EMBO/soda-nel huggingface dataset:
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 2

Figure 2: CHK1-inhibition kills primary murine HSPCs.

(A) LSK (Lin- Sca1+ cKit+) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13.5 or the bone marrow of adult mice and treated for 48h with PF-477736 [1.5 μM]. Survival was assessed using AnnexinV-staining and flow cytometry. Bars represent means ± S.E.M. from n=6 wild type and n=3 Vav-BCL2 fetal liver LSK cells (left) and n=3 bone marrow LSK cells (right).

(B, C) WT and Vav-BCL2 derived E14.5 total fetal liver cells (B) or total bone marrow cells (C) were treated for 72h (B) or up to 48h (C) with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).

Data information: Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 3

Figure 3: CHK1-inhibition kills human cord-blood derived CD34+ HSPCs in a BCL2 regulated manner.

(A) MACS-purified CD34+ human cord-blood derived HSPC were treated with PF-477736 or CHIR-124 ± the caspase-inhibitor QVD (50 μM). Cell death was assessed using AnnexinV/7-AAD staining and flow cytometry.

(B) CD34+ cells were transduced with a BCL2-encoding lentivirus or a control virus, along with IRES-GFP with a MOI of 10 for two consecutive days, followed by CHK1i application for 48 h. Survival was assessed using Nicoletti staining and flow cytometry.

(C) Percentage of living GFP+ transduced human CD34+ HSPCs, cultured in the absence or presence of CHK1i for 48 hours.

(D) Colony formation potential of human CD34+ HSPCs transduced with empty control or BCL2 encoding virus. Colonies were counted 10 days post seeding of 1000 CD34+ cells per plate/sample. "Myeloid colonies" include granulocyte/monocyte, monocyte, granulocyte and GEMM (granulocyte, erythroid, monocyte/macrophage and megakaryocyte) colonies.

Data information: Bars graphs in (A)-(D) represent means ± S.E.M. (independent experiments with 4 individual cord-blood derived CD34+ HSPCs). Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 4

Figure 4: Deletion of Chk1 in HSC prevents fetal hematopoiesis.

(A) Representative pictures of E13.5 and E18.5 embryos of the indicated genotypes. Scale bar E13.5 = 1 mm, E18.5 = 5 mm.

(B) Quantification of fetal liver cell number at E13.5. Bars represent means ± S.E.M. (N=4 for Chk1fl/+, N=3 for Chk1fl/+ Vav-Cre and N=4 for Chk1fl/- Vav-Cre).

(C) Representative pictures of isolated fetal livers at E13.5. Scale bar = 1 mm.

(D) Representative H&E-staining of fetal liver sections from E13.5 embryos. Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl/- Vav-Cre fetal livers. Scale bar = 500 µM.

(E) Single cell suspensions of fetal livers from Chk1fl/+, Chk1fl/+ Vav-Cre and Chk1fl/- Vav-Cre embryos were stained with antibodies recognizing ckit, CD71, Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry. Large 'EryA' erythroblasts (CD71high Ter119high FSChigh), smaller, more mature 'EryB' erythroblasts (CD71high Ter119high FSClow); most mature erythroblast subset is EryC (CD71low Ter119high FSClow). Bars represent means ± S.E.M. ctrl N=5 (pooled N=4 Chk1fl/+ and N=1 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre.

Data information: Asterisks indicate significant differences: *p < 0.05, **p < 0.01 using unpaired Student´s t- test.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 5

Figure 5: CHK1-deficiency causes stem cell loss in the fetal liver.

(A) Representative dot-plots of the LSK cell phenotype of Chk1fl/+ and Chk1fl/- Vav-Cre embryos observed in E13.5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells, as well as LKlow and LSKlow cells, found in the fetal liver on E13.5. Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre.

(B) Colony formation potential of FACS-sorted LSK cells (left panel, N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre) and total fetal liver cells (right panel, N=3 for Chk1fl/+ and N=6 for Chk1fl/- Vav-Cre) in methyl-cellulose assays. Bars represent means ± S.E.M.

