Fig 1: Expression of neurotrophin receptors in human aortic smooth muscle cells (HASMCs). A, Representative agarose gel to demonstrate typical levels of tropomyosin-related kinase (Trk) isoform A (TrkA; 401 bp product), B (TrkB; 575 bp product), and C (TrkC; 455 bp product) and of p75NTR (192 bp product) mRNA expression in HASMCs. Human umbilical vein endothelial cells (HUVECs) were examined in parallel for comparison. B, Flow cytometry analysis of HASMCs using smooth muscle actin (SMA) to visualize smooth muscle cells (SMCs) as well as 2 different antibodies against TrkB (H-181 or F-1) or against an epitope within the C-terminus present in all Trk isoforms (pan-Trk). The percentage of positive cells (in 1×106 total cells) is indicated within the graphs. C, Immunofluorescence confocal microscopy analysis of HASMCs stained with antibodies directed against TrkB (green signal). Scale bars indicate 20 µm. Phalloidin was used to visualize the cytoskeleton (red signal) and DAPI (4',6-diamidino-2-phenylindole) to stain cell nuclei (blue signal). Immunohistochemical analysis of TrkB expression in internal mammary artery and aorta using antibodies against SMA (D) or CD68 (E) as markers for SMCs or macrophages, respectively, on immediately neighboring sections. Scale bars indicate 50 µm. FITC indicates fluorescein isothiocyanat; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IMA, internal mammary artery; and MW, molecular weight.
Fig 2: Expression of neurotrophin receptors in human arterial vessels.mRNA levels of tropomyosin-related kinase (Trk) isoform A (TrkA; A), B (TrkB; B), and C (TrkC; C) and of the low affinity receptor p75NTR (D) were determined using reverse transcription polymerase chain reaction in tissue samples of the A. mammaria interna (IMA; n=9) and the aortic wall (n=7). The results of the statistical analysis are indicated within the graphs. A representative agarose gel showing results in paired samples of IMA and aorta from 2 patients are shown (E). Paraffin-embedded cross-sections through the IMA or aorta were stained using Masson trichrome (MTC) stain (upper row; smooth muscle cells in red) or antibodies against TrkB (middle row; positive immunosignals in brown) (F). The results after omission of the first antibody (negative control; bottom row) are also shown. Scale bars indicate 50 µm. GAPDH indicates glyceraldehyde 3-phosphate dehydrogenase.
Fig 3: Effects of brain-derived neurotrophic factor (BDNF) stimulation on human aortic smooth muscle cells (HASMCs) and importance of protein tyrosine phosphatase 1B (PTP1B).HASMCs were cultivated until subconfluency, incubated with PTP1B inhibitor (50 µmol/L) or dimethyl sulfoxide (DMSO) for 1 hour before being stimulated with recombinant human BDNF (10 and 20 ng/mL) for an additional 24 hours. RNA was isolated and changes in mRNA expression of cyclin D1 (CCND1; A), smooth muscle a-actin (ACTA2; B), smooth muscle myosin heavy chain (MYH11; C), COL1A1 (D), tropomyosin kinase B (TrkB; E), and p75NTR (F) examined using real-time polymerase chain reaction. *P<0.05, **P<0.01, and ****P<0.0001 vs DMSO; # P<0.05 and ## P<0.01 vs PTP1B alone and § P<0.05, §§ P<0.01, §§§ P<0.001, and §§§§ P<0.0001 vs BDNF (10 ng/mL), as determined using 1-way ANOVA (A, D, E, and F) or Kruskall-Wallis test (B and C) followed by multiple comparisons tests. G, Confocal microscopy images after immunostaining of HASMCs with antibodies against p75NTR (green signal). Scale bars indicate 20 µm. DAPI indicates 4',6-diamidino-2-phenylindole.
