Journal: Science Advances

Article Title: TGFβ-dependent signaling drives tumor growth and aberrant extracellular matrix dynamics in NF1-associated plexiform neurofibroma

doi: 10.1126/sciadv.adu0772

Figure Lengend Snippet: ( A ) Principal components analysis (PCA) of bulk RNA-seq data from PNF-bearing Nf1 flox/flox ;PostnCre + nerve tissue and normal control nerve from wild-type (WT) ( Nf1 flox/flox ;PostnCre – ) mice ( n = 6 per group). ( B ) Top 20 upstream regulators with positive activation z scores in PNF-bearing nerve tissue in comparison to normal control ( n = 6 per group). Nodes are ranked by activation z score; node size reflects the –log 10 -adjusted P ( P adj) value (range: 1.2 to 22.3). Nodes related to TGFβ signaling are highlighted in red text and blue dots. IL-1β, interleukin-1β; mTOR, mammalian target of rapamycin; IFN-γ, interferon-γ; STAT1, signal transducer and activator of transcription 1. ( C ) Key components of the canonical TGFβ signaling pathway are shown, including ligands (TGFβ1, 2, and 3), receptors (TGFβRI and II), and SMAD effectors (SMAD2, 3, 4, and 7). Nodes are ranked by activation z score, with blue nodes indicating positive and red nodes (SMAD7) indicating negative activation. Node size reflects –log 10 -adjusted P value (range: 4.3 to 19.9). ( D ) Gene set enrichment analysis (GSEA) reveals enrichment of a TGFβRI-activated mutant mouse gene signature in PNF-bearing nerve tissue from Nf1 flox/flox ;PostnCre mice compared to WT controls ( n = 6 per group). Black bars indicate the rank of individual genes comprising the signature. The green curve corresponds to the running enrichment score ( q = 1.71 × 10 –3 ). ( E ) Box-and-whisker plots show Col1a1 , Col3a1 , and Mmp2 transcript expression from RNA-seq data in murine PNF versus normal nerve ( n = 6 per group). Dots represent individual samples. Whiskers extend to 1.5× the interquartile range. The center line represents the median. The box spans the 25th to 75th percentiles. Outliers are plotted as individual points beyond the whiskers. P values indicate unpaired, two-tailed t tests between groups.

Article Snippet: Immunoblots were carried out using antibodies specific to p-SMAD2 (Ser 465/467 ) (#CST-3108, Cell Signaling Technology), pERK1/2 (Thr 202 /Tyr 204 ) (#CST-9101, Cell Signaling Technology), p-AKT (Ser 473 ) (#CST-4060, Cell Signaling Technology), p-SMAD3 (Ser 423/425 ) (#ab52903, Abcam), actin (A5441, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#CST-5174, Cell Signaling Technology), LAMB1 (#PA5-27271, Thermo Fisher Scientific), LAMC (#PA5-79579, Thermo Fisher Scientific), NID1 (ab254325, AbCam), and TGFβ (#NBP1-03276, Novus Biologicals; #sc-130348, Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Control, Activation Assay, Comparison, Mutagenesis, Whisker Assay, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: TGFβ-dependent signaling drives tumor growth and aberrant extracellular matrix dynamics in NF1-associated plexiform neurofibroma

doi: 10.1126/sciadv.adu0772

Figure Lengend Snippet: ( A ) Concentration-time profiles of galunisertib in plasma and nerve tissue samples from n = 3 mice at each time point. Plasma samples were assayed at 1, 2, 4, 8, and 24 hours, while tissue samples were harvested at 4 and 24 hours after a single 75 mg/kg dose of galunisertib administered by oral gavage. ( B ) p-SMAD2 (Ser 465/467 ), phosphorylation of extracellular signal–regulated kinase 1/2 (p-ERK1/2; Thr 202 /Tyr 204 ), and AKT phosphorylation (p-AKT; Ser 473 ) were detected by Western blot in sciatic nerve tissues at 1, 4, and 12 hours after administration of vehicle or galunisertib. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as the loading control. Bar plots depict quantitative analysis of p-SMAD2 ( C ), p-ERK1/2 ( D ), and p-AKT ( E ) signal intensity in arbitrary densitometry units normalized to GAPDH as the loading control. Dots represent individual data points. P values denote statistical significance as determined by one-way analysis of variance (ANOVA), followed by Šidák’s multiple comparisons test between groups as annotated on the graphs. Error bars represent the SEM. n = 3 independent biological replicates per group, except for the 12-hour time point for which n = 2 replicates are shown. Vehicle-treated samples were harvested at the 12-hour time point.

Article Snippet: Immunoblots were carried out using antibodies specific to p-SMAD2 (Ser 465/467 ) (#CST-3108, Cell Signaling Technology), pERK1/2 (Thr 202 /Tyr 204 ) (#CST-9101, Cell Signaling Technology), p-AKT (Ser 473 ) (#CST-4060, Cell Signaling Technology), p-SMAD3 (Ser 423/425 ) (#ab52903, Abcam), actin (A5441, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#CST-5174, Cell Signaling Technology), LAMB1 (#PA5-27271, Thermo Fisher Scientific), LAMC (#PA5-79579, Thermo Fisher Scientific), NID1 (ab254325, AbCam), and TGFβ (#NBP1-03276, Novus Biologicals; #sc-130348, Santa Cruz Biotechnology).

Techniques: Concentration Assay, Clinical Proteomics, Phospho-proteomics, Western Blot, Control