EMBO/soda-nel huggingface dataset:
MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin (2020)
Figure 4

Figure 4. smyhc1 mutants exhibit skeletal abnormalities in adulthood.

(A) Gross morphology of smyhc1 mutant adults. Most smyhc1-/- adult fish display dorsal tail curvature, while smyhc1R673H/+ adults have a shortened body axis and variable spinal curves.

(B) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1-/- adults, in contrast to the compression and fusion of vertebrae seen in smyhc1R673H/+ adults. Distal tail regions are highlighted and enlarged.

(C) Quantification of skeletal phenotype severity in adults. Representative examples of fish in each of the four phenotypic groups (normal, mild, moderate, and severe) are shown in Figure EV3. Scale bars represent a length of 5 mm.
MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin (2020)
Figure 5

Figure 5. Motor deficits in smyhc1 mutants are variable during development.

(A) Time course of light-triggered larval movements were manually counted at indicated times between 24 and 48 hpf. The smyhc1-/- larvae (n = 36) were paralyzed up to 48 hpf. smyhc1R673H/+ mutants (n = 15) twitched slightly less than their smyhc1+/+ siblings (n = 21) at 34hpf. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

(B) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software (Noldus Ethovision). At 6 dpf, both smyhc1-/- (n = 24) and smyhc1R673H/+ (n = 11) larvae travelled significantly less than smyhc1+/+ larvae (n = 13). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.

(C) Diagram of swim tunnel used to quantify adult swimming behavior. Water flow was gradually increased until the fish fatigued and were collected at the grate.

(D) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization (mpf). smyhc1+/+ (n = 6), smyhc1-/- (n = 5), smyhc1R673H/+ (n = 5).

Data Information: Wilcoxon-rank sum test used to calculate significance. P-values comparing each group displayed above data. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.
MYH3-associated distal arthrogryposis zebrafish model is normalized with para-aminoblebbistatin (2020)
Figure 7

Figure 7. Para-aminoblebbistatin ameliorates the smyhc1R673H larval phenotype.

(A) Representative morphologies of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 2 dpf after 24 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. All para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis.

(B) Representative images of smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae at 3dpf after 48 hour treatment with 0.25% DMSO (control) or 25 μM para-aminoblebbistatin. All larvae treated with para-aminoblebbistatin develop a slight dorsal tail curve and pericardial edema regardless of genotype. Para-aminoblebbistatin-treated smyhc1R673H/+ (n=9) and smyhc1R673H/R673H (n=3) larvae are indistinguishable from smyhc1+/+ unlike the untreated larvae which have markedly curved or distorted body axis.

EXPANDED VIEW FIGURE LEGENDS
Pyruvate Metabolism Guides Definitive Lineage Specification During Hematopoietic Emergence (2021)
Figure 3

Figure 3. Increasing pyruvate flux into mitochondria in HE cells favors definitive hematopoietic differentiation

(a) Pyruvate is transported into mitochondria via the mitochondrial pyruvate carrier (MPC, inhibitor: UK5099) and converted to acetyl-coA by pyruvate dehydrogenase complex (PDH, inhibitor: 1-AA). Pyruvate dehydrogenase kinases (PDKs, inhibitor: DCA) negatively regulate PDH activity.

(b-e) FACS-sorted HE cells were subcultured with or without UK5099 (10 µM) or DCA (3 mM). Subculture day 3 representative GPA/CD43 plots (b, d) and subculture day 6 representative CD45/CD43 plots (c, e) are shown (see Fig. EV5a, g, l and n for corresponding bar graphs).

(f) Ratio of CFU-E to CFU-G, M, GM colonies ± SEM relative to the control condition obtained from HE cells subcultured with the indicated compounds for 6 days, see Fig. EV5s for percentages (n=5 biological replicates with 1-2 technical replicates each, paired t-test).

(g) Fold change in the expression of HBE1 or HBG1-2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non-treated cells (n=3 biological replicates, paired t-test).

(h) Percentages of CD45+CD56+ ± SEM cells obtained following 35-day co-culture of 3-day subcultured HE cells with OP9-DL1 stroma. During the 3-day subculture, HE cells were treated with the indicated compounds. (n=3 biological replicates, one-way ANOVA test, see Fig. EV5v for plots).

(i-k) Pregnant mice were injected with UK5099 or DCA at E9.5 and fetal livers were analyzed at E14.5 by flow cytometry. FL, fetal liver. Levels of T and B cells (i) and LT-HSCs (j) as percentages in fetal liver are shown for control (n=10 biological replicates), UK5099-treated (n=14 biological replicates) and DCA-treated (n=16 biological replicates) conditions (one-way ANOVA test). (k) The ratio of BFU-E to CFU-GM colonies obtained from sorted LT-HSCs are shown (see also data in Appendix Fig. S6f) (one-way ANOVA test). CFU, colony forming unit; BFU, burst forming unit; E, erythroid; M, macrophage; G, granulocyte.

