Journal: The Journal of clinical endocrinology and metabolism

Article Title: Placental sonic hedgehog pathway regulates foetal growth via insulin-like growth factor axis in preeclampsia.

doi: 10.1210/jc.2019-00335

Figure Lengend Snippet: Figure 3. SHH pathway and trophoblast syncytialization. (A) Downregulation of the SHH pathway in primary CTBs. Cyclopamine decreased the expression of PTCH1 (left) and GLI2 (middle) at mRNA (n 5 6) and protein levels (n 5 3, right). (B) Cyclopamine-induced alteration of syncytium markers (n 5 6). (C) Fluorescent immunocytochemistry of CTBs treated with rSHH (200 ng/mL, 48 h, n 5 4) and cyclopamine (5 mM, 48 h, n 5 4). Data are presented as mean 6 SEM. Scale bars, 50mm. Representative images are shown in (C).

Article Snippet: Cyclopamine (5 mM, catalog no. sc-200929, Santa Cruz Biotechnology), an SMO inhibitor, and/or recombinant SHH (rSHH) protein (200 ng/mL, catalog no. 1314-SH, R&D Systems, Minneapolis, MN) were used to examine the potential roles of the SHH pathway in placental development.

Techniques: Expressing, Immunocytochemistry

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Placental sonic hedgehog pathway regulates foetal growth via insulin-like growth factor axis in preeclampsia.

doi: 10.1210/jc.2019-00335

Figure Lengend Snippet: Figure 5. Noncanonical SHH pathway and trophoblast syncytialization. (A) mRNA expressions of syncytium markers and b-hCG protein levels in primary CTBs treated with forskolin, an ADCY activator (20 mM, n 5 6). (B) Western blot analysis of CREB phosphorylation in CTBs treated with forskolin (20 mM, n 5 3), rSHH (200 ng/mL, n 5 3), and cyclopamine (5 mM, n 5 3). (C) Expressions of ADCY family in CTBs treated with cyclopamine (5 mM, 48 h, n 5 6). (D) Fluorescent immunocytochemistry of CTBs treated with forskolin (20 mM, 48 h, n 5 4). Data are presented as mean 6SEM. Scale bars, 50mm. Representative images are shown in (B) and (D). *P , 0.05, **P , 0.01, ***P , 0.001. NS, not significant.

Article Snippet: Cyclopamine (5 mM, catalog no. sc-200929, Santa Cruz Biotechnology), an SMO inhibitor, and/or recombinant SHH (rSHH) protein (200 ng/mL, catalog no. 1314-SH, R&D Systems, Minneapolis, MN) were used to examine the potential roles of the SHH pathway in placental development.

Techniques: Western Blot, Phospho-proteomics, Immunocytochemistry

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Placental sonic hedgehog pathway regulates foetal growth via insulin-like growth factor axis in preeclampsia.

doi: 10.1210/jc.2019-00335

Figure Lengend Snippet: Figure 6. Canonical and noncanonical SHH pathway in regulation of the placental IGF1R pathway. (A) Close correlation between the SHH pathway and the IGF axis in control and preeclampsia placentas (n 5 10 in each group). (B–D) Expressions of major components of the IGF axis in CTBs treated with (B) forskolin (20 mM, 24 h, n 5 5), (C) cyclopamine (5 mM, 24 h, n 5 6), and (D) GLI2 siRNAs (48 h, n 5 6). (E) Western blot analysis of IGF1R expression in CTBs (48 h, n 5 3) treated with forskolin (20 mM), cyclopamine (5 mM), GLI2 siRNAs, rSHH (200 ng/mL), and SQ22536 (100 mM). (F) Pathway analysis of microarray data demonstrating that the SHH pathway was closely associated with ADCY activation pathway, CREB phosphorylation pathway, and IGF1R pathway. (G) Schema of the SHH pathway that regulates the IGF1R pathway in the placenta. Data are presented as mean 6 SEM. Representative images are shown in (E). NS, not significant.

Article Snippet: Cyclopamine (5 mM, catalog no. sc-200929, Santa Cruz Biotechnology), an SMO inhibitor, and/or recombinant SHH (rSHH) protein (200 ng/mL, catalog no. 1314-SH, R&D Systems, Minneapolis, MN) were used to examine the potential roles of the SHH pathway in placental development.

Techniques: Control, Western Blot, Expressing, Microarray, Activation Assay, Phospho-proteomics

Journal: Molecular Cancer

Article Title: Hedgehog signaling drives glial cell plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine neoplasms

