EMBO/soda-nel huggingface dataset:
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 5

Figure 5. Transcriptome analysis of EVL-deficient retinal endothelial cells. CD31 and CD34 double positive endothelial cells were isolated from wild-type or EVL-/- P5 retinas by FACS and analyzed by RNA-sequencing. Two independent experiments; in total 18 retinas from WT mice and 18 retinas from EVL-/- mice from three different litters, each.

(A) RNA levels (FPKM) of VASP, EVL and Mena in endothelial cells from wild-type or EVL-/- retinas. Error bars represent SEM.

(B) Pathway analysis (metascape.org) was used to identify differentially expressed gene sets in EVL-/- retinal endothelial cells. Selected gene sets involved in vessel branching are shown.

(C) Pathway analysis of all under-represented gene sets in EVL-/- endothelial cells.

(D) Analysis of mRNA levels of Esm1, Ptgs2 (cyclooxygenase 2), serpine 1 and paxillin in EVL-deficient MLEC relative to WT controls set to 100. n = 3-4 independent experiments, error bars represent SEM, one sample t-test, *p<0.05; **p<0.01.

(E) ESM1 protein expression in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against the tip cell marker ESM1 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.

(F) Analysis of retinal ESM1 protein levels normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; three different litters.
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 6

Figure 6. EVL deficiency impairs VEGF-induced endothelial cell proliferation and sprouting.

(A) Proliferation of WT and EVL-/- MLEC was assessed by BrdU incorporation. The number of BrdU positive cells (red) was normalized to the total number of cells (white). Eight fields of view per condition and genotype for each of 5 independent WT and EVL-/- cell batches; error bars represent SEM; **p<0.01, ***p<0.001, #p=0.171 non-significant vs. Basal EVL-/-, one-way ANOVA with Bonferroni's multi comparison test; scale bar = 50 µm.

(B) Cumulative length of CD31-positive sprouts from WT and EVL-/- aortic rings embedded in collagen after 5 days without (basal) or with VEGF stimulation (30 ng/ml). 7 WT and 6 EVL-/- adult animals from three different litters, each; 3 aortic rings per animal and condition; error bars represent SEM; **p<0.01; ***p<0.01; #p=0.543 non-significant vs. basal EVL-/-, one-way ANOVA with Bonferroni's multi comparison test; scale bar, 250 µm.

(C) Sprouting of WT and EVL-/- primary MLEC spheres in the absence (Basal) or presence of VEGF (50 ng/ml); asterisks indicate sprouts; scale bars, 100 µm.

(D) Solid bars represent proportion of WT and EVL-/- MLEC spheres forming sprouts in the absence (−) or presence (+) of VEGF. n-numbers are indicated; n.s. non-significant (p=0.557); ***p<0.001, Fisher's exact test.

(E) Quantification of MLEC sphere sprout length without or with VEGF. Dots represent individual sprouts. Horizontal lines represent mean, error bars represent SEM. n = 20-30 beads (from three independent experiments). ***p<0.001, n.s. non-significant (p=0.543), one-way ANOVA with Bonferroni's multi comparison test.
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 7

Figure 7. EVL deficiency impairs VEGFR2 internalization.

(A) VEGFR2 protein levels in wild-type (WT) and EVL-deficient (EVL-/-) endothelial cells. Actin was used as loading control. The lower panel shows quantification of VEGFR2 to actin ratios from five independent cell batches; error bars represent SEM; n.s. non-significant (p=0.388), unpaired Student's t-test.

(B) Antibody feeding assay to monitor VEGFR2 internalization after VEGF stimulation. Left panel: schematic diagram showing the two step staining procedure of surface and internalized VEGFR2 antibodies, which are displayed in yellow and green in the merged confocal images of WT and EVL-/- endothelial cells (middle panel), respectively; scale bar, 10 µm. The bar graphs show quantification of the number of VEGFR2 clusters and the ratio of internalized to total VEGFR2 ratios normalized to wild-type; n = 4-6 independent cell batches, error bars represent SEM; **p<0.01, ***p<0.001, unpaired Student's t-test.
EVL regulates VEGF receptor-2 internalization and signaling in
developmental angiogenesis (2020)
Figure 8

Figure 8. EVL deficiency impairs VEGFR2 signaling.

(A) Western blots and quantification of VEGFR2 phosphorylation levels relative to total VEGFR2 levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 5 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test.

(B) Western blots and quantification of ERK1/2 phosphorylation levels relative to total ERK levels in WT and EVL-/- endothelial cells under basal and VEGF-stimulated conditions (80 ng/ml); n = 6 independent cell batches, error bars represent SEM; *p<0.05, one-way ANOVA with Bonferroni's multi comparison test.

(C) ERK1/2 phosphorylation in P5 retinas of wild-type (WT) and global EVL-/- mice. Retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and antibodies directed against phospho-ERK1/2 (red). Representative images from three independent experiments are shown. Scale bars, 100 µm.

