Journal: bioRxiv

Article Title: Transcriptional landscape of the dorsal raphe serotonin neurons rendering stress resiliency

doi: 10.1101/2024.03.21.586199

Figure Lengend Snippet: (a) The graph represents the expression changes of the SSRI treated mice compared to the naïve mice on the horizontal axis and the expression changes of the CSDS susceptible mice compared to the CSDS resilient mice on the vertical axis. Light blue dots were the genes whose expression changes were significantly increased in CSDS susceptible mice and decreased in SSRI treated mice, whereas the orange dots vice versa. (b) The serotonin neuron enrichment of significant genes in (a). Red dots were the genes whose expressions were enriched 2-fold or more in the TRAP sample and blue dots were the genes whose expressions were depleted 2-fold or more in the TRAP sample compared to Input. (c) Time course of SSRI treatment (top), immunofluorescence image (middle) and quantification of S100a10 (bottom) in DRN of naïve mice and SSRI treated mice. Scale bar, 200 μm (left panel), 20 μm (middle and right panels). n = 6. ** P < 0.01 and *** P < 0.001, two-tailed t-test. (d) Time course of CSDS (top), immunofluorescence image (middle) and quantification of S100a10 (bottom) intensity in DRN of CSDS resilient and susceptible mice. Scale bar, 200 μm (left panel), 20 μm (middle and right panels). n = 12-17. * P < 0.05, two-tailed t-test. (e) S100a10 mRNA expression in Lenti-X 293T cells treated with the negative-control plasmid or the S100a10 knockdown plasmid. The expression was normalized by hGAPDH. n = 4. **** P < 0.0001, two-tailed t-test. (f) Schematic representation of AAV injection and time course of experiments. (g) Immunofluorescence image (top) and quantification of S100a10 intensity (bottom) in DRN of control and knockdown mice. See Materials & Methods for the sequence used for each knockdown. Scale bar, 20 μm. n = 15-19 (for KD #1 experiments), 11 (for KD #2 experiments). *** P < 0.0001, **** P < 0.0001, two-tailed t-test. (h) Total distance in Open Field Test (OFT), (i) time spent in open arm in Elevated Plus Maze test (EPM) and (j) total immobility time in Tail Suspension Test (TST). n = 15-19 (for KD #1 experiments), 11 (for KD #2 experiments). * P < 0.05, two-tailed t-test. All data presented as means ± s.e.m.

Article Snippet: Then sections were incubated overnight at 4 ◦C with goat polyclonal anti-S100a10 antibody (1:200; AF2377, R&D Systems, Minneapolis, MN, USA), rabbit polyclonal anti-5HT 1B Receptor antibody (1:100; ab13896, abcam, Cambridge, UK), sheep polyclonal anti-tryptophan hydroxylase (TPH) antibody (1:500; AB1541, Merck Millipore, Burlington, MA, USA), and rabbit polyclonal Anti-STAT6 (phospho Y641) antibody (1:100; ab28829, abcam) diluted in the blocking buffer.

Techniques: Expressing, Immunofluorescence, Two Tailed Test, Negative Control, Plasmid Preparation, Knockdown, Injection, Control, Sequencing, Suspension

Journal: bioRxiv

Article Title: Transcriptional landscape of the dorsal raphe serotonin neurons rendering stress resiliency

doi: 10.1101/2024.03.21.586199

Figure Lengend Snippet: (a) Immunofluorescence image (top) and quantification of 5-HT 1B R (bottom) in DRN of control mice and S100a10 knockdown mice. Images were taken in areas of the DRN where AAV vector was well expressed (mainly ventral part of the DRN). Scale bar, 20 μm. n = 14-19 (for KD #1 experiments), 17 (for KD #2 experiments). * P < 0.05, two-tailed t-test. (b) Htr1b mRNA expression in HEK 293T cells treated with the negative-control plasmid or the Htr1b knockdown plasmid. The expression was normalized by hGAPDH. n = 4. **P < 0.01, two-tailed t-test. (c) Schematic representation of AAV injection and time course of experiments. (d) Immunofluorescence image (left) and quantification of 5-HT 1B R intensity (right) in DRN of control and knockdown mice. Scale bar, 20 μm. n = 13-14. **P < 0.01, two-tailed t-test. (e) Total distance in OFT, (f) time spent in open arm in EPM, (g) total immobility time in TST. n = 13-14. *P < 0.05, two-tailed t-test. All data presented as means ± s.e.m.