(C) Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 (N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre), to discriminate LT- and ST-HSC from MPP within the LSK-subset. Bars represent means ± S.E.M.

Data information: Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 6

Figure 6 CHK1-deficient HSPCs accumulate DNA-damage during compensatory proliferation triggering cell death.

(A) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of fetal liver cryosections of the indicated genotypes at E13.5 and quantification. Scale bar 100x = 200 µM, 400x = 50 µM. The percentage of TUNEL+ area/field was assessed across three random fields from two control (Chk1fl/+) and two knock-out embryos (Chk1fl/- Vav-Cre) using ImageJ, as reported by us before (60). Bars represent means ± S.D.

(B) Total fetal liver cell suspensions of Chk1fl/+ (+/+), Chk1fl/+ Vav-Cre (+/-) and Chk1fl/- Vav-Cre (-/-) E13.5 embryos were processed for Western blot analysis using the indicated antibodies. Each lane represents samples isolated from an individual littermate embryo.

(C) DAPI-negative viable LK- and LSK-cells of the indicated genotypes (littermates) were sorted from E13.5 fetal livers and immediately fixed in 70% EtOH. Fixed cell suspensions were stained for ɣH2A.X and DAPI intracellularly. Shown here are representative dot-plots and quantification of N=4 embroys per genotype; Bars represent means ± S.E.M.

(D, E) Total E13.5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK (top panel) from LSK (lower panel) cells. Shown here are representative dot-plots of Ki67 and DAPI staining in LK and LSK cells, quantified in (E). Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre.

Data information: Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 7

Figure 7: Apoptosis inhibition cannot restore hematopoiesis in the absence of CHK1.

(A) Representative dot-plots and quantification of E13.5 fetal liver LSKhi/LSKlow cell phenotypes observed in embryos of the indicated genotypes. N=21 for Chk1fl/+, N=5 for Chk1fl/- Vav-Cre, N=16 for Chk1fl/+ Vav-BCL2, N=5 for Chk1fl/- Vav-Cre Vav-BCL2, N=2 for p53-/- Chk1fl/+ and N=4 for p53-/- Chk1fl/- Vav-Cre.

(B) Flow cytometry-based analysis of LSK cells stained with antibodies specific for CD34 and Flt3, to discriminate LT- and ST-HSC from MPP within the LSK subset in fetal livers of embryos of the indicated genotypes. Left: N=25 for Chk1fl/+, N=7 for Chk1fl/+ Vav-Cre and N=7 for Chk1fl/- Vav-Cre. Right: N=16 for Chk1fl/+ Vav-BCL2, N=6 for Chk1fl/+ Vav-Cre Vav-BCL2 and N=5 for Chk1fl/- Vav-Cre Vav-BCL2.

(C) Analysis of the colony formation potential of E14.5 total fetal liver cells of the indicated genotypes in methylcellulose. N=3 for controls (pooled; 1 Chk1fl/+, 2 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre and N=3 for Chk1fl/- Vav-Cre Vav-BCL2.

(D) Western blot analysis of fetal liver cells of the indicated genotypes (each lane represents an embryo) at E14.5 using the indicated antibodies.

Data information: Bars usually represent means ± S.E.M. Bars depicting p53-/- Chk1fl/+, and p53-/- Chk1fl/+ Vav-Cre show means ± S.D. due to the lower number of mice analyzed. Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test.
Checkpoint kinase 1 is essential for fetal and adult hematopoiesis (2019)
Figure 8

Figure 8: Acute depletion of Chk1 in the hematopoietic system leads to cell loss in vitro and retention of CHK1 expression in vivo.

(A) Three independent Chk1fl/fl mT/mG (coloured) and mT/mG (grey) Hoxb8-FL progenitor cell lines were transduced with a retroviral construct carrying Cre (pMIP-CRE-IRES-PURO) and monitored over time for GFP-expression. Shown here are three technical replicates per genotype. Additionally, Chk1fl/fl mT/mG Hoxb8-FL progenitor cells were FACS-sorted for GFP+ and dsRED+ cells 48h after transduction with the Cre-construct. Sorted cells were processed for western blotting using the indicated antibodies.