(D) Analysis of endothelial ERK1/2-phosphorylation normalized to wild-type littermates. Error bars represent SEM; ***p<0.001, unpaired Student's t-test; two different litters.

Expanded View Figure Legends
Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion (2021)
Figure 2

Figure 2 Traction force maps and distributions of signaling components in fan shaped cells.

A: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 1 cells. Corresponding speeds are 10.8µm/min and 9.4µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint, as extracted from the corresponding stress kymographs.

B: Stress maps quantifying the magnitude of the force per area using a color scale with blue/red corresponding to small/large stresses and the direction of forces using vectors. Fan-shaped cells were rotated so that the vertical (x) axis is the direction of motion (see Methods). Shown are the overall stress T, the stress in the direction of motion Tx and the stress perpendicular to motion Ty for the type 1 cell with the LimE marker, and the corresponding Tx and Ty kymographs along the cells outlines.

C: Snapshots of LimE-GFP, GFP-myo, and corresponding kymographs for type 2 cells. Corresponding speeds are 5.6µm/min and 8.7µm/min. White dots in the fluorescent kymographs indicate the location of the two poles of force at each timepoint.

D: Stress maps of T, Tx and Ty for the type 2 cell with the LimE marker, and corresponding Tx and Ty kymographs along the cells outlines.

E: Left three panels: Major and minor axis of the cells, Lmaj and Lmin, and total force F as a function of the cell area A. Right panel: Force perpendicular to the motion, Fy, as a function of the force parallel to the motion, Fx. The dashed line represents a linear fit with a slope of 1.87 (r2=0.88). The plots present averaged values for each cell based on the duration of each recording (cell type 1: blue markers, cell type 2: orange markers, less stable cells: gray markers (see Methods)). The basal area and speed of type 2 cells was larger than for type 1 cells: 628 (502/692) μm2 and 6.0 (5.4/8.2) μm/min (N=12 biological replicates) vs. 326 (258/461) μm2 and 10.8 (9.4/12.3) μm/min (N=161 biological replicates) (p=1.9x10-6 and 2.6x10-7) while the median ratio between the pole-pole distance and the cell's length was 0.75 (0.70/0.79, N=161 biological replicates) for type 1 cells and 0.84 (0.77/0.90, N=12 biological replicates) for type 2 cells (p=2.2 10-3).

Data information: The arrows indicate the direction of motion and black dots in the kymographs correspond to the location of maximum stress. All scale bars in the figure: 10μm.
Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion (2021)
Figure 3

Figure 3 Traction force maps and distributions of signaling components in oscillatory cells.

A: Sequence of snapshots of the LimE-GFP distribution and stress T for an oscillatory cell together with snapshots of the GFP-myo distribution for a separate oscillatory cell. Corresponding speeds are 7.0µm/min and 4.6µm/min. The sequences show an entire cycle of oscillation (spreading and contraction). White arrows represent the direction of motion before the cell spreads and contracts during which no net motion exists.

B: Top: Basal area (black line) and limE fluorescent intensity (green line) as a function of time, for the same cell as in A. Inset: Temporal CF of the area and the limE fluorescent intensity (blue) and of the area change rate and the limE fluorescent intensity (magenta). Bottom: Kymograph of the LimE-GFP intensity along the cell outline.

C: Top: Basal area (black line) and myosin fluorescent intensity (green line) as a function of time for a separate cell. Inset: Temporal CF of the area and the myosin fluorescent intensity (blue) and the area change rate and the myosin fluorescent intensity (magenta). Bottom: Kymograph of the GFP-myo intensity along the cell outline.

D: Top: Basal area (black line) and total force F (blue line) as a function of time, for the same cell as in A. Inset: Temporal cross-correlation function (CF) of the area and F (blue) and of the area change rate and F (magenta). Bottom: Kymograph of T along the cell outline.

E: Median shift in the CF of the area and F (33.8 (22.5/42.2) s; N=45 biological replicates), the area and the LimE-GFP intensity (-11.2 (-19.6/ -10.3) s; N=21 biological replicates), and the area and the GFP-myo intensity (52.5 (41.3/62.8) s; N=11 biological replicates).

F: Median shift in the CF of the area change rate and F (-3.7 (-11.2/3.8) s; N=42 biological replicates), the area change rate and the LimE-GFP intensity (16.9 (7.5/30.0) s; N=20 biological replicates), and the area change rate and the GFP-myo intensity (3.8 (0/7.5) s; N=11 biological replicates). Data bellow the dashed line indicates cells for which no significant correlation was found.

G: Time-averaged total force F as a function of the cell area A.