(l-p) HE cells co-cultured with OP9-DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice. Bone marrow (BM) and thymi were harvested on week 12. (l) The percentages ± SEM of human CD4+CD8+ double positive thymocytes in huCD45+ cells from the thymus are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). The percentages ± SEM of human B cells (m), CLPs (n) from the BM at week 12, as well as myeloid cells from PB at week 8 (o) and myeloid cells from BM at week 12 (p) in huCD45+ cells are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). PB, peripheral blood.

Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001
Alveolar Macrophage-Derived Extracellular Vesicles Inhibit Endosomal Fusion of Influenza Virus (2020)
Figure 2

Figure 2. AM-EVs inhibit influenza replication in AECs in vitro and in vivo.

A, B. Luc-WSN/33 replication co-incubated with MH-S EVs was assessed in (A) human A549 and (B) mouse MLE-12 AECs. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.

C. MLE-12 AECs incubated with MH-S EVs infected with PR/8 influenza. Replication was quantified by RT-qPCR at 12 h post infection. Data represent mean M gene transcripts normalized to β actin from 3 independent experiments.

D. Schematic outline for AM depletion in lungs of mice with o.p. Clo followed by intrapulmonary delivery of AM-EVs PR/8 infection.

E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT-qPCR. Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments (4-5 mice per group).

Data information: Error bars = SD. One-way ANOVA (A, B) and unpaired Student's t-test (C) (* p value of < 0.05, *** p < 0.001, **** p < 0.0001). For (E), the comparison between conditions was performed with one-way ANOVA (multiple comparisons) on values obtained from individual mice across all experiments.
Alveolar Macrophage-Derived Extracellular Vesicles Inhibit Endosomal Fusion of Influenza Virus (2020)
Figure 4

Figure 4. Patient-derived influenza isolates exhibit strain-dependent sensitivity to the inhibitory effects of AM-EVs.

A. MLE-12 AECs co-incubated with the listed WT strains (MOI = 0.4) plus AM-EVs (grey bars, EV:cell = 1). Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain (white bars), each normalized to β actin from 2-4 independent experiments per strain.

B. Schematic flow diagram of the generation of luciferase-expressing patient-derived influenza viruses. The generation of the novel Luc-CA/09 strain is shown as a representative example.

C. MHS EVs were co-incubated with the listed Luc-expressing strains plus AM-EVs (grey bars, EV:cell = 2) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain (white bars) from 3 independent experiments per strain.

Data information: Error bars = SD. One-way ANOVA (A, C) (** p value of < 0.01, *** p < 0.001, **** p < 0.0001).
Alveolar Macrophage-Derived Extracellular Vesicles Inhibit Endosomal Fusion of Influenza Virus (2020)
Figure 5

Figure 5. AM-EVs prevent the fusion of influenza virus with endosomal membranes in susceptible strains.

A. MDCK-SIAT1 cells were pretreated with MH-S EVs (EV:cell = 2, 'EV pretreat') or control for 8 h, washed, and subsequently incubated with Luc-WSN/33 (MOI = 1). Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments.

B, C. MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (B) RNA isolation or (C) immunofluorescence microscopy. (B) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition, each normalized to β actin from 3 independent experiments. (C) Cells in both conditions were fixed and stained with anti-NP or isotype control Ab. Images are representative of two independent experiments. Scale bars = 50 μm

D. A549 cells were incubated with WSN/33 or HK/14 + AM-EVs for 40 min, washed, fixed, and permeabilized. Cells were subsequently stained with Anti-NP (green), Anti-Rab-7 (purple) Abs, and DAPI (blue). Images shown are representative of 8 independent experiments. Scale bars = 5 μm

E, F. Co-localization analysis of (E) NP:DAPI and (F) NP:Rab7 for each condition was performed on Z-stack confocal images images of WSN/33 (black) or HK/14 (red) infected A549s. Data represent the mean NP:DAPI co-localization ratios and the mean NP:Rab7 Mander's co-localization coefficient for individual cells from these 8 experiments.

Data information: Error bars = SD. One-way ANOVA (B) and unpaired Student's t-test (A, E, F) (* p value of < 0.05, **** p < 0.0001).
Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress (2019)
Figure 3

Figure 3 - The DNA damage response is largely driven by extrinsic signals.