(B) Chk1fl/fl Hoxb8-FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP (pMIG-CRE-IRES-GFP). Cells were FACS-sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies.

(C) Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).

(D) Quantification of Chk1-del PCR-product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± S.E.M. from n=5 mT/mG Vav-CreERT2 Chk1fl/fl mice.

(E, F) (E) Percentage of living LK or LSK-cells and (F) percentage of GFP+ LK or LSK-cells found in the bone marrow of mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 post first TAM administration. Bars in (E)/(F) represent means ± S.E.M. (Day 8 N=8/8, WT/Chk1fl/fl, Day 15 N=7/8).

(G) GFP+ (CRE) and Tomato+ (CRE-) total bone marrow cells were sorted on day 8 after first TAM-administration and processed for Western Blot using the indicated antibodies. Sorted cells from three animals - mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) - were pooled per lane.

(H) Thymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for western analysis using the indicated antibodies.

Data information: Asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001 using unpaired Student´s t- test.

Expanded View Figure Legends
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 1

Figure 1. Helix-7 dynamically interacts with the miRNA seed region. A) Cartoon representation of the Ago2-miRNA crystal structure. Ago2 is colored gray except for helix-7, which is yellow. miRNA guide colored red. B) Crystal structure of the Ago2-miRNA-target RNA ternary complex. Target RNA is colored blue. C) Close-up view of the unpaired complex shows helix-7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7. D) Close-up view of the Ago2-miRNA-target complex shows helix-7 docks into the minor groove of the guide:target duplex, directly contacting base pairs at positions g6 and g7.
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 2

Figure 2. Helix-7 is not required for target recognition. A) Plot of g2-g8 target RNA bound (0.1 nM) versus Ago2-miR122 concentration for wild type (WT), helix-7 double point mutant (MI-AA), and Ηhelix-7 Ago2. Average values from at least three independent experiments ± standard deviation (SD) are plotted. Top panel shows target RNA paired to seed region of the guide RNA (miRNA-122). B) Dissociation of a 32P-labeled target RNA (0.1 nM) from the Ago2-miR122 complex (1 nM) was monitored in the presence of unlabeled target RNA (100 nM). Fraction of the target RNA bound to Ago2-miR122 is plotted as a function of time for WT and Ηhelix-7 Ago2. Average values from at least three independent experiments ± SD were fit to single exponential decays.
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 3

Figure 3. Helix-7 minimizes dwell time on off-targets. A) Cartoon schematic of our single-molecule FRET assay. B) Guide:off-target pairs examined listed with increasing values of N (the length of the matched region between miRNA and target RNA). N = 7 is the fully seed paired target. C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber. D) Dwell time histograms obtained for N = 4, 5 and 6, with wild type Ago2; histograms were fit with single exponential decay. The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution. The bin size is 0.5 sec for N =4 and 1 sec for N =5 and 6. E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching. F) Dwell time histogram for different N's with the MI-AA helix-7 mutant. The dwell time histogram for N = 6 fit with double exponential decay (green; R2 = 0.98); The dwell time for N = 6 with single exponential fit was 15.2 ± 3.41 sec, R2 = 0.96). G) Bar plot for dwell times of MI-AA Ago2 as a function of N. H) Dwell time histogram for different N's with the ∆helix-7 mutant. The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays (green; R2 = 0.97 for N = 5 and R2 = 0.96 for N = 6); The dwell time for N = 5 and N = 6 with single exponential fit was 9.1 ± 0.91 sec (R2 = 0.86) and 22.3 ± 5.08 sec (R2 = 0.90) respectively). I) Bar plot for dwell times of ∆helix-7 Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 4

