Journal: Cell Reports

Article Title: Variable Outcomes in Neural Differentiation of Human PSCs Arise from Intrinsic Differences in Developmental Signaling Pathways

doi: 10.1016/j.celrep.2020.107732

Figure Lengend Snippet: Inherent Tendency to Ventralization Is Largely Rescued by a Brief, Specific Phase of Wnt Signaling Activation (A) Diagram of Hedgehog pathway components targeted by small molecules in (B) and (C). (B) Treatment with purmorphamine (1 μM) between 7 and 17 dpi results in a more ventralized gene expression profile at 33 dpi (line iPSC22.1, n = 2). (C) Treatment with Hedgehog inhibitor cyclopamine (1 μM) between 7 and 17 dpi has no observable effect on dorso-ventral gene expression in a highly ventralized line at ∼35 dpi (iPSC14.1, n = 3). (D) Summary diagram of Wnt pathway components targeted by small molecules in (E)–(G). (E) Treatment with Wnt inhibitor IWP2 (2 μM) between 0 and 12 dpi results in a more ventralized gene expression profile at 33 dpi (line iPSC22.1, n = 2). (F) Treatment with Wnt activator CHIR99021 (1 μM) between 13 and 17 dpi significantly increases cortex-associated gene expression and decreases MGE-associated expression at ∼35 dpi in differentiations of 4 ventral-prone lines compared to vehicle treatment (GMESC01.1, iPSC01.1, iPSC06.1, and iPSC22.1) (one-sample Student’s t test, mu = 0, n = 7, FDR-corrected p values: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). Top panels show combined trend; bottom panels show breakdown by PSC line. (G) Clustering of ∼35-dpi differentiations from ventral-prone lines treated between 7 and 17 dpi with either vehicle or 1 μM CHIR99021. Treatment stimulating Wnt/β-catenin signaling results in shift in classificationof ventralized differentiations to more dorsalized clusters. (H) Model for outcome of differentiation of distinct cell lines. All error bars represent standard error. See also Figure S7 .

Article Snippet: For experiments manipulating signaling pathways, the induction or maintenance medium were supplemented with 1 μM purmorphamine (Tocris Bioscience), 1 μM cyclopamine (StemCell technologies), 1 μM CHIR99021 (Sigma), or 2 μM IWP2 (StemCell Technologies) during the indicated time window ( & ) and replaced daily.

Techniques: Activation Assay, Gene Expression, Expressing

Journal: Cell Reports

Article Title: Variable Outcomes in Neural Differentiation of Human PSCs Arise from Intrinsic Differences in Developmental Signaling Pathways

doi: 10.1016/j.celrep.2020.107732

Figure Lengend Snippet:

Article Snippet: For experiments manipulating signaling pathways, the induction or maintenance medium were supplemented with 1 μM purmorphamine (Tocris Bioscience), 1 μM cyclopamine (StemCell technologies), 1 μM CHIR99021 (Sigma), or 2 μM IWP2 (StemCell Technologies) during the indicated time window ( & ) and replaced daily.

Techniques: Recombinant, Gene Expression, RNA HS Assay, In Vivo, Expressing, Software, Imaging

Journal: Cells

Article Title: An Autocrine Wnt5a Loop Promotes NF-κB Pathway Activation and Cytokine/Chemokine Secretion in Melanoma

doi: 10.3390/cells8091060

Figure Lengend Snippet: Wnt5a increased p65 phosphorylation. ( A ) 1205Lu and MeWo cells were stimulated for 30 min with recombinant Wnt5a (rWnt5a) or Wnt5a conditioned media (Wnt5a-CM). Protein extracts were blotted with the indicated antibodies. ( B ) 1205Lu cells were stimulated with Wnt5a-CM (Wnt5a +) or Control CM (Wnt5a −), which were collected in the presence of either IWP-2 (20 µM) or DMSO. Protein extracts were blotted with the indicated antibodies. ( C ) 1205Lu cells were stimulated with Wnt5a-CM (Wnt5a) for the indicated times, and the proteins extracts were then blotted with the indicated antibodies. ( D ) The indicated melanoma cell lines were stimulated with Wnt5a-CM (Wnt5a) for 30 min, and the proteins extracts were then blotted with the indicated antibodies. GAPDH or Vinculin were used as loading controls. The blots displayed are representative of three independent experiments. Bar graphs show the mean ± SD (from three independent experiments) of P-p65 levels, normalized to the level of total p65, detected after stripping the membrane. In panel C , to evaluate changes in total p65, the data were normalized to GAPDH levels. Results are expressed as the fold change relative to control-treated cells (lane 1 in A – C ). In panel D , each cell line has its own control-treated lane. 1205Lu, SK-Mel28, and WM9 are both BRAF and PTEN mutant; A375 and WM983A are BRAF mutant; SK-Mel2 is Ras mutant; MeWo is p53 mutant. The statistical significance was tested by a student’s t-Test (treated sample vs. paired control in B,D) or ANOVA, followed by Dunnett’s Multiple Comparison Test ( A , C ), using log transformed fold change (FC) values. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3.

Article Snippet: The final concentrations of the reagents were: rWnt5a (R&D Systems, Minneapolis, MN, 645-WN/CF), 400 ng/mL; IWP-2 (Cayman Chemicals, Ann Arbor, MI, 04614560-30), 20 µM; hTNFα (R&D Systems, 210-TA-020), 10 µg/mL; Cycloheximide (Sigma, St. Louis, MO, C7698-1G), 10 µg/mL; BAY11-7082 (Cayman Chemicals, 10010266), 10 µM; LiCl (Sigma, L9650), 10 mM; LY294002 (Calbiochem, Burlington, MA, 440202), 10 µM; JSH-23 (Abcam, UK, 144824), 10 µM; Tunicamycin (Cayman Chemicals, 11089-65-9), 4 µM; and Box5 (EMD Millipore, Burlington, MA, 681673), 200 µM.

Techniques: Recombinant, Stripping Membranes, Mutagenesis, Transformation Assay