Fig 4: Pathways and genes differentially expressed in selumetinib-treated xenografts.a Schematic of workflow used to identify compensatory pathways in SHH xenograft tumors following MEK inhibitor treatment. A portion of this schematic, including mouse, syringe, and cell icon images, was created with BioRender.com. b Representative H&E images of FFPE sections from control (left) and selumetinib-treated (right) UI226 SHH MB xenografts. Scale bars: 1500 µm (upper) and 600 µm (lower). c Volcano plot depicting the log2 fold change and p-values in RNA-seq data with points in red identifying the 576 significantly differentially expressed genes (FDR < 0.05). d Normalized counts for representative differentially expressed transcripts from RNA-seq data. IFIT2 (p = 6.33E–16), IGFN1 (p = 0.0002), RIMBP2 (p = 0.00049) and JAK2 (p = 0.0041) are increased while CD271 (p = 0.047) expression is decreased in selumetinib-treated UI226 SHH MB xenografts relative to vehicle controls. p < 0.05 *, p < 0.01**, p < 0.001***. Expression differences were calculated using the DESeq results function. Differentially expressed genes were identified using a q-value (Benjamini-Hochberg corrected p-value) cut-off of 0.05, which coincides with a p-value of 0.00168. e GSEA demonstrating that genes associated with a downregulated MEK signature, increased JAK/STAT3, TNFalpha, and apoptosis signaling are enriched in genes sets that are upregulated in selumetinib-treated xenografts. padj < 0.05* for all signatures. f Most significantly downregulated genes in selumetinib-treated xenografts. Differentially expressed genes were identified using a q-value (Benjamini-Hochberg corrected p-value) cut-off of 0.05, which coincides with a p-value of 0.00168. padj < 0.05* for all genes. g Representative images of IHC staining for pSTAT3 (Tyr705) in FFPE sections from two independent control (upper) and selumetinib-treated (lower) UI226 SHH MB xenografts. Scale bar: 150 µm. h Quantitative analyses of IHC pSTAT3 (Tyr705) staining in vehicle control (white) and selumetinib-treated (blue) xenografts. The proportion of pSTAT3+ cells for each sample was calculated using QuPath and expressed as the total number of pSTAT3+ cells relative to the total number of cells in each image (pSTAT3- nuclei (hematoxylin-stained) and pSTAT3+ cells). Significance was calculated using a two-tailed t-test. Error bars: SEM. p = 0.011.
Fig 5: Multiplex digital spatial profiling of 56 proteins on MEK and/or JAK/STAT3 inhibitor-treated UI226 tumors in vivo.Representative immunofluorescent images depicting 12 ROIs from the vehicle control (a), pacritinib (b), selumetinib (c) and pacritinib + selumetinib (d) treated samples utilized for analyses of 56 different proteins. Samples were stained for CD271 (yellow), MAP2 (green), Ki67 (pink), and Syto13 (blue) for tumor visualization. e Heat map depicting CD271 levels across 12 ROIs in vehicle control, pacritinib, selumetinib, and pacritinib + selumetinib treated samples. f Boxplots depicting quantification of CD271 levels by mean fluorescent intensity across vehicle control, pacritinib, selumetinib and pacritinib + selumetinib treated samples. Bars represent minimum and maximum counts. Significance was determined using ANOVA and a Tukey’s test for multiple comparisons. p < 0.05*, p < 0.001***. For vehicle vs sel, vehicle vs pac/sel and pac vs pac/sel, p < 0.0001. For sel vs pac/sel, p = 0.0393. Volcano plots displaying significantly differentially expressed proteins (based on signal-to-noise-ratio for each target relative to negative control IgG probes comparing the vehicle control to the pacritinib (g), selumetinib (h), and pacritinib + selumetinib-treated (i) tumor. For each volcano plot, significance of a specific protein was determined using a two-tailed t-test. FDR: false discovery rate. Of note, 36 of the 56 proteins reached threshold levels based on signal-to-noise ratio and were used for further analyses. j–s Boxplots depicting select differentially expressed proteins based on signal-to-noise-ratio in vehicle control, pacritinib, selumetinib, and pacritinib + selumetinib treated samples. Bars represent minimum and maximum counts. ns: not significant. Significance was determined using ANOVA and a Dunnett’s test for multiple comparisons. p < 0.05*, p < 0.01**, p < 0.001***. pMEK: vehicle vs sel, p < 0.0001; total ERK1/2: vehicle vs sel, p < 0.0001; pERK1/2: vehicle vs pac, p = 0.0023; vehicle vs pac/sel, p = 0.001; BRAF: vehicle vs sel, p < 0.0001; NF1: vehicle vs pac, p = 0.0116; vehicle vs sel, p = 0.0070; vehicle vs pac/sel, p < 0.0001; P53: vehicle vs pac/sel, p < 0.0001; OLIG2: vehicle vs sel, p = 0.0004; PLCG1: vehicle vs all groups, p < 0.0001; pAKT: vehicle vs all groups, p < 0.0001.
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