Figure 4. Helix-7 discourages non-Watson Crick pairing at g6 and g7. A-C) Plots of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 (wild type and Ηhelix-7) concentration for targets with an increasing number of G:U wobble pairs in the seed 3' end. D) Plot of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 concentration for a target with mismatches at positions g6-g8. E) Bar plots of the Kd of wild type (left) Ηhelix-7 (right) Ago2-miR122 for indicated target RNAs. For A-E, average values from at least three independent experiments ± SD are plotted.
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 5

Figure 5. ΗHelix-7 Ago2 is sensitive to inhibition by off-target RNAs. A) Ago2-miRNA122 (1 nM) was incubated with a 32P-labeled, seed-matched target RNA (0.1 nM) in the presence of increasing concentrations of unlabeled a competitor RNA (top panel). Fraction target RNA bound to Ago2 is plotted as a function of off-target competitor RNA concentration. B) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA. For A-B, average values from at least three independent experiments ± SD are plotted.
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 6

Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted. B) Observed binding rates (kon, obs) plotted as a function of Ago2-miR122 concentration. Average values ± SD are plotted. C) Arrival time distribution of single Ago2-miRNA complexes binding immobilized targets in a microfluidic chamber. The value is the average of three independent measurements.
Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition (2017)
Figure 7

Figure 7. Structure of a helix-7 Ago2 mutant. A) Superposition of wild type (white) and MI-AA (gray) Ago2. Helix-7 colored yellow; ordered region of the guide RNAs colored in red. B) Close-up view shows helix-7 tilts away from the seed region in the MI-AA mutant. C) Side-by-side comparison of the seed region in wild type (left) and MI-AA (right) Ago2 crystal structures.
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 1

Figure 1. CHIKV infection outcomes are more severe in MARCO-/- mice and viremia and dissemination are enhanced.

(A-B) WT or MARCO-/- C57BL/6 mice were inoculated subcutaneously (s.c.) in the left rear footpad with 103 PFU of CHIKV or CHIKV E2 K200R. The percent of starting body weight (A) and disease score (B) were recorded daily over 14 days. Mean ± SEM. N=7-12. Data are pooled from three experiments. Two-way ANOVA with Bonferroni's multiple comparison test; ****P < 0.0001.

(C-E) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of CHIKV. Serum collected on days 1, 2, 3, 5, 7, and 14 was analyzed by focus formation assay (FFA), mean ± SD (C) and by RT-qPCR, mean ± SD (D). (E) Viral tissue burdens were analyzed at 1 day post inoculation (dpi) by FFA. Mean ± SEM. Data are pooled from two experiments, n=5-10. Two-way ANOVA with Bonferroni's multiple comparisons test (C-D) or Mann-Whitney test (E); ****P < 0.0001.

(F-G) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of ONNV (F) or RRV (G). Tissues and serum were collected at 1 dpi and analyzed by plaque assay. Mean ± SEM. Data are pooled from two experiments, n= 8-10. Mann-Whitney test; ****P < 0.0001.
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 2

Figure 2. Depletion of Kupffer cells impedes CHIKV clearance from the circulation.

(A) WT and Clec4F-DTR+ C57BL/6 mice were treated with diphtheria toxin (DT) either intraperitoneally (i.p.) 24 h prior to inoculation (n=10, data pooled from two experiments, black dots) or intravenously (i.v.) 48 and 24 h prior to inoculation (n=4, one experiment, grey dots) to deplete KCs and inoculated i.v. with 108 CHIKV particles. Viral genomes in the inoculum and serum at 45 minutes (min)-post inoculation were determined by RT-qPCR. Mean ± SD. Data are pooled from three experiments, n= 14. Mann-Whitney test; ****P < 0.0001.

(B) WT or Clec4F-DTR+ C57BL/6 mice were treated i.v. with DT, PBS-loaded liposomes (PLL) or clodronate-loaded liposomes (CLL) prior to i.v. inoculation of 108 CHIKV particles. Viral genomes were quantified as in (A). Mean ± SD. One experiment, n=4. Mann-Whitney test; **P < 0.01, ****P < 0.0001.