A-E Spearman's correlation between green (A-D) or yellow (E) fluorescence range and red fluorescence range over the lifetime of the cell (105 < n < 153). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (A-D) or a C-term translational reporter of SSBa fused to mCitrinewt (E). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (A and E), transcriptional reporters of ruvC (B) and radA (C), and mCherrydes expressed from a constitutive control promoter (D). Colors (A-D) indicate subpopulations segregated by VMR of P2P1recA-GFPdes: gray (0<VMR≤1), dark green (1<VMR≤10), and light green (VMR>10). Colors (E) indicate subpopulations segregated by VMR of RecA-mCherrywt: gray (0<VMR≤1), plum (1<VMR≤10), and magenta (VMR>10). See also Movie EV3.

F-J Pearson's correlation between green (F-I) or yellow (J) fluorescence and red fluorescence, measured 3 hours after MMC exposure (203 < n < 314). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (F-I) or a C-term translational reporter of SSBa fused to mCitrinewt (J). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (F and J), transcriptional reporters of ruvC (G) and radA (H), and mCherrydes expressed from a constitutive control promoter (I). See also Movie EV3. Positive correlations between recA and other DNA-damage markers both under normal growth conditions, where spontaneous DNA-damage events occur at the subpopulation level, and in the presence of an alkylating agent that induces double-strand breaks in the entire population, validate recA as a reliable proxy for DNA damage in single cells.
Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress (2019)
Figure 4

Figure 4 - RecA pulsing is predictive of cell fate in M. smegmatis.

A, B Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm. See also Movie EV4.

C, D Representative time traces of individual lineages of cells fed with fresh medium (C) or spent medium (D). Different styles are used for lineages with different fates: death and lysis (L); SYTOX-positive (SX+); and survival (S). Line breaks represent division events in the same lineage, where a single random daughter cell is tracked for clarity. Vertical bars represent fresh (white) or spent (dark shading) 7H9 medium, CIP (yellow), and SX (blue).

E, F Comparison of single-cell growth rates prior to CIP exposure for "D" cells that die and "S" cells that survive and regrow after CIP washout (E), and lag time of regrowth (F). Cells were cultured in fresh (EXP) or spent (STAT) flow medium. Gray and black lines indicate mean ± SD. Significance by Kruskal-Wallis and Dunn's multiple comparison test (E) and unpaired Mann-Whitney test (F): ns, not significant; ****p < 0.0001; 6 < n < 84. The data shown are from 2 independent experiments. Single-cell fate is independent of the growth rate before exposure to the drug. However, cells cultured in spent medium (STAT) grow at significantly slower rates, survive at higher frequencies, and recover faster after CIP washout compared to cells cultured in fresh medium (EXP).

G, H Representative GFPdes (G) and mCherrywt (H) fluorescence time-traces for single-cell lineages of cells cultured in fresh medium (EXP) and cells cultured in spent medium (STAT), and having different fates. Vertical bars are color-coded as in C and D.

I, J Single-cell fate as a function of VMR of P2P1recA-GFPdes (I) and RecA-mCherrywt (J) fluorescence prior to CIP exposure. Significance by Chi-square test of independence for cells that die (n = 84) and cells that survive (n = 54). The data shown are from 2 independent experiments. Cells that do not experience DNA damage prior to drug exposure (RecA-non-pulsing cells) survive more than cells that do experience DNA damage (RecA-pulsing cells).
Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress (2019)
Figure 5

Figure 5 - M. tuberculosis RecA-mCherrywt expression dynamics under normal growth conditions.

A Spearman's correlation between single-cell RecA-mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell (n = 360). The data shown are from 3 independent experiments. Dashed lines separate different levels of dispersion, and colors denote subpopulations that are underdispersed (gray), dispersed (plum), or highly dispersed (magenta).

B RecA-mCherrywt fluorescence of subpopulations segregated by fluorescence VMR (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001.

C Single-cell time traces of RecA-mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 360): 40% (0<VMR≤1); 44% (1<VMR≤10); 16% (VMR>10). The data shown are from 3 independent experiments.

D Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Bars shaded in plum indicate moderately pulsing cells (n = 185) and magenta bars indicate highly pulsing cells (n = 57). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 80% and 100% of the generation time.

E, F Single-cell size (E) and growth rate (F) averaged over the lifetime of the cell (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; **p = 0.002; ****p < 0.0001. Cells experiencing more DNA damage (VMR>10) are slightly larger and grow at slower rates. See also Movie EV5.
Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal
and identity (2019)
Figure 1

Figure 1. Expression of Bhlhe40 and Bhlhe41 in myeloid cells and cell-surface phenotype of DKO alveolar and peritoneal macrophages.

(A) Expression of Bhlhe40 (grey) and Bhlhe41 (orange) in the indicated cell populations, data are from the Immgen Database (microarray data set). CMP - common myeloid progenitors. MΦ - macrophages.

