Journal: Heliyon

Article Title: Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

doi: 10.1016/j.heliyon.2023.e19244

Figure Lengend Snippet: Primers for RT‒qPCR.

Article Snippet: Thereafter, the membrane was treated overnight at 4 °C with the appropriate primary antibodies: PCNA (1:5000; Proteintech; 10205-2-AP), Ki67 (1:1000; Abclonal; A2094), BCL2 (1:2000; Proteintech; 12789-1-AP), BAX (1:5000; Proteintech; 60267-1-Ig), SHH (1:1000; Proteintech; 20697-1-AP), PTCH1 (1:1000; Affinity; AF5202), SMO (1:3000; Proteintech; 66851-1-Ig), GLI1 (1:3000; Proteintech; 66905-1-IG), and GAPDH (1:5000; Abcam; ab9485).

Techniques:

Journal: Heliyon

Article Title: Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

doi: 10.1016/j.heliyon.2023.e19244

Figure Lengend Snippet: Itraconazole inhibited the HH signaling pathway in HemECs and HUVECs. HemECs and HUVECs were treated with itraconazole (HemECs: 0.5, 2 μM; HUVECs: 5, 20 μM) for 24 and 48 h. The protein levels of SHH, PTCH1, SMO, and GLI1 in HemECs and HUVECs were detected through western blotting ( A , B ). The mRNA levels of SHH, PTCH1, SMO, and GLI1 in HemECs and HUVECs were measured through RT‒qPCR ( C , D ). * P < 0.05, ** P < 0.01, compared with untreated cells.

Article Snippet: Thereafter, the membrane was treated overnight at 4 °C with the appropriate primary antibodies: PCNA (1:5000; Proteintech; 10205-2-AP), Ki67 (1:1000; Abclonal; A2094), BCL2 (1:2000; Proteintech; 12789-1-AP), BAX (1:5000; Proteintech; 60267-1-Ig), SHH (1:1000; Proteintech; 20697-1-AP), PTCH1 (1:1000; Affinity; AF5202), SMO (1:3000; Proteintech; 66851-1-Ig), GLI1 (1:3000; Proteintech; 66905-1-IG), and GAPDH (1:5000; Abcam; ab9485).

Techniques: Western Blot

Journal: Heliyon

Article Title: Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

doi: 10.1016/j.heliyon.2023.e19244

Figure Lengend Snippet: rhSHH attenuated the effects of itraconazole on angiogenesis and downregulation of the HH signaling pathway. HemECs and HUVECs were treated with 2 μg/ml rhSHH, 2 μM itraconazole, or 2 μg/ml rhSHH +2 μM itraconazole for 24 h. HUVECs were treated with 2 μg/ml rhSHH, 20 μM itraconazole, or 2 μg/ml rhSHH +20 μM itraconazole for 24 h. Vascular angiogenesis of HemECs and HUVECs was determined using the tube formation assay ( A , B ). Scale bar = 0.1 mm. The protein levels of VEGFA were detected using ELISAs ( C ). The protein and mRNA levels of SHH, PTCH1, SMO, and GLI1 in HemECs ( D ) and HUVECs ( E ) were determined through western blotting and RT‒qPCR, respectively. * P < 0.05, ** P < 0.01, compared with untreated cells. # P < 0.05, ## P < 0.01, compared with the itraconazole group.

Article Snippet: Thereafter, the membrane was treated overnight at 4 °C with the appropriate primary antibodies: PCNA (1:5000; Proteintech; 10205-2-AP), Ki67 (1:1000; Abclonal; A2094), BCL2 (1:2000; Proteintech; 12789-1-AP), BAX (1:5000; Proteintech; 60267-1-Ig), SHH (1:1000; Proteintech; 20697-1-AP), PTCH1 (1:1000; Affinity; AF5202), SMO (1:3000; Proteintech; 66851-1-Ig), GLI1 (1:3000; Proteintech; 66905-1-IG), and GAPDH (1:5000; Abcam; ab9485).

Techniques: Tube Formation Assay, Western Blot

Journal: Cell Reports

Article Title: A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

doi: 10.1016/j.celrep.2019.07.025

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-SMO , Santa Cruz Biotechnology , Cat#sc-166685; RRID: AB_2239686.

Techniques: Recombinant, Transgenic Assay, Produced, Blocking Assay, CRISPR, Construct, Plasmid Preparation, Expressing, Derivative Assay, Software

Journal:

Article Title: Basal Cell Carcinomas Arise From Hair Follicle Stem Cells in Ptch1+/- Mice

doi: 10.1016/j.ccr.2010.11.007

Figure Lengend Snippet: (A to C) Representative images of immunostaining with anti-Smo antibody (brown) and counterstained with hematoxylin (blue) show absence of staining in ear or tail and positive staining in dorsal skin follicles.

Article Snippet: For immunofluorescent staining, antigen retrieved slides were blocked with 2% FBS followed by incubation with primary antibodies: rabbit anti-YFP (1:100), anti-Smo (1:100, LS-A2668, MBL, Woburn, MA) or mouse anti-cyclin B1 (1: 200, Invitrogen, Camerillo, CA) overnight at 4° C. The secondary antibody conjugated with Alexa Fluor 488 or 555 (1:1000, Molecular Probes, Eugene, OR) was used together with DAPI (1:5000, Molecular Probes) to detect primary antibodies followed by mounting with ProLong Gold Antifade Reagents (Molecular Probes).

Techniques: Immunostaining, Staining