Article Snippet: Then sections were incubated overnight at 4 ◦C with goat polyclonal anti-S100a10 antibody (1:200; AF2377, R&D Systems, Minneapolis, MN, USA), rabbit polyclonal anti-5HT 1B Receptor antibody (1:100; ab13896, abcam, Cambridge, UK), sheep polyclonal anti-tryptophan hydroxylase (TPH) antibody (1:500; AB1541, Merck Millipore, Burlington, MA, USA), and rabbit polyclonal Anti-STAT6 (phospho Y641) antibody (1:100; ab28829, abcam) diluted in the blocking buffer.

Techniques: Immunofluorescence, Control, Knockdown, Plasmid Preparation, Two Tailed Test, Expressing, Negative Control, Injection

Journal: bioRxiv

Article Title: Transcriptional landscape of the dorsal raphe serotonin neurons rendering stress resiliency

doi: 10.1101/2024.03.21.586199

Figure Lengend Snippet: (a) IPA comparison analysis of upstream pathways. (b) A schematic illustration of the IL4-STAT6 signaling pathway. (c) Immunofluorescence image (left) and quantification of S100a10 (right) in DRN of SSRI-treated mice at each time points. Scale bar, 200 μm (top pannel), 20 μm (middle and bottom panels). n = 9-10. **P < 0.01, compared to 0 day, one-way ANOVA, Dunnett’s post hoc test. (d) Immunofluorescence image (left) and quantification of pSTAT6 (phosphor Y641) (right) in DRN of SSRI-treated mice at each time point. Scale bar, 20 μm. n = 9-10. *P < 0.01, compared to 0 day, one-way ANOVA, Dunnett’s post hoc test. (e) ELISA measurements for IL-4 (left) and IL-13 (middle) levels of DRN at each time point after SSRI treatment. Total protein levels of DRN samples measured by BCA assay (right). n = 9-10. Data presented as means ± s.e.m. n = 9-10. *P < 0.01, compared to 0 day, one-way ANOVA, Dunnett’s post hoc test. (f) An illustration of the STAT6 mutant used in the following experiments. (g) Luciferase activity of 48 h posttransfection of hSTAT6 YF or pcDNA in either unstimulated HepG2 cells or cells that had been treated with IL-4 6 h prior to harvest. We conducted two independent assays (n = 3 each) and the luciferase activity was normalized by each batch of the IL-4 (+) YF (-) group. ****P < 0.0001, compared to IL-4 (-) YF (-), ####P < 0.0001, compared to IL-4 (+) YF (-), respectively by One-way ANOVA, Tukeys post hoc test. (h) Schematic representation of AAV injection and time course of experiments. (i) Total distance in OFT, (j) time spent in open arm in EPM, (k) total immobility time in TST. n = 14-15. ****P < 0.0001, two-tailed t-test. (l) Schematic representation of drug injection and time course of experiments. (m) Total distance in OFT, (n) time spent in open arm in EPM, (o) total immobility time in TST. n = 11-12. ***P < 0.001, compared to saline, one-way ANOVA, Dunnett’s post hoc test. (p) Immunofluorescence image (top) and quantification of S100a10 (bottom) in DRN of IL4-injected mice at each time points. Scale bar, 20 μm. n = 11-12. *P < 0.05, compared to saline, one-way ANOVA, Dunnett’s post hoc test. All data presented as means ± s.e.m.

Article Snippet: Then sections were incubated overnight at 4 ◦C with goat polyclonal anti-S100a10 antibody (1:200; AF2377, R&D Systems, Minneapolis, MN, USA), rabbit polyclonal anti-5HT 1B Receptor antibody (1:100; ab13896, abcam, Cambridge, UK), sheep polyclonal anti-tryptophan hydroxylase (TPH) antibody (1:500; AB1541, Merck Millipore, Burlington, MA, USA), and rabbit polyclonal Anti-STAT6 (phospho Y641) antibody (1:100; ab28829, abcam) diluted in the blocking buffer.

Techniques: Comparison, Immunofluorescence, Enzyme-linked Immunosorbent Assay, BIA-KA, Mutagenesis, Luciferase, Activity Assay, Injection, Two Tailed Test, Saline

Journal: The FASEB Journal

Article Title: Cellular Landscape of Synovial Chondromatosis Synovium Revealed by Single‐Cell RNA Sequencing

doi: 10.1096/fj.202500034RR

Figure Lengend Snippet: Transcriptomic Changes and Pseudotime Analysis of Fibroblasts in Synovial Chondromatosis. (A) Volcano plot of differential genes in fibroblasts for the SC group vs. the normal group. (B) Elevated protein expression of S100A10 in the synovium of patients with synovial chondromatosis. Scale bar = 80 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Bar plot of Gene Ontology (GO) enrichment analysis. (D) Bar plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. (E) Monocle pseudotime trajectory plots of each fibroblast cell type. (F) Monocle pseudotime trajectory plot of fibroblasts presenting the beginning and end pseudotime profiles. (G) The fibroblasts trajectories in the SC and normal groups. (H) The heatmap shows the expression levels of genes grouped into three gene modules according to the pseudotime axis. (I) Pseudotime kinetics of the top six genes among the five fibroblast subclusters. Each dot represents a cell, different colors represent different clusters, and the ordinate represents the expression level of each gene. (J) RNA velocity analysis distinguished several sets of velocity vectors across the pseudotime.

