Journal: Cellular signalling

Article Title: Coordinated D -cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

doi: 10.1016/j.cellsig.2017.12.007

Figure Lengend Snippet: Canonical Shh signaling drives proliferation in iMEFs. (A) Wild-type (WT) and Gli2−/−3−/− iMEFs were cultured ± SHH ligand and ± the SMO antagonist vismodegib (Vis). Cell number is shown relative to vehicle-treated cells. SHH increased proliferation in WT iMEFs, but not in Gli2−/−3−/− iMEFs. This increase in proliferation was blocked by the addition of vismodegib (n = 5). (B) Histogram of fluorescence intensity of cells treated with the cell tracing reagent CellTrace™ Far Red and analyzed by flow cytometry. SHH treatment resulted in a leftward shift in fluorescence compared to vehicle-treated cells, indicating an increase in cellular divisions (n = 1). Significance: * = p < 0.05. Error bars show SEM.

Article Snippet: The SMO inhibitor, vismodegib, was purchased from LC Laboratories (CAS No 879085–55–9) and dissolved in DMSO.

Techniques: Cell Culture, Fluorescence, Flow Cytometry

Journal: Cellular signalling

Article Title: Coordinated D -cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

doi: 10.1016/j.cellsig.2017.12.007

Figure Lengend Snippet: Foxd1 is a target of canonical Sonic Hedgehog signaling. (A) Gene expression was determined for WT iMEFs cultured ± SHH ligand and ± the SMO antagonist vismodegib (Vis). Expression changes are shown as a fold change from vehicle-treated cells. SHH treatment resulted in an increase in Gli1 and Foxd1 expression, which was blocked by the addition of vismodegib (n = 6). (B) Expression of Gli1 and Foxd1 is increased in Ptch1−/− iMEFs relative to wild-type (inset) and significantly reduced by vismodegib treatment. (C) Expression of Gli1 and Foxd1 is increased in iMEFs overexpressing a constitutively active form of human Smoothened (SMOM2). Expression changes are shown as a fold change from cell overexpressing GFP (n = 5). Insert shows SMO expression. (D) Expression of Gli1 and Foxd1 is not significantly changed in Gli2−/−3−/− iMEFs treated with SHH ligand (n = 4). Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Error bars show SEM.

Article Snippet: The SMO inhibitor, vismodegib, was purchased from LC Laboratories (CAS No 879085–55–9) and dissolved in DMSO.

Techniques: Gene Expression, Cell Culture, Expressing

Journal: Cellular signalling

Article Title: Coordinated D -cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway

doi: 10.1016/j.cellsig.2017.12.007

Figure Lengend Snippet: SHH suppresses Cdkn1c expression. (A) Gene expression was determined for wild-type (WT) iMEFs cultured ± SHH ligand and ± the SMO antagonist vismodegib (Vis). Expression changes are shown as a fold change from vehicle-treated cells. SHH treatment resulted in an increase in Ccnd1 and a decrease in Cdkn1c expression, both of which were blocked by the addition of vismodegib. Expression changes are shown as a fold change from vehicle-treated cells. (n = 9). (B) Expression of Ccnd1 is increased in Ptch1−/− iMEFs relative to WT (inset). Vismodegib treatment Ptch1−/− iMEFs results in decreased Ccnd1 expression and increased Cdkn1c expression (C) Expression of Gli1 and Foxd1 is not significantly changed in Gli2−/− 3−/− iMEFs treated with SHH ligand (n = 4). (C) In iMEFs overexpressing a constitutively active form of human Smoothened (SMOM2), expression of Ccnd1 was increased while Cdkn1c expression was decreased. Expression changes are shown as a fold change from cells overexpressing GFP (n = 5). (D) To determine the temporal sequence of gene expression changes, WT cells were harvested at 6, 12, 24, 48, and 72 h after treatment ± SHH ligand. Gli1 expression (white arrow) is significantly increased by 6 h followed by increased Foxd1 expression (black arrow) by 24 h, and Cdkn1c expression (gray arrow) is not significantly decreased until 48 h post-treatment. Expression changes are shown as a fold change from vehicle-treated cells at respective time points (n = 5). Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Error bars show SEM.

Article Snippet: The SMO inhibitor, vismodegib, was purchased from LC Laboratories (CAS No 879085–55–9) and dissolved in DMSO.

Techniques: Expressing, Gene Expression, Cell Culture, Sequencing