Journal: Neurotrauma Reports

Article Title: Sonic Hedgehog Signaling Promotes Peri-Lesion Cell Proliferation and Functional Improvement after Cortical Contusion Injury

doi: 10.1089/neur.2020.0016

Figure Lengend Snippet: Shh is reduced in CSF following TBI in human patients. CSF was collected from external ventricular drains in patients who had suffered a severe TBI or from a control group undergoing routine lumbar puncture. The concentration of Shh was determined by ELISA and was significantly lower in patients with TBI compared with the control group. Graph shows distribution of the data with the median indicated by the horizontal bars (control, n = 7; TBI, n = 14; ** p < 0.01). CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; Shh, sonic hedgehog; TBI, traumatic brain injury.

Article Snippet: The Shh concentration was determined by enzyme-linked immunosorbent assay (ELISA; Human Shh N-terminus Quantikine ELISA Kit, DSHH00, Bio-Techne) according to the manufacturer's instructions.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of visualized experiments : JoVE

Article Title: Using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer.

doi: 10.3791/58957

Figure Lengend Snippet: Materials

Article Snippet: SHH , RnD_Systems_Own , 1314-SH-025 , Ligand.

Techniques: Imaging, Inverted Microscopy, Microarray, Electron Microscopy

Journal: Mechanisms of development

Article Title: A new role for Hedgehogs in juxtacrine signaling

doi: 10.1016/j.mod.2013.12.002

Figure Lengend Snippet: Direct transfection of FL-HH cDNA generates a Gli1 response. (A) 3T3 cells were transfected with full-length HH constructs and after 48 h culture in 0.5% FBS medium, mRNA for HH-responsive genes Gli1 and Ptch1 was quantified by qPCR. mRNA expression levels are shown as a % of positive control (N-Shh) expression. Protein levels of Gli1 were confirmed by Western blot (B) of lysates from transfected 3T3 cells. Quantified ratio of Gli1/GAPDH protein is shown below GAPDH for each lane. (C) Light2 cells transfected with full length HH constructs were assayed for luciferase activity after 48 h culture in 0.5% FBS medium. Luciferase activity is shown normalised to 100% of positive control (N-Shh). (D) CH310T1/2 cells and TM3 cells (E) were transfected with full length HH constructs and Gli1 and Ptch1 mRNA assayed by qPCR after 48 h culture in 0.5% FBS medium. (F) Gli1 protein induction in transfected TM3 cells was confirmed by Western blot on cell lysates following HH transfection and 48 h culture in 0.5% FBS medium.

Article Snippet: Full-length cDNA expression constructs for human SHH, IHH, DHH in pCMV-XL5 were purchased from Origene. pcDNA3.

Techniques: Transfection, Construct, Expressing, Positive Control, Western Blot, Luciferase, Activity Assay

Journal: Mechanisms of development

Article Title: A new role for Hedgehogs in juxtacrine signaling

doi: 10.1016/j.mod.2013.12.002

Figure Lengend Snippet: Conditioned media from cells transfected with full length HH demonstrates isoform-specific autoprocessing and secretion. (A) Light2 cells were assayed for luciferase activity after 48 h treatment with conditioned media (1:4 in medium with 0.5% FBS, with and without cyclopamine as indicated) generated from 293T cells transfected with full length HH cDNA and cultured for 48 h in medium with 2% FBS. EcR Shh was used as a positive control for secreted HH. (B) Gli1 protein levels were determine by Western blot on 3T3 cells after 48 h treatment with conditioned media (1:4 in medium with 0.5% FBS). Quantified ratio of Gli1/GAPDH protein is shown below GAPDH for each lane. (C) HH production and secretion was determined by Western blot of cell lysates (CL) and conditioned media (CM) from 293T transfected cells for HH and cultured in medium with 2% FBS for 48 h, probed with 5E1 anti-HH or anti-DHH primary antibody as indicated. TCA denotes TCA precipitated CM.

Article Snippet: Full-length cDNA expression constructs for human SHH, IHH, DHH in pCMV-XL5 were purchased from Origene. pcDNA3.

Techniques: Transfection, Luciferase, Activity Assay, Generated, Cell Culture, Positive Control, Western Blot

Journal: Mechanisms of development

Article Title: A new role for Hedgehogs in juxtacrine signaling

doi: 10.1016/j.mod.2013.12.002

Figure Lengend Snippet: HH fusion constructs demonstrate the HH C-terminus defines auto-processing and secretion events. (A) Schematic representation of fusion cDNA constructs. (B) Luciferase assays were performed on Light2 cells treated for 48 h with conditioned media from 293T cells transfected with full length SHH and HH fusion constructs (1:4 in media with 0.5% FBS). Luciferase activity is shown as a % of activity for full length SHH. (C) Gli1 protein levels were determined by Western blot of cell lysates from 3T3 cells treated with conditioned media from 293T cells transfected with HH and HH fusion constructs (1:4 in media with 0.5% FBS). (D) The presence of secreted HH was determined Western blot of conditioned media from transfected 293T cells cultured for 48 h in media with 2% FBS. (E) HH proteins were detected by Western blot using the 5E1 anti-HH antibody on cell lysates from transfected 293T cells cultured for 48 h in media with 2% FBS. (F) Luciferase assays were performed on Light2 cells co-plated for 48 h in media with 0.5% FBS with transfected 293T cells. Luciferase activity is shown as a % of activity for 293T cells transfected with full length SHH.

Article Snippet: Full-length cDNA expression constructs for human SHH, IHH, DHH in pCMV-XL5 were purchased from Origene. pcDNA3.

Techniques: Construct, Luciferase, Transfection, Activity Assay, Western Blot, Cell Culture

Journal: Mechanisms of development

Article Title: A new role for Hedgehogs in juxtacrine signaling

doi: 10.1016/j.mod.2013.12.002

Figure Lengend Snippet: Prostate cancer cell lines express variable amounts of HH ligand capable of inducing a juxtacrine signaling response in coplating cultures. (A) RNA obtained from LnCAP prostate cancer cells grown in normal growth conditions, and also for 2 weeks in androgen deprived (AD) conditions to confluence or to sub-confluent levels was used in qPCR to determine endogenous SHH, DHH (B) and IHH (C) mRNA levels, as well as Gli1, Gli2 and Gli3 mRNA expression (D). (E) Luciferase assays were performed on Light2 cells co-plated for 48 h in androgen deprived media with LnCAP cells previously grown for 2 weeks in AD media. Luciferase activity is shown relative to Light2 cells plated alone.

Article Snippet: Full-length cDNA expression constructs for human SHH, IHH, DHH in pCMV-XL5 were purchased from Origene. pcDNA3.

Techniques: Expressing, Luciferase, Activity Assay

Journal: Mechanisms of development

Article Title: A new role for Hedgehogs in juxtacrine signaling

doi: 10.1016/j.mod.2013.12.002

Figure Lengend Snippet: Schematic models of the three alternative modes of HH processing, secretion and presentation. Paracrine signaling by processed HH is representative of what is known for mammalian SHH. Full length HH secretion has been observed for both IHH (also in association with vLDL) and here for SHH. Juxtacrine full length HH signaling has been observed here for DHH but may also be utilised by SHH and IHH.

Article Snippet: Full-length cDNA expression constructs for human SHH, IHH, DHH in pCMV-XL5 were purchased from Origene. pcDNA3.

Techniques: