EMBO/soda-nel huggingface dataset:
Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features (2020)
Figure 4

Figure 4. Netrin-1 is neuroprotective in progressive and acute mouse models of PD.

(A) Experimental design in a unilateral 6OHDA mouse model of PD. Netrin-1 inducible mice and WT littermates were lesioned with 6-OHDA in two sites of the right striatum (2 x 1µg). After two weeks, mice received i-p injections of tamoxifen to induce netrin-1 expression (O/E). Amphetamine-induced ipsilateral rotation behaviour was assessed at week two and week six post lesion then brains were taken to perform IHC analyses.

(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.

(C) Optical density of TH-positive fibres in the striatum. Individual values and mean ± SD, n= 10 animals, unpaired t-test, *P < 0.05.

(D) TH-positive cell count in the SN. Individual values and mean ± SD, n= 10 animals, unpaired t-test.

(E) Experimental design in the SNCA Tg MPTP acute mouse model of PD. WT and SNCA Tg mice were injected with netrin- 1 human recombinant protein in one site of the SN (2µg). MPTP (18mg / kg) was then injected (i-p) every 2 h for a total of four doses over on 8 h period in 1 day.

(F) TH staining by immunohistochemistry (Scale bar, 2000µm) in the SN (left) and striatum (right) of WT and SNCA Tg mice, 6 days after treatment.

(G) TH-positive cell count bar graph. All experiments were repeated 3 times. We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. **P< 0.01; N.S., not significant.

(H-J) Motor behavioural assays (Rotarod rod test (H), Cylinder test (I), Grid test (J)) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.
Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features (2020)
Figure 5

Figure 5. Netrin-1 is neurorestorative in the rat 6-OHDA preclinical model of PD.

(A) Experimental design in a unilateral 6-OHDA rat model of PD. Rats were lesioned unilaterally with 6-OHDA into three sites of the right striatum (3 x 2 µg). After two weeks, rats received intrastriatal injections of recombinant human netrin-1, recombinant human GDNF or PBS (VEH). Amphetamine-induced behaviour was assessed every two weeks for 12 weeks then brains were taken to perform IHC analyses.

(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.

(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom

(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.
Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features (2020)
Figure 6

Figure 6. Netrin-1 is reduced in the SN of PD patients

(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown.

(B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 1

Figure 1. FRS2 knockdown activates TGF signaling in primary human aortic smooth muscle cells (HASMCs).

(A-B) qRT-PCR analysis of TGF ligands, TGF receptors, and TGF target expression in control and FRS2 knockdown HASMCs. (NS: not significant compared to control, *p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student’s t test). -actin was used for sample loading normalization. Histogram of qRT-PCR results are representative of three independent experiments.

(C) Left: Immunoblot analysis of TGFRs, phosphorylated Smad2 (p-Smad2), and phosphorylated Smad3 (p-Smad3) in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of p-Smad2, p-Smad3, TGFR1, and TGFR2 were normalized to Smad2/3 or GAPDH and expressed as a fraction of a control value. Results are expressed as means ± SD (*p<0.05; ***p<0.001 compared to control; unpaired two-tailed Student’s t test).

Source data are available online for this figure.
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 2

Figure 2. FRS2 knockdown increases smooth muscle marker gene expression via the TGF pathway in primary human aortic smooth muscle cells (HASMCs).

(A) Left: Immunoblot analysis of smooth muscle marker gene expression in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of SM -actin, SM22, and SM-calponin were normalized to -tubulin and expressed as a fraction of a control value. Results are expressed as means ± SD (**p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student’s t test).

(B) qRT-PCR analysis of SMC transcription factor gene expression in control and FRS2 knockdown HASMCs. (*p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student’s t test. N=3). -actin was used for sample loading normalization.

(C) Collagen gel contraction assays were used to determine the contractile ability of control or FRS2 knockdown HASMCs (*p<0.05 compared to control; unpaired two-tailed Student’s t test. N=3).

(D-F) Upper panels: Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 (10 m), TGFR2 or Smad2 shRNA lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to -tubulin, HSP90, or Smad2 and expressed as a fraction of a control value.

Source data are available online for this figure.
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 3

Figure 3. FRS2 knockdown increases smooth muscle marker gene expression via the let-7-TGF pathway in primary human aortic smooth muscle cells (HASMCs).

(A) Quantitative real-time PCR analysis of mature let-7 family in control and FRS2 knockdown HASMCs. SNORD47 was used to normalize the variability in template loading. Histogram of qRT-PCR results are three independent experiments.

(B) Upper panel: Immunoblots of SM-calponin, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs transduced with or without let-7b lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to GAPDH or Smad2 and expressed as a fraction of a control value.

(C) Phase-contrast and immunofluorescence staining of smooth muscle markers (red) in HASMCs. Nuclei were counterstained with DAPI (blue). Scale bar: 12 m. Images are representative of three independent experiments.

(D) HASMCs were cultured in the growth medium (M231 + SMGS) at day 0 then switched from growth conditions to differentiation medium (M231 + SMDS) for 8 days. Quantitative real-time PCR analysis of mature let-7 family in HASMCs. SNORD47 was used to normalize the variability in template loading. Histogram of qRT-PCR results are three independent experiments.

(E) Control and FRS2 knockdown HASMCs were cultured in the growth medium (M231 + SMGS) at day 0 then switched from growth conditions to differentiation medium (M231 + SMDS) for 6 days with or without let-7b lentiviruses. Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs with or without let-7b lentiviruses. Blots are representative of three independent experiments.

Source data are available online for this figure.
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 4

Figure 4. FGFR1 signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis.

(A) Coronary arteries dissected from the human heart. Left main (LM), left anterior descending (LAD), and left circumflex (LCX) branches. Scale bar: 1 cm.

(B) Elastic-Van Gieson (EVG) staining of human coronary arteries demonstrating various degrees of atherosclerosis.

(C-D) Representative images of immunofluorescence staining for CD31 (green) and SM -actin (red) or SM-MHC (red) in No/mild, moderate, and severe disease human left main coronary arteries. No: no-disease. Nuclei were stained with DAPI (blue). (b)&(d)&(f) are high-magnification view from (a)&(c)&(e). Images are representative of ten No/mild, nine moderate and ten severe disease human left main coronary artery samples. L: lumen. Scale bar: 16 m.

(E&G) Representative images of immunofluorescence staining for p-FGFR1 (red) or FGFR1 (red) in the same patient cohort. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m.

(F&H) Percentage of medial p-FGFR1+ SMC and FGFR1+ SMC (***p<0.001 compared to No/mild disease, NS: not significant compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction).

A full table of p-values for this figure is shown in Appendix Table S1.
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 5

Figure 5. TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis.

(A&C&E) Representative images of immunofluorescence staining for TGF (red), p-Smad2 (red), or p-Smad3 (red) from patients with No/mild, moderate, or severe disease. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m.

(B&D&F) Medial TGF area and percentage of medial p-Smad2, and p-Smad3 positive SMCs (***p<0.001 compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction).

A full table of p-values for this figure is shown in Appendix Table S1.
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 6

Figure 6. FGFR1 and TGFβ signaling activity in smooth muscle cells in a mouse atherosclerosis model.

(A) Dissected mouse aorta demonstrating lipid-rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe-/- mice. (b) & (d) Cross-section of brachiocephalic artery from (a) & (c) stained with Oil Red O. L: lumen. Scale bar: 4 mm. 3 mice per group.

(B) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti-SM -actin, anti-Notch3, and anti-SM-MHC antibodies. Nuclei were counterstained with DAPI (blue). L: lumen. Scale bar: 62 m. 3 mice per group.

(C-F) Analysis of brachiocephalic artery of Apoe-/- mice maintained for 4 months on either normal or high fat diet using anti-CD31 (green), anti-p-FGFR1 (red), anti-FGFR1 (red), anti-p-Smad2 (red), and anti-p-Smad3 (red) antibodies. Nuclei counterstained with DAPI (blue). Scale bar: 62 m. L: lumen. M: Media. 6 mice per group.

(G-J) Quantification of the number of medial smooth muscle cells expressing p-FGFR1, FGFR1, p-Smad2, and p-Smad3 (***p<0.001 compared to ND, NS: not significant compared to ND; unpaired two-tailed Student’s t test.). ND: Normal diet. HFD: High fat diet.

