Journal: Cancers

Article Title: Granulocyte-Macrophage Colony Stimulating Factor Receptor Contributes to Plexiform Neurofibroma Initiation

doi: 10.3390/cancers17050905

Figure Lengend Snippet: Gene and protein expression of GM-CSF alpha (GM-CSFR-α) and beta-common (GM-CSFR-β c ) receptors in 7-month-old mice. ( a ) UMAP of PNF from 7-month-old Nf1 f/f ; DhhCre mice compared to aged-match wild-type (WT) control DRG. ( b ) Dot plot showing gene expression in various cell types (y-axis: Identity). ( c ) Representative pictures of immunostaining for GM-CSFR-α and GM-CSFR-β c in WT DRG and PNF tissue sections. ( d ) Frequency of non-immune (CD45-) and immune (CD45+) cells in WT DRG (n = 6) and PNF (n = 8)and ( e ) subtypes of CD45+ immune cells expressing GM-CSFR-α ((WT DRG (n = 4) and PNF (n = 4)) and GM-CSFR-β c ((WT DRG (n = 6) and PNF (n = 8)).Unpaired t -test or 2-way ANOVA multiple comparison test, * p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001.

Article Snippet: For immunohistochemistry (IHC), the procedure was as for immunofluorescence (IF), except that following overnight incubation with anti-GM-CSFR beta rabbit polyclonal antibody (Bioss; cat# bs-3689R, Woburn, MA, USA) or anti-GM-CSFR alpha rabbit polyclonal antibody (Bioss; cat# bs-1457R, USA) at 4 °C, the sections were subsequently incubated with biotinylated goat anti-rabbit secondary antibody (VectorLabs; cat#BA-1000, Newark, CA, USA) for 1 h and rinsed three times in TBST for 5 min each.

Techniques: Expressing, Control, Gene Expression, Immunostaining, Comparison

Journal: Cancers

Article Title: Granulocyte-Macrophage Colony Stimulating Factor Receptor Contributes to Plexiform Neurofibroma Initiation

doi: 10.3390/cancers17050905

Figure Lengend Snippet: Effects of GM-CSFR-α and GM-CSFR β c genetic deletion on survival and nerve pathology. ( a ) Kaplan–Meier survival curve of Nf1 f/f ; DhhCre (control) (n = 9) and mice bearing deletion of the receptor GM-CSFR-α (GM-CSFR-α-/-; Nf1 f/f ; DhhCre (n = 18)) or GM-CSFR-β c (GM-CSFR-β c -/-; Nf1 f/f ; DhhCre (n = 16)). ( b ) representative pictures of aged-matched gross dissection of spinal cord from each mouse (Nf1 f/f ; DhhCre (control) (n = 5), GM-CSFR-α-/-; Nf1 f/f ; DhhCre (n = 6) and GM-CSFR-β c -/-; Nf1 f/f ; DhhCre (n = 6)) and quantification of tumor number and size (diameter). White arrows indicate PNF. ( c ) Representative images of tissue sections stained with H&E and ( d ) electromicrographs of saphenous nerve showing the ultrastructure of an intact Schwann cell Remak bundle (blue margin) in WT mice compared to the disrupted Remak structure (blue arrows) in tumor mice in the presence or loss of GM-CSF receptors. ( e ) High percentage (6 or more) of grouped axons indicate a normal Remak bundle. Two-way ANOVA multiple comparison test, * p < 0.05, **** p < 0.00001.

Article Snippet: For immunohistochemistry (IHC), the procedure was as for immunofluorescence (IF), except that following overnight incubation with anti-GM-CSFR beta rabbit polyclonal antibody (Bioss; cat# bs-3689R, Woburn, MA, USA) or anti-GM-CSFR alpha rabbit polyclonal antibody (Bioss; cat# bs-1457R, USA) at 4 °C, the sections were subsequently incubated with biotinylated goat anti-rabbit secondary antibody (VectorLabs; cat#BA-1000, Newark, CA, USA) for 1 h and rinsed three times in TBST for 5 min each.