Data information: (B-D) The peaks in the CF are indicated by star symbols, the 95% confidence interval is gray-shaded, and the sign of the peak in the CF defined whether the quantities were correlated (largest peak occurred for positive CF values) or were anti-correlated (largest peak occurred for negative CF values) (see Methods). (E-F) P-values higher that 0.05 are considered not significant, *P<0.05, **P<0.01, ***P< 0.001, ****P<0.0001 as determined by the Wilcoxon-Mann-Whitney test using the ranksum function in MATLAB. All scale bars in the figure: 10μm.
Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion (2021)
Figure 4

Figure 4 Traction force maps and distributions of signaling components in amoeboid cells.

A: Sequence of snapshots of the LimE-GFP distribution and stress T for an amoeboid cell and the GFP-myo distribution for a separate amoeboid cell. Corresponding speeds are 7.4µm/min and 4.5µm/min. The sequences show both the phases of protrusion and retraction. White arrows represent the direction of motion.

B: Spatially averaged LimE-GFP intensity as a function of time (green line), together with the basal area (black line) for the first cell in A. Inset: CF of the area and LimE-GFP intensity (blue) and of the area change rate and the LimE-GFP intensity (magenta).

C: Basal area (black line) and total force F (blue line) as a function of time corresponding to the first cell in A. Inset: Temporal cross-correlation function (CF) of the area and the total force F (blue) and of the area change rate and F (magenta). The correlation within the shaded region is below the 95% confidence interval.

D: Median shift in the CF of the area and F (blue symbols; N=66 biological replicates), the LimE-GFP intensity (brown symbols, N=18 biological replicates) and the GFP-myo intensity (yellow symbols, N=7 biological replicates). Values of these shifts were determined as 11.3 (3.8/15) s, -11.2 (-15.0/-7.5) s, and 7.5 (1.9/12.2) s.

E: Median shift in the CF of the area change rate and F (blue symbols, N=52 biological replicates), the LimE-GFP intensity (brown symbols, N=17 biological replicates) and the GFP-myo intensity (yellow symbols, N=7 biological replicates). Data below the dashed line indicates cells for which no significant correlation was found. Shift values are -11.2 (-15.0/-9.4) s, 11.3 (7.5/11.3) s, and -26.3 (-30.0/-14.1) s.

F, G and H: Kymographs for LimE-GFP intensity, T, and edge velocity along the cell outlines for the first cell of A.

I, J and K: kymographs for GFP-myo intensity, T, and edge velocity along the cell outlines for the second cell of A.

L and M: Time-averaged total force F as a function of the cell's area and time-averaged Fy as a function of time-averaged Fx (slope of fit shown as a dashed line: 0.86, r2=0.94). Markers indicate the different strains: AX2 LimE-GFP (>), AX2 GFP-myo (<), AX2 lifeAct-GFP (^) and engineered cells (o).

Data information: (B, C) The peaks in the CF are indicated by star symbols, the 95% confidence interval is gray-shaded, and the sign of the peak in the CF defined whether the quantities were correlated (largest peak occurred for positive CF values) or were anti-correlated (largest peak occurred for negative CF values) (see Methods). (D-E) P-values higher that 0.05 are considered not significant, *P<0.05, **P<0.01, ***P< 0.001, ****P<0.0001 as determined by the Wilcoxon-Mann-Whitney test using the ranksum function in MATLAB. All scale bars in the figure: 10μm. Arrows indicate direction of motion.
Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycetes-specific transcription factor (2021)
Figure 1

Figure 1: AosR is a novel redox responsive transcription factor.

a. Schematic outline of the strategy used for the identification of rv1332/aosR.

b. Single-cell suspensions of Rv, Rv∆aosR, and Rv∆aosR::aosR strains were used to infect pΦ isolated from Balb/C at 1:10 MOI. Cells were lysed at indicated time points and the intracellular survival was enumerated. Data is presented as mean CFU log10/ml ± SEM and is representative of two biologically independent experiments, each performed in triplicates (n=3).

c. Balb/C mice were aerosolically challenged with Rv, Rv∆aosR, and Rv∆aosR::aosR strains. Each data point represents log10 CFU obtained from the infected lung of mice and e­rror bar depicts the median with interquartile range for each group.

d. Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group.

e. Bacterial strains were treated with indicated oxidizing agents for 24 h and the survival was monitored. Control represents untreated cultures. Data is presented as mean CFU log10/ml ± SEM and is representative of two biologically independent experiments, each performed in triplicates (n=3).

