Journal: Nature communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

doi: 10.1038/ncomms13648

Figure Lengend Snippet: Figure 3 | Expression of the SCN4B/b4gene in human breast cancer cell lines and contribution to cancer cell invasiveness. (a) The expression of SCN4B gene was studied by RT–qPCR in human mammary epithelial non-cancer MCF-10A and cancer MCF7, MDA-MB-468, MDA-MB-435s and MDA-MB-231 cell lines. Results are expressed as relative to that of HPRT-1 gene (n ¼ 7–12) and presented as mean values±s.e.m. *, significantly different from MCF-10A at Po0.05 (MW). (b) The expression of SCN4B/b4 protein was studied by densitometric analysis of western blot experiments in same cells as in a. Results are given as the amount of SCN4B/b4 protein relative to that of HSC70 (n ¼ 5) and presented as mean values±s.e.m. *, significantly different from MCF-10A at Po0.05 (MW). The image on top shows a representative western blotting experiment. (c) The expression of SCNxB genes was analysed in MDA-MB-231-Luc cells by reverse transcription–PCR. Plasmids encoding human SCNxB genes were used as positive controls for PCR primers. (d) Representative western blotting experiments showing protein expression for b1 (SCN1B), b2 (SCN2B) and b4 (SCN4B) in MDA-MB-231-Luc cells. (e) Cells were transfected with scrambled siRNA (siCTL) or with siRNA directed against the expression of the SCN1B gene (siSCN1B), the SCN2B gene (siSCN2B) or the SCN4B gene (siSCN4B). The efficacy of siRNA transfection was assessed by western blotting experiments 48 h after transfection. HSC70 was used as a control for sample loading. (f) Representative images of fixed and haematoxylin-stained MDA-MB-231-Luc cells on invasion inserts. Cancer cells were transfected with scrambled siCTL or with specific siRNA. Scale bars, 50 mm. (g) Summary of cancer cell invasiveness results (n ¼ 8) for MDA-MB-231-Luc cells transfected with siCTL or siSCNxB. Results were expressed relative to siCTL and presented as mean values±s.e.m. ***, statistically different from siCTL at Po0.001 (Student’s t-test). (h) Representative image of a zebrafish embryo injected in the yolk sac with MDA-MB-231-Luc cells stained with CM-Dil and showing sites of colonization. Scale bars, 500 mm. Below is a magnification of the highlighted region containing human cancer cells (see arrows) colonizing organs of the embryo. (i) Zebrafish colonization index of siCTL or siSCN4B cells. Numbers in brackets indicate the number of embryos examined for each condition, from three different experiments. Results are presented as mean values±s.e.m. **, statistically different from siCTL at Po0.01 (Student’s t-test).

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (siSCN1B, sc-97849), SCN2B (siSCN2B, sc-96252), SCN4B (siSCN4B, sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Transfection, Control, Staining, Injection

Journal: bioRxiv

Article Title: NG2 glia are required for maintaining microglia homeostatic state

doi: 10.1101/667402

Figure Lengend Snippet: a-b , western blots (a) and quantification (b) of PDGFRα and NG2 in slices treated with CP673451 and Crenolanib for 9 days. c , immunofluorescence of PDGFRα and NG2 on slices treated with CP673451 and Crenolanib for 9 days. Scale bar: 50 μm. d , quantification of PDGFRα + and NG2 + cell numbers shown in c. e , immunofluorescence of NG2 on slices incubated with 1 μM, 500 nM and 100 nM CP673451 or Crenolanib for 9 days. Scale bar: 50 μm. f , quantifications of NG2 + cell numbers shown in e. g , immunofluorescence of NG2 on slices incubated with 500 nM CP673451 and Crenolanib for 3, 5 and 7 days. Scale bar: 50 μm. h , quantifications of NG2 + cell numbers shown in g . * P<0.05; ** P<0.01; *** P< 0.001. n.s, not significant.

Article Snippet: PDGFR inhibitors CP673451 (HY-12050) and Crenolanib (HY-13223) were purchased from MedChemExpress, dissolved in dimethyl sulfoxide (DMSO, Sigma, 472301) and kept at −80 °C in aliquots.