(C) WT or CD169-DTR+ mice were treated i.p. with DT prior to i.v. inoculation of 108 CHIKV particles. Viral genomes were quantified as in (A). Mean ± SD. Data are pooled from two experiments, n=7-8. Mann-Whitney test; ***P < 0.001.

(D) Livers from WT, Clec4F-DTR+ or CD169-DTR+ mice treated i.p. with DT, or WT mice treated i.v. with PLL or CLL were analyzed by IHC to visualize F4/80+ macrophages (brown). Two representative sections for each group are shown.

(E) F4/80+ cells in 10 randomly selected high-powered fields (HPF) per liver section were counted to calculate average F4/80+ cells per HPF. Mean ± SD. Two experiments, n=2-4 mice per group. One-way ANOVA with Bonferroni's multiple comparisons test; ****P < 0.0001
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 3

Figure 3. Depletion of KCs is not sufficient to enhance early CHIKV dissemination. (A) WT or Clec4F-DTR+ C57BL/6 mice were treated i.v. with DT prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus was quantified at 24 hpi by FFA. Mean ± SEM. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.

(B) WT C57BL/6 mice were treated i.v. with PLL or CLL 42 h prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; ***P < 0.001.
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 4

Figure 4. The draining lymph node limits arthritogenic alphavirus dissemination.

(A) WT or LTalpha-/- mice were treated i.v. with PBS-loaded liposomes (PLL) or clodronate-loaded liposomes (CLL) 42 h prior to s.c. inoculation of the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was analyzed by FFA. Mean ± SEM. Date are pooled from two experiments, n=8-10. Mann-Whitney test; *P < 0.05, ***P < 0.001, ****P < 0.0001.

(B) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=10. Two-way ANOVA with Bonferroni's multiple comparisons test; ***P < 0.001, ****P < 0.0001.

(C) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of RRV. Viral genomes in the dLN and serum were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8-9. Mann-Whitney test; ***P < 0.001, ****P < 0.0001.
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 5

Figure 5. Macrophages in the dLN are not required for CHIKV accumulation in the dLN or limiting viral dissemination.

(A) CD169-DTR+ mice were treated i.p. with PBS or DT prior to collection of the popliteal LN. Frozen LN sections were stained for CD169+ macrophages (green), B220+ B cells (blue) or ERTR-7+ stromal cells (red). Image shown is representative of three biological replicates. Scale bars: 200 μm.

(B) WT or CD169-DTR+ C57BL/6 mice were treated i.p. with DT prior to s.c. inoculation in the left rear footpad with 108 particles of WT CHIKV. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.

(C) WT or CD169-DTR+ C57BL/6 mice were treated i.p. with DT prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SEM. Data are pooled from two experiments, n=9-10. Mann-Whitney test; **P < 0.01.
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 6

Figure 6. MARCO+ LECs capture lymph-borne CHIKV particles.

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the rear feet with 5*104 PFU of CHIKV-E2-mCherry and the popliteal dLNs were collected at 2 hpi.

(A) Frozen dLN sections were stained for Lyve-1+ LECs (white), MARCO (green), B220 (blue) and mCherry+ CHIKV particles (red). Scale bars: 200 μm.

(B) Representative histograms of confocal images showing voxel intensities for Lyve1 and mCherry.

(C) Percent of Lyve1+ voxels with mCherry signal; dots represent individual sections. Data are representative of two experiments, n=6-9. One-way ANOVA with Tukey's multiple comparisons test; ****P<0.0001.

(D) Higher magnification confocal image of a WT lymph node section stained as in (A). Scale bar: 20 μm.

(E) Higher magnification confocal image of a MARCO-/- lymph node stained for CD11b+ cells (white), mCherry+ CHIKV particles (red), B220+ B cells (blue), and CD169+ macrophages (green). Scale bar: 20 μm.