Journal: PLoS ONE

Article Title: Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0084551

Figure Lengend Snippet: (A) As examined as both qRT-PCR and Western immunoblotting, enforced expression of GLI1 in Huh7 cells (left) increased Cav-1 expression (right); (B) Both qRT-PCR and Western immunoblotting revealed that knockdown of GLI1 in SNU449 cells (left) resulted in down-regulation of Cav-1 (right); (C) The results of Western immunoblotting showed that ectopic expression of GLI1 induced down-regulation of E-cadherin and up-regulation of N-cadherin, Fibronectin and Vimentin in Huh7 cells. When we repressed the Cav-1 up-regulation induced by GLI1 overexpression in Huh7 cells, E-cadherin expression was increased and the expression of N-cadherin, Fibronectin and Vimentin was attenuated apparently.

Article Snippet: The 18s rRNA TaqMan probe (Hs99999901_s1), Cav-1 TaqMan probe (Hs00971716_m1) and GLI1 TaqMan probe (Hs01110766_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Over Expression

Journal: PLoS ONE

Article Title: Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0084551

Figure Lengend Snippet: (A) The representative IHC staining of Cav-1 in HCC tissues (a) and adjacent normal tissues (b). There was Cav-1 protein expressing in both cell membrane (as labelled by black arrows) and cytoplasm (as labelled by a white arrow); (B) The representative IHC staining of GLI1 in HCC tissues (a) and adjacent normal tissues (b). Most of GLI1 protein located in cell nucleus, as shown by black arrow head; (C) The negative control staining in HCC tissues (a) and adjacent liver tissues (b); (D) HCC patients with higher level of Cav-1 had the worse prognosis after hepatic resection than ones with lower level of Cav-1 (HR = 4.49, P<0.0001).

Article Snippet: The 18s rRNA TaqMan probe (Hs99999901_s1), Cav-1 TaqMan probe (Hs00971716_m1) and GLI1 TaqMan probe (Hs01110766_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Immunohistochemistry, Expressing, Membrane, Negative Control, Staining

Journal: PLoS ONE

Article Title: Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma

doi: 10.1371/journal.pone.0084551

Figure Lengend Snippet: (A) The size of xenografts from Huh7 GLI1 group was significantly larger than one from Huh7 Vector group (P = 0.002); (B) The representative IHC staining of Cav-1 in xenografts from both Huh7 GLI1 group (a) and Huh7 Vector group (b). Cav-1 protein was detected in both cell membrane (as labelled by a black arrow) and cytoplasm (as labelled by a white arrow). Cav-1 expression in xenografts from Huh7 GLI1 group was apparently more than one in xenografts from Huh7 Vector group; (C) The representative IHC staining of E-cadherin in xenografts from both Huh7 GLI1 group (a) and Huh7 Vector group (b). E-cadherin protein located mainly in cell membrane, as labelled by black arrows. There were less E-cadherin expression in xenograft tissues from Huh7 GLI1 group than ones from Huh7 Vector group; (D) The representative IHC staining of N-cadherin in xenografts from both Huh7 GLI1 group (a) and Huh7 Vector group (b). N-cadherin expression expressing basically in cytoplasm (labelled by white arrows) was increased clearly in xenograft parenchymal tissues from Huh7 GLI1 group than ones from Huh7 Vector group. Cytoplasmic N-cadherin expression was also found in xenograft mesenchymal tissues from both groups, as labelled by black arrows.

Article Snippet: The 18s rRNA TaqMan probe (Hs99999901_s1), Cav-1 TaqMan probe (Hs00971716_m1) and GLI1 TaqMan probe (Hs01110766_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Immunohistochemistry, Membrane, Expressing

Journal: The Prostate

Article Title: β 2 ‐adrenergic receptor signaling drives prostate cancer progression by targeting the Sonic hedgehog‐Gli1 signaling activation

doi: 10.1002/pros.24060

Figure Lengend Snippet: Antibodies’ references and immunohistochemistry and Western blot analysis procedures

Article Snippet: Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β 2 ‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table ), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions.

Techniques: Immunohistochemistry, Western Blot, Incubation

Journal: The Prostate

Article Title: β 2 ‐adrenergic receptor signaling drives prostate cancer progression by targeting the Sonic hedgehog‐Gli1 signaling activation

doi: 10.1002/pros.24060

Figure Lengend Snippet: Shh‐Gli1 signaling is required for ISO‐mediated proliferation of PCa cells. A, Colony formation assays and quantification of cells treated with DMSO, GANT61 (10 μM), or ISO (1 μM) in the absence or presence of GANT61 (10 μM). Data represent the mean ± SD of three experiments. * P < .05. B and C, Western blotting and relative quantification of mRNA expression of Gli1 in PC‐3 and LNCaP cells transduced with a control shRNA (shNC) and shRNA targeting Gli1 (shGli1). D, Colony formation assays and quantification of transduced cells targeting Gli1 without or with 1 μM ISO treatment. β 2 ‐AR, β 2 ‐adrenergic receptor; DMSO, dimethyl sulfoxide; ISO, isoproterenol; mRNA, messenger RNA; SD, standard deviation; shRNA, small hairpin RNA [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β 2 ‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table ), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Transduction, shRNA, Standard Deviation