A full table of p-values for this figure is shown in Appendix Table S1.
Smooth muscle FGF/TGFβ cross-talk regulates atherosclerosis progression (2016)
Figure 7

Figure 7. Smooth muscle cell FRS2 knockout inhibits atherosclerosis plaque development after 16 weeks of high fat diet

(A) Representative photomicrographs of Oil Red O-stained atherosclerotic lesions in the aortic arch, of Apoe-/- or Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet. 3 mice per group. Scale bar: 5 mm.

(B) (Left) Microphotographs of aortas (en face) from Apoe-/- and Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet after staining with Oil Red O. (Right) Lesion area quantification. All data shown as mean ± SD. (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student’s t test). 12 mice per group.

(C-D) Representative cross-sections of brachiocephalic arteries Apoe-/- and Frs2SMCKO/Apoe-/- mice stained with hematoxylin and eosin (H&E) (C) and Movat (D). (b)&(d) are high-magnification view from (a)&(c). NC: necrotic core. Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.

(E) Histological analysis of atheosclerotic plaque with anti-Ki67 antibody. Nuclei were counterstained with DAPI (blue). (b)&(d) are high-magnification images from (a)&(c). Scale bar: 62 m (low-magnification images); 10 m (high-magnification images). Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.

(F) Quantification of plaque cellularity; Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12 (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student’s t test).

(G) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe-/- and Frs2SMCKO/Apoe-/- mice. (*p<0.05, **p<0.01 compared to Apoe-/-; unpaired two-tailed Student’s t test).

(H) Measurement of Ki67+ cells (*p<0.05, ***p<0.001 compared to Apoe-/-; unpaired two-tailed Student’s t test).

A full table of p-values for this figure is shown in Appendix Table S1.
Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation (2022)
Figure 2

Figure 2. TOP1 expression is increased in neuroinflammatory conditions.

A A schematic overview of the in vitro experiments.

B, C Kinetic change of mRNA expression of Top1, Btbd1, Tdp1, and Srsf1 in microglia following LPS/IFNγ stimulation (n = 6, technical replicates).

D, E The protein expression of TOP1 was detected by Western blotting (n = 3, biological replicates).

F-H Immunostaining of TOP1 (red) in stimulated microglia (4 h after LPS/IFNγ stimulation) or resting microglia, co-stained with Hoechst (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed.

G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti-F4/80 (green), TOP1 (magenta), and nuclei with Hoechst (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.

I A magnified image of the selected region (white box) in (G); bar = 50 μm; the yellow arrowhead indicates the representative F4/80+ cell with increased TOP1 expression in the nucleus in (G).

J The mean fluorescent intensity of TOP1 expression in the white matter in (G) was graphed; n = 3 to 4 mice per group with 3-5 sections from each mouse.

K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak (scored 3) or control mice were examined by Western blotting.

L The quantification of TOP1 protein expression of Western blot bands in (K) with normalization to GAPDH and β-actin (n = 3 biological samples).

M, N Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples).

O Microglia were sorted from LPS challenged mice (i.p) after 4 h or naive mice, and the mRNA expression of TOP1 was graphed (n = 5 or 6 biological samples).

P Brain sections from healthy control and MS brain tissues, including the active MS lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained with anti-HLA-DR, anti-TOP1, and co-stained with Hoechst (bar = 50 μm).

Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C), and the rest were performed using the student's unpaired two-tailed t-test.
Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation (2022)
Figure 3

Figure 3. Camptothecin impedes the inflammatory response in stimulated primary microglia.

A Cytotoxicity of CPT on stimulated microglia was determined by measuring the release of LDH in the supernatants (n = 4 technical replicates).

B Supernatants were collected after 24 h LPS/IFNγ stimulation and measured for nitrite, as an index of nitric oxide (NO) production (n = 4 technical replicates).

C, D Immunostaining of iNOS (red) and Phalloidin (green) in microglia (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 μm).

E The kinetics of mRNA expression of Nos2, Tnfa, Il1b, Il6, and Il10 after 1, 3, 6, and 24 h of LPS/IFNγ stimulation (n = 3 to 6 technical replicates).

F Cytokine release (TNF, IL-6, MCP-1, and IL-10) to the supernatants were measured by cytometric beads assay after 24 h LPS/IFNγ stimulation (n = 2 or 5 technical replicates).

G, H The production of TNF-α within microglia was determined by flow cytometry (n = 3 to 5 technical replicates).

