the formation of γH2AX and BRCA1 foci in shFBL and shNC HCT116 cells treated with 5 μM MMC or mock treatment. The scale bar represents 10 μm. The (E) and (F) panel displays quantification of the (D) panel: indicating the number of γH2AX or BRCA1 foci per cell (n≥100 cells). Data are shown as the mean ± SD. The statistical significance was determined using one-way ANOVA, with ns indicating no statistical significance, *** indicating P < 0.001.
Figure 3(B). Co-IP analyses of GFP- tagged FBL and SFB-tagged YBX1 in HCT116 cells using indicated antibodies. HCT116 cells were transfected with vectors encoding GFP-FBL and SFB-YBX1. Cells were lyzed with NETN300 buffer. Cell extracts were examined by IP and western blot with indicated antibodies.(C). Co-IP analyses of endogenous FBL and YBX1 in HCT116 cells using indicated antibodies. Cells were lyzed with NETN300 buffer, and lysates were examined by IP and western blot with indicated antibodies. GAPDH was used as the protein-loading control.(E) The N-terminal GAR domain is essential for the interaction between FBL and YBX1. Upper panel, a schematic representation of wild-type FBL with the GAR domain and methyltransferase domain (MTase). Lower panel, Co-IP analyses of GFP-tagged FBL deletion mutants and SFB-tagged YBX1 in HCT116 cells.(F) The B/A repeat domain of YBX1 is essential for the interaction between YBX1 and FBL. Upper panel, a schematic representation of wild-type YBX1 with the Alanine/proline rich domain, Cold shock domain and B/A repeat domain. Lower panel, co-IP analyses of GFP-tagged YBX1 deletion mutants and SFB-tagged FBL in HCT116 cells.
Figure 4(B) HCT116 cells were treated with or without 5 μM MMC overnight or 25 μM cisplatin for 2 hours, cells were then harvested and separated into nuclear and cytoplasmic fractions. The protein level of YBX1 in each fraction was detected by Western blotting assays. GAPDH and Histone 3 (H3) were used as loading controls for cytoplasmic and nuclear fractions, respectively.(C) Subcellular localization of YBX1/FBL was confirmed by immunofluorescence staining following MMC (5 μM, overnight) or cisplatin (25 μM, 2 hours) treatment in shFBL and shNC cells. Scale bar, 10 μm.(D) The endogenous association between FBL and YBX1 in HCT116 cells was examined by co-immunoprecipitation assays with 5 μM MMC treatment. Cells were lyzed with NETN300 buffer, and nuclear lysates were examined by IP and western blot with indicated antibodies. GAPDH was used as the protein-loading control.(E) The distribution of YBX1 proteins in the nuclear and cytoplasmic fractions of control or FBL KD HCT116 cells after 5 μM MMC incubation overnight were determined by western blotting assays. H3 was used as a nuclear control. GAPDH was used as a cytoplasmic control.(F) The protein contents of YBX1 in the nuclear and cytoplasmic fractions in FBL KD HCT116 cells reconstituted with wild-type FBL or deletion mutants of FBL with 5 μM MMC treatment overnight were detected by western blotting assays with indicated antibodies.
Figure 5(A) A Volcano plot of differentially expressed gene analysis results for RNA-seq data comparing FBL KD HCT116 cells (n = 3) to control cells (n = 3). The dots for significant upregulated or downregulated genes were colored in red or blue, respectively.(E) The mRNA and protein levels of BRCA1 and RAD51 were validated by RT-qPCR and western blot assays using FBL KD HCT116 cells or control cells. Ct values were normalized to GAPDH. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using Student's t-test. ***P < 0.001.(F) The mRNA and protein levels of BRCA1 were validated by RT-qPCR and Western blotting assays using YBX1-silenced HCT116 cells or control cells. Ct values were normalized to GAPDH. The bar graphs present data as mean ± SD from three independent experiments. The statistical significance was determined using Student's t-test. ***P < 0.001.
Figure 6(C) HE staining of tumor tissues. Scale bar, 50 µm. (D Representative images of immunohistochemical staining showing BRCA1 and γH2AX expression levels in MMC-treated and untreated xenograft tumors driven by control or FBL KD HCT116 cells. panel displays quantification of the (D) panel: showing BRCA1 and γH2AX expression levels. Scale bar, 25 µm. Graphs represent the mean ± SD (n = 3 mice in each group). The statistical significance was determined using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.(F, G) Immunofluorescence for TUNEL staining of tumor tissues. Quantification of the percentage of apoptotic cells is shown on the (G) panel. Scale bar, 50 µm. Graphs represent the mean ± SD (n = 3 mice in each group). The statistical significance was determined using one-way ANOVA. *P < 0.05, ***P < 0.001.