Journal: The Prostate

Article Title: β 2 ‐adrenergic receptor signaling drives prostate cancer progression by targeting the Sonic hedgehog‐Gli1 signaling activation

doi: 10.1002/pros.24060

Figure Lengend Snippet: ISO antagonizes the apoptosis induced by targeting Shh‐Gli1 signaling A, Flow cytometry apoptosis assays of cells treated with DMSO, GANT61 (10 μM), or ISO (1 μM) in the absence or presence of GANT61 (10 μM) for 24 hours. Data represent the mean ± SD of three experiments. * P < .05. B, Flow cytometry apoptosis assays of PC‐3 and LNCaP cells transduced with a control shRNA (shNC) or shRNA targeting Gli1 (shGli1) treated with DMSO or ISO (1 μM) for 24 hours. Data represent the mean ± SD of three experiments. * P < .05. β 2 ‐AR, β 2 ‐adrenergic receptor; DMSO, dimethyl sulfoxide; ISO, isoproterenol; SD, standard deviation; shRNA, small hairpin RNA [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β 2 ‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table ), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions.

Techniques: Flow Cytometry, Transduction, shRNA, Standard Deviation

Journal: The Prostate

Article Title: β 2 ‐adrenergic receptor signaling drives prostate cancer progression by targeting the Sonic hedgehog‐Gli1 signaling activation

doi: 10.1002/pros.24060

Figure Lengend Snippet: Stimulation of β 2 ‐AR signaling promotes, whereas knockdown of β 2 ‐AR suppresses, Shh‐Gli1 signaling activity in vivo and in vitro. A, Gli1, Shh and Ptch1 expression levels were detected by Western blotting and quantification of PC‐3 and LNCaP cells treated with DMSO or 1 μM ISO for 24 hours. B, Gli1, Shh, and Ptch1 expression levels were detected by Western blotting and quantification in PC‐3 and LNCaP cells transduced with a negative control shRNA (shNC) and shRNA targeting β 2 ‐AR (shβ 2 ‐AR) treated with DMSO or 1 μM ISO for 24 hours. C and D, Representative immunohistochemistry images and quantification of Gli1, Shh, and Ptch1 expression levels in xenografts. Scale bar, 100 μm. E, Western blotting assay of the expression of downstream genes of Shh signaling and quantification in PC‐3 and LNCaP cells stably expressing shNC or shGli1 following treatment with DMSO or 1 μM ISO for 24 hours. β2‐AR, β2‐adrenergic receptor; DMSO, dimethyl sulfoxide; ISO, isoproterenol; Ptch1, patched1; SD, standard deviation; shRNA, small hairpin RNA [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β 2 ‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table ), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions.

Techniques: Activity Assay, In Vivo, In Vitro, Expressing, Western Blot, Transduction, Negative Control, shRNA, Immunohistochemistry, Stable Transfection, Standard Deviation

Journal: The Prostate

Article Title: β 2 ‐adrenergic receptor signaling drives prostate cancer progression by targeting the Sonic hedgehog‐Gli1 signaling activation

doi: 10.1002/pros.24060

Figure Lengend Snippet:

Article Snippet: Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β 2 ‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table ), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions.

Techniques:

Journal: Open Medicine

Article Title: GPC2 deficiency inhibits cell growth and metastasis in colon adenocarcinoma

doi: 10.1515/med-2022-0421

Figure Lengend Snippet: Prediction of pathways involved in GPC2: (a and b) the genes co-expressed with GPC2 in COAD tissues were enriched in (a) notch signaling and (b) hedgehog signaling and (c) After transfection of shNC, shGPC2 into HCT-8 and SW620 cells, the protein levels of PTCH1, PTCH2, GLI1, NOTCH1, HES1, and DLL4 were detected by Western blotting ( n = 3). KEGG: Kyoto Encyclopedia of Genes and Genomes. ** P < 0.01, *** P < 0.001.

Article Snippet: After separated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, the proteins were transferred onto polyvinylidenefluoride membranes, which were subsequently incubated with 5% skimmed milk at 25°C for 2 h. The membrane was incubated with GPC2 antibody (Biorbyt, catalog number: orb157203), PTCH1 antibody (Biorbyt, catalog number: orb538088), PTCH2 antibody (Biorbyt, catalog number: orb416229), GLI1 antibody (Proteintech, catalog number: 66905-1-Ig), NOTCH1 antibody (Biorbyt, catalog number: orb256723), HES1 antibody (Biorbyt, catalog number: orb36445), DLL4 antibody (Biorbyt, catalog number: orb97478), or glycerine aldehyde phosphate dehydrogenase antibody (Proteintech, catalog number: 10494-1-AP) at 4°C overnight, followed by horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG(H + L) (Proteintech, Catalog number: SA00001-2) or HRP-conjugated goat anti-mouse IgG(H + L) (Proteintech, Catalog number: SA00001-1) at 25°C for 2 h. Finally, Super Sensitive ECL Solution (Beijing Bai’aolaibo Technology Co., Ltd, Catalog number: MT0076) was used to display the results.