doi: 10.1186/s12943-026-02611-y

Figure Lengend Snippet: Patient-derived PanNET tumoroids express HH signaling proteins and respond to HH pathway activation and inhibition. A Combined fluorescence and phase-contrast images of 21-day-old human PanNET tumoroids (PanNET3) stained for chromogranin A (CHGA) and HH proteins (PTCH1, SHH). B Four patient-derived PanNET tumoroid lines (PanNET1–4) were exposed to recombinant human SHH N-terminal peptide (100 ng/mL) or the SMO inhibitor vismodegib (VISMO, 20 µM) for 72 h and changes in mRNA expression were analyzed by RT-qPCR. mRNA changes were normalized to HPRT1 expression and DMSO vehicle control. ( n = 4 unique patient lines). * = p < 0.05, ** = p < 0.01, **** = p < 0.0001 by Two-way ANOVA with Sidak post-test. C EdU labeling showing proliferation of dissociated PanNET3 cells following 5-day exposure to: the HH agonists SHH-N (100 ng/mL) and SAG (10 nM); inhibitors of the canonical HH signaling pathway vismodegib (20 µM) and sonidegib (10 nM); or inhibitors of the GLI1/2 effectors GANT61 (10 µM) and itraconazole (ITZ, 1 µM). D Quantitation of the percentage of EdU-positive PanNET tumor cells following 5-day treatment. ( n = 3 replicates from one patient tumoroid line). * = p < 0.05, by One-way ANOVA with Tukey post-test. E SHH or SAG were co-administered with the respective pharmacologic inhibitors and EdU uptake was evaluated after 7 days. F Immunofluorescent images of CHGA, SHH, and PTCH1 expression in a second PanNET tumoroid line (PanNET5). G , H EdU labeling was evaluated in PanNET5 tumoroids following 7-day treatment with SHH-N (200 ng/mL) and SAG (20 nM); inhibitors of the canonical HH signaling pathway vismodegib (20 µM) and sonidegib (10 nM); or inhibitors of the GLI1/2 effectors GANT61 (10 µM) and itraconazole (ITZ, 5 µM). I Crystal violet staining of human BON-1 PanNET cells after 48 h treatment. J BrdU incorporation in BON-1 cells after 48 h treatment with HH agonists SHH-N and SAG, ( K ) SMO inhibitors vismodegib and sonidegib, and ( L ) inhibitors of GLI1/2 signaling. M Immunofluorescent images of CHGA, SHH, and PTCH1 expression in tumoroids derived from a metastatic ileal NET (IL-NET-met1). N , O EdU labeling was assayed in the IL-NET-met1 tumoroid line following 7-day treatment with the same drug concentrations used for PanNET5. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 by One-way ANOVA with Dunnett post-test

Article Snippet: Drug compounds used in the studies include: human recombinant SHH N-terminal peptide (R&D Systems, Cat# 1845-GMP), SAG (Tocris, Cat# 4366), vismodegib (Tocris, Cat# 7710), sonidegib (Tocris, Cat# 7826), GANT61 (Tocris, Cat# 3191), and itraconazole (Tocris, Cat# 5981).

Techniques: Derivative Assay, Activation Assay, Inhibition, Fluorescence, Staining, Recombinant, Expressing, Quantitative RT-PCR, Control, Labeling, Quantitation Assay, BrdU Incorporation Assay

Journal: Molecular Cancer

Article Title: Hedgehog signaling drives glial cell plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine neoplasms

doi: 10.1186/s12943-026-02611-y

Figure Lengend Snippet: Loss of Men1 in enteric glial cells stimulates GLI1/2-dependent transcriptional reprogramming. A Combined fluorescence and phase contrast images of 5-day-old primary enteric glial cell (EGC) cultures from Sox10-CreER T2 ; LSL-tdTomato mice and CreER T2 negative controls. Top panel shows TdTomato+ EGCs after 48 h exposure to 4-hydroxytamoxifen 4-OHT (2 µM). B TdTomato + EGCs were sorted by FACS to enrich for a pure SOX10 + cell population. C Combined fluorescence and phase contrast images of FACS-enriched SOX10-tdTomato + EGCs. D Fluctuations in HH pathway mRNA levels were evaluated in SOX10-tdTomato + EGCs 72 h following siRNA-mediated Men1 silencing. siRNA treatment consisted of four pooled siRNAs targeting the Men1 gene ( si -Men1 , 25 nM) or non-targeting (si-NT, 25 nM) controls. ( n = 5). E Immunofluorescence images of SHH expression in si-NT and si-Men1 treated EGCs (SHH = red pseudo-color, DAPI = blue). Inset shows higher power image. F Western blot analysis of si-NT and si-Men1 EGCs after 72 h treatment. SHH-FL = 55 kDa full length peptide; SHH- N = 22 kDa N-terminal peptide. ( n = 3). G Quantitation of protein expression in panel (F) normalized to GAPDH loading control. ( n = 3). H Relative fold-change in glial lineage transcripts and ( I ) neuroendocrine and neural progenitor transcripts in si-NT and si-Men1 treated EGCs. ( n = 6). J qPCR analysis of HH pathway genes and ( K ) neuroendocrine and neural progenitor transcriptsin si-NT and si-Men1 EGCs after 72 h treatment with GANT61 (10 µM) or vismodegib (VISMO 20 µM). ( n = 3). For all plots, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 by Two-way ANOVA with Sidak post-test. L Immunofluorescence images of si-NT and si-Men1 EGCs after 96 h siRNA knockdown and 72 h treatment with vehicle or GANT61. Menin = green, GFAP = magenta, SHH = yellow. M Significant GSEA pathways in enteric glial cells following 5-days of si- Men1 knockdown compared to non-targeting control. GSEA was performed on Men1 -depleted cells and cells co-treated with ( N ) si- Men1 , si- Gli1 , and si- Gli2 , and ( O ) cells co-treated with si- Men1 and GANT61 (10 µM). P – R KEGG pathway enrichment analysis comparing the same groups as shown in panels ( M – O ). S Heatmap showing significant DEGs mapped to the cell cycle, HH signaling, epigenetic regulation, neural stem cell (NSC) reprogramming, neuronal and neuroendocrine differentiation

Article Snippet: Drug compounds used in the studies include: human recombinant SHH N-terminal peptide (R&D Systems, Cat# 1845-GMP), SAG (Tocris, Cat# 4366), vismodegib (Tocris, Cat# 7710), sonidegib (Tocris, Cat# 7826), GANT61 (Tocris, Cat# 3191), and itraconazole (Tocris, Cat# 5981).

Techniques: Fluorescence, Immunofluorescence, Expressing, Western Blot, Quantitation Assay, Control, Knockdown