Figure 1C-E NF2 and WWC1-3 regulate the kinase activity of LATS1, but not MST1 or MAP4K4. LATS1, MST1 or MAP4K4 was immunoprecipitated from WT or KO HEK293A cells and subjected to in vitro kinase assay using purified GST-YAP or GST-LATS2 as substrates. Phosphorylation-specific signals of YAP reflect the activity of LATS1, and phosphorylation-specific signals of LATS2 reflect the activity of MST1 and MAP4K4.H NF2 and WWC2 interact with LATS1 as indicated by BioID labeling. HEK293A cells stably expressing BirA-NF2, WWC2, MOB1A, or SAV1 were labeled with biotin overnight, and endogenous kinases were immunoprecipitated and subjected to immunoblotting using Streptavidin-HRP. No biotinylation of MAP4K4 was detected.
Figure 2B-D NF2 is required for the activation of LATS1/2 by RAP2A. WT and NF2 KO HEK293A cells were transfected with or without HA-RAP2A. Immunoprecipitated LATS1 (B), MAP4K4 (C) or MST1(D) was then subjected to in vitro kinase assays.E, F NF2 is required for the activation of LATS1/2 by MAP4K kinases. MST1/2 dKO or MST1/2;NF2 3KO cells were transfected with siRNAs against STRIP1/2. Immunoprecipitated MAP4K4 (E) or LATS1 (F) was then subjected to in vitro kinase assays.
Figure 3B Both HPO1 and HPO2 are required for LATS1/2 activation by serum deprivation. Control or KO HEK293A cells were serum starved for 0, 1 or 2 hours, and cell lysates were subjected to immunoblotting to analyze LATS1 and YAP phosphorylation (phostag gel). Genotypes (in B and C) labeled in blue, orange, or red indicate deficiencies in HPO1, HPO2, or both, respectively.C Both HPO1 and HPO2 are required for LATS1/2 activation by actin disrupting agent LatB. Control or KO HEK293A cells were treated with 0.2 μg/ml LatB for 30 min, and cell lysates were subjected to immunoblotting to analyze phosphorylation of LATS1, YAP, NF2, and MOB1. Asterisk indicates S13 phosphorylated NF2.
Figure 4A Survival curves showing life expectancies of mice with genetic deletions of HPO1 and/or HPO2 genes. n≥3.B Concurrent deletion of Nf2 and Wwc1/2 at developmental stage driven by Alb-Cre results in liver dysfunction and perinatal death. Gross liver images, H&E, IHC, and IF images of 3-day-old livers from WT and Nf2;Wwc1/2 compound KO mice were shown. Scale bars: 1 cm (gross liver image), 50 μm (H&E and IHC), 20 μm (IF).E, F Activities of Hippo pathway kinases in Nf2;Wwc1/2 deficient livers. LATS1, MST2, and MAP4K4 were immunoprecipitated from control or Nf2F/F;Wwc1F/F;Wwc2F/F (Ad-Cre) mouse livers (4 each), and kinase assays were performed (E) and quantified (F). Four biological replicates were shown in F, error bars indicate SEM.
Figure 5Phenotypes of 1-week-old livers with HPO1 and/or HPO2 gene deletions. liver/body weight ratio (B) of WT, Nf2-/-, Sav1-/-, Wwc1-/-;Wwc2-/-, Mst1-/-;Mst2Δ/Δ and compound KO mice. Scale bar in A: 1cm. Each data point in B stands for one mouse, data shown are mean + SEM.C HPO1 and HPO2 inactivation results in rapid iCCA. H&E and CK19 staining for Nf2-/- (8-month-old), Nf2-/-;Sav1-/- (1-week-old) and Nf2-/-;Wwc1-/-;Wwc2+/- (1-week-old) livers. Nf2-/- liver displayed bile duct hyperplasia at 8 months, while Nf2-/-;Sav1-/- and Nf2-/-;Wwc1-/-;Wwc2+/- livers at 1 week exhibited massive expansion of cholangiocytes with enlarged nuclei, irregular shape, and invasive behaviors. Nf2-/-;Sav1-/- also exhibited cholestasis, as indicated by asterisk. Scale bar: 20 μm (H&E and IHC).E Hippo pathway signature score of 1-week-old livers.