Techniques: Transfection, Western Blot

Journal: Neuro-Oncology

Article Title: Activation of the Hedgehog pathway in pilocytic astrocytomas

doi: 10.1093/neuonc/noq026

Figure Lengend Snippet: Clinical and pathological features and the molecular analyses of pilocytic astrocytomas

Article Snippet: For primary cell cultures, qRT-PCR reactions were performed with the TaqMan Fast Universal PCR Master Mix (Applied Biosystems), cDNA template, and primers (TaqMan Gene Expression Assay, Applied Biosystems) for hPTCH (Hs00970979_m1), hGLI1 (Hs00171790_m1), and hGAPDH (Hs99999905_m1), respectively.

Techniques: Paraffin-embedded Immunohistochemistry

Journal: Neuro-Oncology

Article Title: Activation of the Hedgehog pathway in pilocytic astrocytomas

doi: 10.1093/neuonc/noq026

Figure Lengend Snippet: Comparison of age at diagnosis and molecular analyses of Hedgehog pathway activation. (A) When grouped according to diagnosis before and after the age of 10 years, median PTCH levels are 5.01 and 1.23, respectively (Student's t -test, P = .013). (B and C) Higher staining indices for PTCH and GLI1 are measured in tumors from younger patients. In specimens from patients diagnosed before and after the age of 10, the median values for PTCH are 14.00 and 6.81, respectively (Student's t -test, P = .033; B) and for GLI1 they are 4.82 and 0.74 (Student's t -test, P = .034; C).

Article Snippet: For primary cell cultures, qRT-PCR reactions were performed with the TaqMan Fast Universal PCR Master Mix (Applied Biosystems), cDNA template, and primers (TaqMan Gene Expression Assay, Applied Biosystems) for hPTCH (Hs00970979_m1), hGLI1 (Hs00171790_m1), and hGAPDH (Hs99999905_m1), respectively.

Techniques: Comparison, Biomarker Discovery, Activation Assay, Staining

Journal: Neuro-Oncology

Article Title: Activation of the Hedgehog pathway in pilocytic astrocytomas

doi: 10.1093/neuonc/noq026

Figure Lengend Snippet: Hedgehog pathway component expression in a pilocytic astrocytoma specimens. (A–C) Representative image of a pilocytic astrocytoma from a tissue microarray stained with hematoxylin and eosin (A) and for GLI1 (arrowheads in B), and PTCH and Ki67 (C). Arrowhead in C demonstrates a PTCH + /Ki67 + cells. Scale bar = 50 µm.

Article Snippet: For primary cell cultures, qRT-PCR reactions were performed with the TaqMan Fast Universal PCR Master Mix (Applied Biosystems), cDNA template, and primers (TaqMan Gene Expression Assay, Applied Biosystems) for hPTCH (Hs00970979_m1), hGLI1 (Hs00171790_m1), and hGAPDH (Hs99999905_m1), respectively.

Techniques: Expressing, Microarray, Staining

Journal: Neuro-Oncology

Article Title: Activation of the Hedgehog pathway in pilocytic astrocytomas

doi: 10.1093/neuonc/noq026

Figure Lengend Snippet: Characterization of Hedgehog pathway responsiveness in primary cell cultures from pilocytic astrocytomas. (A) Astrocytic tumors were grown as spheres under stem cell culture conditions. Cells from a Hh-responsive astrocytoma (GII A), a Hh-unresponsive GBM (GIV GBM), and 4 pilocytic astrocytomas (GI PA-6, GI PA-10, GI PA-15, and GI PA-18) were cultured for 36 hours either alone (control), with 50 nM SAG, or with 200 nM SANT1. In triplicate wells for each cell line and culture condition, the GLI1 levels were normalized to GAPDH and expressed relative to the untreated control cultures. (B) To confirm a dose-dependent response to Hh pathway stimulation, the GI PA-18 cells were cultured in triplicate wells either alone (control), with 50 nM SAG, or with 200 nM SAG. After 40 hours, the GLI1 levels were normalized to GAPDH and expressed relative to the untreated control cultures. (C) To determine whether Hh signaling could be inhibited following pathway activation, the GI PA-18 cells were cultured in the presence of SAG (50 nM) for 7 days and then re-plated in triplicate wells containing either 50 nM SAG or 200 nM SANT1. After an additional 48 hours in culture, the GLI1 levels were normalized to GAPDH and expressed relative to the SAG-treated cultures. GI, WHO grade I; GII, WHO grade II; GIV, WHO grade IV; A, astrocytoma; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; PA, pilocytic astrocytoma; SAG, smoothened agonist; SANT, smoothened antagonist; * P ≤ .05.

Article Snippet: For primary cell cultures, qRT-PCR reactions were performed with the TaqMan Fast Universal PCR Master Mix (Applied Biosystems), cDNA template, and primers (TaqMan Gene Expression Assay, Applied Biosystems) for hPTCH (Hs00970979_m1), hGLI1 (Hs00171790_m1), and hGAPDH (Hs99999905_m1), respectively.

Techniques: Stem Cell Culture, Cell Culture, Control, Activation Assay