(B) Flow cytometry detection of the Bhlhe40 (grey) and Bhlhe41 (orange) transcripts by the PrimeFlow RNA assay in alveolar (right) and peritoneal (left) macrophages. A probe specific for the Cd8a mRNA (black line) was used as a negative control. Results representative of at least four independent experiments.

(C, D) Cell surface phenotype and numbers of alveolar (C; digested lung) and peritoneal (D) macrophages in WT and DKO mice. Additional gating for CD11b+ cells was applied for peritoneal macrophages as indicated. Levels of CD11b expression on WT and DKO AMs are shown, and median fluorescent intensity for CD11b expression was quantified (C, bottom left; 3 mice per genotype). Absolute numbers of CD11chiCD11bloSiglecFhi alveolar macrophages from digested lungs (C) and CD11b+F4/80hi peritoneal macrophages (D) from WT and DKO mice are shown (bottom; 4 mice per genotype). Representative results of two independent experiments. Horizontal lines indicate the mean and error bars (s.d.). *** P < 0.001 (Student's t-test).
Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal
and identity (2019)
Figure 2

Figure 2. Competitive disadvantage of Bhlhe40/Bhlhe41-deficient cells in AM compartment.

.

(A) Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells in the indicated BM (LSK, CMP), splenic (monocytes), liver (Kupffer cells), peritoneal and alveolar macrophage populations of lethally irradiated recipients ≥ 6 weeks after transfer of a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice. P values < 0.05 (paired Student's t-test) between LSK and other populations are shown for frequencies of WT cells. Three mixed BM chimeras analyzed; error bars represent s.d. Representative results of three independent experiments.

(B, C) Representative flow-cytometric analysis of lung (B) and peritoneal (C) macrophages as in A (left). The expression of CD45.1 and CD45.2 on donor and recipient cells is shown next to the indicated gates (right).

(D) Frequency of WT donor (CD45.1), DKO donor (CD45.2) and recipient (CD45.1/2) cells among the BM LSK cells, splenic monocytes and alveolar macrophages of the recipients that were irradiated with lung shielding, treated with one dose of busulfan as described in Materials and Methods, transferred with a 1:1 mixture of lineage-depleted BM cells from WT and DKO mice and analyzed ≥ 8 weeks after transfer. Representative flow cytometry results for alveolar macrophages (left) and quantification (right) is shown. Single experiment with three chimeras. Error bars represent s.d.

(E) Surface phenotype of cells from the digested lungs (right) and quantification of donor contribution to the AM compartment (left) of AM-deficient Csf2rb-/-Csf2rb2-/-recipients (CD45.2) that were subjected to intranasal transfer of a 1:1 mixture of sorted fetal liver monocytes from WT (CD45.1/2) and DKO (CD45.2) E18.5 embryos at days 1 or 2 after birth and analyzed ≥ 7 weeks after the transfer. Cell surface phenotype of lung cells from a Csf2rb-/-Csf2rb2-/- mouse that was not subjected to the transfer is shown for comparison. Single experiment with seven recipients. ** P < 0.01 (paired Student's t-test)

(F) Frequency of donor- and recipient- derived AMs in WT and DKO recipients transferred at days 1 or 2 after birth intranasally with WT CD45+ cells sorted from neonatal lungs and analyzed ≥ 7 weeks after the transfer. Results for quantification (lower panel) are pooled from 3 independent experiments with a total of 8 WT and 9 DKO recipients; various combinations of CD45.1 and CD45.2 were used to distinguish donor and recipient cells. Horizontal lines indicate the mean and error bars (s.d.). ** P < 0.01 (Student's t-test).
Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal
and identity (2019)
Figure 3

Figure 3. Impaired proliferation of DKO AMs.

(A) Mixed WT:DKO BM chimeras established as in Figure 2A were injected with EdU on three consecutive days. EdU incorporation was analyzed one day after the last injection. Results pooled from two independent experiments; 9 BM chimeras were analyzed in total. *** P < 0.001 (Student's t-test).

(B, C) Steady-state WT and DKO mice were injected with EdU and analyzed as in A. Representative flow cytometry plots of EdU incorporation by AMs (C) and their quantification (results pooled from two independent experiments, 7 WT and 6 DKO mice were analyzed in total) (B) is shown. Horizontal lines indicate the mean, error bars - s.d. *** P < 0.001 (Student's t-test).

(D) WT (CD45.1) and DKO (CD45.2) AMs were sorted, mixed in a 1:1 ratio, labeled with CellTrace Violet and cultured in the presence of GM-CSF. The ratio of CD45.1 and CD45.2 cells and CellTrace Violet dilution was analyzed prior to culture (day 0) and after 7 days of culture as described in Materials and Methods. Results representative of two independent experiments.

(E) RNA-seq analysis of Maf and Mafb expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom). Error bars represent SEM; two biological replicates per genotype.