Figure 6B Phenotypes of 3-week-old livers. Gross images, H&E staining and IF images of livers from mice with HPO1 or HPO2 inactivation. Mice were euthanized at 3 weeks old. IF: green for CK19, red for YAP/TAZ, Ki67 or HNF4α, blue for nuclei. Scale bars: 1 cm (gross liver image), 50 μm (H&E), 50 μm (IF).F Temporal analysis of expression of Hippo pathway components. Mouse liver protein samples were obtained from wild-type mice at different postnatal days (P1 to P28) and probed with antibodies against different Hippo pathway components.
Figure 7A Tumor formation in mouse livers with long term HPO1 or HPO2 inactivation. Gross images (black arrow heads indicate HCC lesions), H&E staining and IF images of livers with HPO1 or HPO2 inactivation at indicated ages. IF: green for CK19, red for YAP/TAZ, Ki67 or HNF4α, blue for nuclei. Scale bars: 1 cm (gross liver image), 50 μm (H&E), 50 μm (IF).
Figure 1(a & b) Interaction between several human C-type lectins (50 μg/mL) and recombinant SARS-CoV-2 RBD (50 μl/well) was determined by ELISA (a), and the interactions of hCLEC2.Fc with SARS-CoV-2 RBDs from various strains (b).
Figure 3Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 (MOI = 0.1 and 1) with or without autologous platelets (PLTs, 4 × 106/mL) for 5 h and 20 h at 37 °C. (a) The image of NETs (arrow heads: aggregated NETs).Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 (MOI = 0.1 and 1) with or without autologous platelets (PLTs, 4 × 106/mL) for 5 h and 20 h at 37 °C. (b) quantitation of NET area by citrullinated Histone H3 (Cit-H3) area.Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 (MOI = 0.1 and 1) with autologous platelets (PLTs, 4 × 106/mL) for 5 h and 20 h at 37 °C. (c) Human platelets were pretreated with anti-CLEC2 mAb (AYP1, 2.5 μg/mL) or isotype control for 15 min at room temperature before incubation with neutrophils and SARS-CoV-2 stimulation.(d) Neutrophils (4 × 105/mL) from wild type (WT) mice were incubated with SARS-CoV-2 (MOI = 1) in the presence of WT platelets (4 × 106/mL) or clec2-/- platelets (4 × 106/mL) for 5 h at 37 °C.Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 with or without autologous platelets (PLTs, 4 × 106/mL) (e & f) Human neutrophils were stimulated with SARS-CoV-2 spike RBD pseudotyped virus (pseudotyped virus) (MOI = 0.1) in presence of autologous platelets at 37°C for 3 h (e). Platelets were incubated with anti-CLEC2 mAb (2.5 μg/mL) at room temperature for 15 min before incubation with neutrophils and pseudotyped virus stimulation at 37°C for 3 h (f). The level of NET formation was determined using Cit-H3 area (μm2).
Figure 4(a & b) Human neutrophils were stimulated with SARS-CoV-2 (MOI = 0.1 and 1) at 37 °C in the presence of isotype control (hIgG1, 3 μg/mL) or human CLEC2.Fc (3 μg/mL) and with or without autologous platelets for 5 h (a) or with or without anti-SARS-CoV-2 spike RBD antibody (3 μg/mL) for 20 h (b). Data are means ± sd from at least three independent experiments. *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).The expression levels of viral copy number (d) Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n= 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). 'd.p.i.' stands for 'day post-infection'.cytokines and chemokines in lung were measured by qPCR (e). Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n= 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). 'd.p.i.' stands for 'day post-infection'.
Figure 5(a) AAV-hACE2 mice were treated with saline and challenged with SARS-CoV-2 (8 × 104 PFU) for 5 days. Lung tissue sections were stained with DAPI (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (Cit-H3) antibody (red), anti-CD42b antibody (yellow) and anti-CD31 antibody (gray). yellow arrow: NETs (DNA+Gr-1+MPO+Cit-H3+); red arrow: immunothrombosis (NETs + thrombus (CD42b+)), Scale bar is 100 μm.b & c) For prophylactic treatment, CLEC2.Fc (or vehicle) was given at 1 h before virus challenge; for therapeutic treatment, CLEC2.Fc was given at 8 hours post-infection. The area of NET (colocalization area of MPO and Cit-H3) (b) and immunothrombosis (colocalization area of Cit-H3 and CD42b) (c) were measured using MetaMorph software. Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n=5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01 (Two-way ANOVA).