Techniques: Control, Dissection, Staining, Comparison

Journal: Cancers

Article Title: Granulocyte-Macrophage Colony Stimulating Factor Receptor Contributes to Plexiform Neurofibroma Initiation

doi: 10.3390/cancers17050905

Figure Lengend Snippet: Loss of GM-CSFR-β c reduced the presence of myeloid cells. Representative pictures of stained tumor tissue sections and their corresponding quantifications of ( a ) toluidine blue staining for mast cells (red arrows) (representative picture from each group, n = 4), ( b ) CD11c+ dendritic cells, ( c ) Iba – 1+ macrophages and ( d ) CD3+ T cells (white arrows indicate immune cell) (representative picture from tumor tissue of PNF control (n = 5) and each GM-CSFR receptor knockout (n = 6) mice). Two-way ANOVA multiple comparison test, * p < 0.05.

Article Snippet: For immunohistochemistry (IHC), the procedure was as for immunofluorescence (IF), except that following overnight incubation with anti-GM-CSFR beta rabbit polyclonal antibody (Bioss; cat# bs-3689R, Woburn, MA, USA) or anti-GM-CSFR alpha rabbit polyclonal antibody (Bioss; cat# bs-1457R, USA) at 4 °C, the sections were subsequently incubated with biotinylated goat anti-rabbit secondary antibody (VectorLabs; cat#BA-1000, Newark, CA, USA) for 1 h and rinsed three times in TBST for 5 min each.

Techniques: Staining, Control, Knock-Out, Comparison

Journal: Cancers

Article Title: Granulocyte-Macrophage Colony Stimulating Factor Receptor Contributes to Plexiform Neurofibroma Initiation

doi: 10.3390/cancers17050905

Figure Lengend Snippet: Altered proteome profile results from lack of GM-CSFR-α and GM-CSFR-β c . ( a ) Images of scanned proteome microarray blots incubated with lysates of tumors from Nf1 f/f ; DhhCre (control) and with GM-CSFR-α or GM-CSFR-β c deletion. ( b ) Heatmap representation of proteome profile. ( c ) Cytokines that robustly increased or decreased after the loss of the GM-CSFR-β c receptor.

Article Snippet: For immunohistochemistry (IHC), the procedure was as for immunofluorescence (IF), except that following overnight incubation with anti-GM-CSFR beta rabbit polyclonal antibody (Bioss; cat# bs-3689R, Woburn, MA, USA) or anti-GM-CSFR alpha rabbit polyclonal antibody (Bioss; cat# bs-1457R, USA) at 4 °C, the sections were subsequently incubated with biotinylated goat anti-rabbit secondary antibody (VectorLabs; cat#BA-1000, Newark, CA, USA) for 1 h and rinsed three times in TBST for 5 min each.

Techniques: Microarray, Incubation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Th17 Cells Induce Th1 Polarizing Monocyte-Derived Dendritic Cells

doi: 10.4049/jimmunol.1203201

Figure Lengend Snippet: A) Monocytes were cultured for 5 days with Th17 cells in the presence of 10μg/ml of functional grade blocking antibody to either CD40L or RANKL. A coculture with 10μg/ml of each functional grade isotype was included as a control. After 5 days, 1μg/ml of LPS was added to the cultures and the next day the cell free supernatant was harvested for IL-12p70 ELISA. Done in triplicate. B) DCTh17 were prepared as in A), but after 12 hours of stimulation with LPS, the cells were treated with BFA for 4 hours. The cells were then FC blocked and intracellularly stained for IL-12p40. The cells shown were previously gated on live/dead aqua−, Thy1.2−, CD11b+ cells. C) DCTh17 were generated with wild-type Th17 or CD40L KO Th17 cells. After 5 days, the cells were stimulated with 1μg/ml of LPS. 24 hours later, the cell free supernatant was tested for IL-12p70 with ELISA. Done in triplicate. D) Th1, Th2 or Th17 cultures were rested in the presence of IL-2 for 2 days and then restimulated with CD3/CD28 microbeads. 2 hours after restimulation the cells were FC blocked and then stained for CD40L. 6 hours after restimulation the cells were FC blocked and then stained for RANKL. Cells shown were gated on CD4+, Thy1.2+, Dapi− cells. Shown with an isotype control for each antibody and cell type. Representative of 2 independent experiments.

Article Snippet: Mice WT C57BL/6, 2D2, CD40L KO, GM-CSFR KO and IL-12p35 KO female mice were purchased from the Jackson Laboratory and used between 6 and 14 weeks of age.

Techniques: Cell Culture, Functional Assay, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay, Staining, Generated