Data information: Data shown are mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett's Multiple Comparison Test for (A), (B), and (D), and the comparisons between the LPS/IFNγ and LPS/IFNγ+CPT group in (E-H) were performed using the student's unpaired two-tailed t-test.
Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation (2022)
Figure 4

Figure 4. Topotecan mitigates microglial activation and CNS inflammation in LPS-challenged mice.

A An overview of the experimental design.

B Microglia in the hippocampus and thalamus were stained with Iba1 (red); bar = 50 μm; microglia in the yellow boxes were presented in (E).

C, D Fluorescent intensity of Iba1 in hippocampus and thalamus in (B); n = 2, 4, 5 mice for naive, LPS, and LPS + TPT group, respectively (2-6 sections per mouse were analyzed).

E Sholl analysis was performed to analyze the morphology of microglia. Microglia marked in yellow boxes in (B) were presented accordingly.

F-I The morphological changes of microglia were objectified by the number of intersections per radius (F and G), furthest branch length (H), and the ramification index presented as total intersections/primary branches (I); n = 2, 4, 5 mice for naive, LPS, and LPS + TPT group, respectively (4 to 7 complete microglia from each mouse were included for analysis); in (H) and (I), the central band represents the median value, the box extends from the 25th to 75th percentiles, and the whiskers plot the smallest and the largest value

J, K Expression of inflammatory genes in the hippocampus and hypothalamus 4 h after the LPS challenge (n = 3 mice).

L, M Cytokine array on brain homogenates after 24 h of LPS challenge (n = 2 mice for the naïve group; n = 3 mice for the LPS and LPS + TPT groups).

N The contact frequency of each mouse to the new toy during the cage explorative experiment was recorded for 5 min (n = 5, 12, 13 mice for the naive, LPS_PBS, and LPS_TPT groups, respectively); data are pooled from two independent experiments with similar results.

O A lethal dose of LPS (18 mg/kg; i.p) was given to mice; mice in the LPS + TPT group received 1 mg/kg TPT (i.p) 1 h before the lethal dose LPS challenge, whereas the control group received PBS injection (n = 14 mice).

Data information: Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses comparing the LPS_PBS group with the LPS_TPT group were performed using the Student's unpaired two-tailed t-test; the survival curve was analyzed with Log-rank.
Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation (2022)
Figure 6

Figure 6. Topotecan prevents disease progression in the EAE model.

A Mice received TPT treatment (1 mg/kg; i.p) at indicated days post-immunization and were randomly assigned to the pre-onset group (day 5, 7, 9), onset group (day 11, 13, 15), and peak group (day 17, 19, 21); mice in the EAE control group received 3 PBS injections randomly from day 5 (i.p).

B Bodyweight change following EAE immunization (n = 9-11 mice per group).

C-G EAE clinical scores among the groups were analyzed using different parameters: area under the curve (C), day of onset (D), cumulative score (E), the average score from day 10 (F), and max score (G); n = 9-11 mice per group; data are from one EAE batch representing three batches with a similar effect.

H EAE mice were treated individually from the day when they were just scored or scored higher than 1; n = 8 mice per group. Data are from one EAE batch representing two batches with a similar effect.

I EAE mice from the onset group or control were euthanized at the peak of EAE (day 18), and the spinal cords were dissected for flow cytometry with an antibody panel for myeloid cells.

J The counts of different myeloid cells in (I), including monocytes (Mono), macrophages (Mac), microglia (Mic), and neutrophils (Neutro), were graphed (n = 5 mice); this experiment was performed twice.

K Spinal cord sections dissected from Day 30 from the EAE batch in (H) were stained with CD11b (red), Fluoromyelin (green), and Hoechst (blue) (bar = 200 μm).

L The CD11b immunofluorescent intensity in (K) was graphed (n = 2 mice for the control group; n = 4 mice for the EAE-PBS and EAE-TPT groups).

M The percentage of demyelinated area in the white matter of the spinal cord was graphed (n = 2 mice for the control group, n = 4 mice for the EAE-PBS and EAE-TPT groups).

Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. The following statistical analyses were used: the EAE scores in (D-G) were analyzed using the Kruskal-Wallis nonparametric test; the AUC in (C) was analyzed using the one-way ANOVA with Dunnett's Multiple Comparison test, and the Student's unpaired two-tailed t-test was used for comparing the EAE-PBS group with the EAE-TPT group in H (AUC), J, L, and M.