Techniques: Western Blot, Immunofluorescence, Incubation

Journal: bioRxiv

Article Title: NG2 glia are required for maintaining microglia homeostatic state

doi: 10.1101/667402

Figure Lengend Snippet: a , mRNA levels of NG2 and PDGFRα after CP673451 and Crenolanib treatment on Oli-neu cells. b , representative images of western blots and quantifications of NG2 and PDGFRα protein levels after CP673451 and Crenoilanib treatment on Oli-neu cells. c , immunofluorescence of PDGFRα and NG2 on hippocampal and cortical slices treated with 500 nM CP673451 and Crenolanib for 9 days. Scale bar: 50 μm. d-g , quantifications of PDGFRα + and NG2 + cell numbers shown in c . * P<0.05; ** P<0.01; *** P< 0.001. n.s, not significant.

Article Snippet: PDGFR inhibitors CP673451 (HY-12050) and Crenolanib (HY-13223) were purchased from MedChemExpress, dissolved in dimethyl sulfoxide (DMSO, Sigma, 472301) and kept at −80 °C in aliquots.

Techniques: Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: NG2 glia are required for maintaining microglia homeostatic state

doi: 10.1101/667402

Figure Lengend Snippet: a , immunofluorescence of NG2, NeuN, GFAP, CC1, MBP, PDGFRβ, CD13 and Iba1 on slices treated with 500 nM CP673451 and Crenolanib for 9 days. Scale bar: 50 μm. Arrowheads: PDGFRβ + cells with NG2 glia morphology. b , quantifications of cell numbers or signal positive areas shown in a . *** P< 0.001. n.s, not significant.

Article Snippet: PDGFR inhibitors CP673451 (HY-12050) and Crenolanib (HY-13223) were purchased from MedChemExpress, dissolved in dimethyl sulfoxide (DMSO, Sigma, 472301) and kept at −80 °C in aliquots.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: NG2 glia are required for maintaining microglia homeostatic state

doi: 10.1101/667402

Figure Lengend Snippet: a , immunofluorescence of NG2 and CD68 on slices treated with 500 nM CP673451 and Crenolanib for 9 days. Scale bar: 50 μm. b , quantifications of NG2 + or CD68 + cell numbers shown in a. c , mRNA levels of TNFa, IL1β, IL6, IL12β, TGFβ1 and TGFβ2 after NG2 glia depletion. *** P< 0.001. n.s, not significant.

Article Snippet: PDGFR inhibitors CP673451 (HY-12050) and Crenolanib (HY-13223) were purchased from MedChemExpress, dissolved in dimethyl sulfoxide (DMSO, Sigma, 472301) and kept at −80 °C in aliquots.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: NG2 glia are required for maintaining microglia homeostatic state

doi: 10.1101/667402

Figure Lengend Snippet: a , volcano plots of RNAseq data comparing CP673451-treated (upper image) or Crenolanib-treated (lower image) with DMSO-treated slices. b , heatmap of genes differentially expressed between both CP673451 vs. DMSO and Crenolanib vs. DMSO (core-DEGs). c , assignment of core-DEGs to specific cell types according to published cell-type enrichment datasets. Core-Down: downregulated genes within the core-DEG collection. d , heatmaps of overlapping genes between the core-DEGs and NG2 glia-enriched (left image) or neuron-enriched genes (right image). e , heatmap of the 65 homeostatic microglia signature genes after NG2 glia depletion. f , heatmap of significantly dysregulated TGFβ-dependent microglia genes after NG2 glia depletion.

Article Snippet: PDGFR inhibitors CP673451 (HY-12050) and Crenolanib (HY-13223) were purchased from MedChemExpress, dissolved in dimethyl sulfoxide (DMSO, Sigma, 472301) and kept at −80 °C in aliquots.

Techniques:

Journal: bioRxiv

Article Title: NG2 glia are required for maintaining microglia homeostatic state

doi: 10.1101/667402

Figure Lengend Snippet: a , volcano plot of RNAseq data comparing CP673451 and Crenolanib treated brain slices. b , Venn diagram showing the overlapping genes between different experiment groups. c , mRNA levels of a subset of homeostatic microglia signature genes after CP673451 and Crenolanib treatment on BV2 microglia. d , heatmap of disease-associated microglia genes after NG2 glia depletion. e , expression levels of LTBPs in the RNAseq dataset.

Article Snippet: PDGFR inhibitors CP673451 (HY-12050) and Crenolanib (HY-13223) were purchased from MedChemExpress, dissolved in dimethyl sulfoxide (DMSO, Sigma, 472301) and kept at −80 °C in aliquots.

Techniques: Expressing