Article Snippet: The primary antibodies used included Collagen Type III (N‐terminal) Polyclonal antibody (22734‐1‐AP, Proteintech, 1:1000), MMP3 Polyclonal antibody (17873‐1‐AP, Proteintech, 1:100), MMP1 Polyclonal antibody (10371‐2‐AP, Proteintech, 1:100), S100A10 Polyclonal antibody (11250‐1‐AP, Proteintech, 1:400), TIMD4 Polyclonal antibody (12008‐1‐AP, Proteintech, 1:200), CNRIP1 Polyclonal antibody (16827‐1‐AP, Proteintech, 1:200), C1QA Monoclonal antibody (67063‐1‐IG, Proteintech, 1:100), CSF1R Polyclonal antibody (25949‐1‐AP, Proteintech, 1:50), FCGR3A Polyclonal antibody (16559‐1‐AP, Proteintech, 1:100), and MRC1 Polyclonal antibody (18704‐1‐AP, Proteintech, 1:100).

Techniques: Expressing

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: Runx2 expression increased after SCI and was related with astrocytes. A Western blot analysis of Runx2 (57 kDa) in lesions, with GAPDH (37 kDa) as an internal loading control. B Quantification of result in panel ( A ). The data were analyzed using one-way analysis of variance and all data are expressed as the mean ± S.D. C–E Immunofluorescence staining of Runx2 (green) and NeuN, GFAP, or Iba-1（red）in the spinal cord at 3 days post-injury. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 200 μm

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: Overexpressing Runx2 inhibited the activation of astrocytes following SCI in vivo. A Western blot analysis of Neurocan (143 kDa) and GFAP (55 kDa)in lesions, with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). The data were analyzed using one-way analysis of variance and all data are expressed as the mean ± S.D. D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the spinal cord at 14 days post-injury. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 200 μm. Runx2-OE (AAV-Runx2)

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Activation Assay, In Vivo, Western Blot, Control, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: The expression of Runx2 in nucleus increased in SVG-P12 after injury. A Western blot analysis of Runx2 (57 kDa) in PC12, with GAPDH (37 kDa) as an internal loading control. B Quantification of result in panel ( A ). D Western blot analysis of Runx2 (57 kDa) in BV2, with GAPDH (37 kDa) as an internal loading control. E Quantification of result in panel ( D ). G Western blot analysis of Runx2 (57 kDa) in SVG-P12, with GAPDH (37 kDa) as an internal loading control. H Quantification of result in panel ( G ). J Western blot analysis of Runx2 (57 kDa) in nucleus and cytoplasm in SVG-P12, with GAPDH (37 kDa) as an internal loading control. K , L Quantification of result in panel ( J ). The data were analyzed by two-tailed unpaired Student’s t -test, and the results represent as the mean ± S.D. C , F , I CCK8 assay was performed for the most suitable concentration in PC12 ( C ), BV2 ( F ), and SVG-P12 ( I ). The data were analyzed using one-way analysis of variance, and all data are expressed as the mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Expressing, Western Blot, Control, Two Tailed Test, CCK-8 Assay, Concentration Assay

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: The effect of Runx2 nuclear import is crucial on modulation of astroglial scar. A Western blot analysis of Runx2 in nucleus (57 kDa) and GFAP (55 kDa), with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the SVG-P12. E Immunofluorescence staining of DAPI (blue), Neurocan (red), and Runx2 (green) in the SVG-P12. The data were analyzed using one‑way analysis of variance, and all data are expressed as the mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 20 μm. DC (plasmid without Runx2), DR (plasmid of Runx2 with NMTS), DM (plasmid of Runx2 without NMTS)

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: Knockdown of Runx2 promoted astrocytes activation. A Western blot analysis of Runx2 in nucleus (57 kDa) and GFAP (55 kDa), with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the SVG-P12. The data were analyzed using one‑way analysis of variance, and all data are expressed as the mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 20 μm

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Knockdown, Activation Assay, Western Blot, Control, Immunofluorescence, Staining