Data information: Statistical significance was analysed using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Source data are provided in the Source Data file.
Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycetes-specific transcription factor (2021)
Figure 3

Figure 3: AosR rewires transcriptome in response to oxidative stress.

a,b. Volcano plots were obtained from DEseq2 analysis across three biological replicates of Rv vs Rv∆aosR. DEGs are highlighted as red dots (log2 fold change<1; p adj<0.1). P values were calculated using DEseq2, which uses Wald test and were adjusted for multiple testing using Benjamini-Hotchberg method. Numbers in top quadrants reveal upregulated (in green) and downregulated (in red) in Rv compared with Rv∆aosR. b. Heat map indicates log2 fold changes of enriched (green) and depleted (red) genes in Rv compared with Rv∆aosR. Color intensity indicates relative higher upregulation and downregulation, respectively.

c. Same as (a) under oxidative conditions.

d. Venn diagram illustrating the common set of genes between RvandRv∆aosR under oxidative conditions compared with Rv∆aosR in normoxic and oxidative conditions.

e. Same as (b) under oxidative conditions. DEGs are clustered according to their functional category. The analysis was performed as explained in Appendix Fig S1.

f. Bar graphs depicting the percentage of enriched (green) and depleted (red) DEGs plotted in (e) belonging to a particular functional category.

g, h. Relative gene expression of selected DEGs measured by qRT-PCR in Rv compared with Rv treated with CHP (g) and Rv as compared with Rv∆aosR under oxidative conditions (h). Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates.

Data information: Statistical significance was analysed using the student's t-test (two tailed, unpaired). *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Source data are provided in the Source Data file.
Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycetes-specific transcription factor (2021)
Figure 4

Figure 4: AosR regulates non-canonical cysteine biosynthesis pathway.

a. Non-canonical pathway for cysteine biosynthesis in Mtb. MoeZ transfers sulfur from a yet unknown source to the C-terminus of CysO to form thiocarboxylated CysO (CysO-SH). CysM, a non-canonical cysteine synthase, utilizes O-phospho-L-serine (OPS) and CysO-SH as substrates to generate CysO-cysteine adduct, which is cleaved by a protease Mec, to release cysteine and regenerate CysO(Burns et al., 2005).

b. Hypothetical model for transcriptional regulation mediated by AosR. Upon oxidative stress, AosR binds to its promoter regulating the entire operon through a positive feedback loop, resulting in higher L-cysteine biosynthesis.

c. Early log-phase cultures of indicated strains were subjected to 50 µM of CHP for 24 h, in the presence or absence of 1 mM L-cysteine, and CFUs were enumerated (mean ±SEM; n=3).

d. Single-cell suspensions of indicated strains were used to infect pΦ from Balb/C at 1:10 MOI. Cells were either supplemented with 0.2 mM L-cysteine or left untreated and the CFUs were enumerated 96 h p.i. (mean ± SEM; n=3).

e. pΦ from Balb/C were infected with indicated strains and bacterial viability was monitored 96 h p.i (mean ± SEM; n=3).

f. 35 µg whole cell lysates (WCL) of Rv, Rv∆aosR and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 24 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane and probed with α-AosR and α-SigA antibodies.

g. Relative expression of indicated genes in Rvtreated with 50 µM CHP for 24 h as compared with Rv as measured by qRT-PCR. Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change ± SD of three independent replicates and is representative of one of two biological replicates. Statistical significance was analysed using student's t-test (two-tailed, unpaired).

h. WCLs of Rv, Rv∆aosR, and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, and probed with indicated antibodies.

i. Early log-phase cultures of indicated strains were subjected to 50 µM of CHP for 24 h and the CFUs (mean ±SEM; n=3) were enumerated.

j, k. pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p.i (mean ± SEM; n=3).

Data information: (c-e & i-k) Statistical significance was analysed using one-way ANOVA followed by a post hoc test (Tukey test; GraphPad prism). *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Source data are provided in the Source Data file.
Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycetes-specific transcription factor (2021)
Figure 5

Figure 5: AosR binds to a stress-inducible auxiliary promoter in conjunction with SigH.

a. Alternate model for the transcriptional regulation mediated by AosR. Upon oxidative stress, AosR regulates auxiliary promoter upstream of mec-cysO-cysM resulting in enhanced L-cysteine biosynthesis.

b. The promoter of sigA and mec was fused to luciferase, and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=6)

c. Outline of interactome experiment.

d. Venn diagram illustrating the overlaps among interacting partners found in different conditions.

e. Table listing the proteins that interact with AosR specifically under oxidative stress.

f. BLI sensorgram demonstrating relative binding affinities of 800nM His-AosR to immobilized His-SigH under oxidizing and reducing conditions.

g. Rv::pN and Rv::pN-F-sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-AosR. 50 µg WCLs were probed with α-FLAG,α-AosR, and α-SigA as controls.

h. Expression of CysO, CysM, AosR and GroEL1 (control) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h.

i. SigH dependent promoter is present upstream of mec. The consensus -35 and -10 regions along with TSP are indicated (Sharp et al., 2016).

j. 100 bp upstream of mec was fused to luciferase, and transformed cells were either left untreated or exposed to 50 μM CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=3)

k. 5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.

Source data are provided in the Source Data file.