vp16  (Santa Cruz Biotechnology)

Journal: Oncogene

Article Title: Partial truncation of the C-terminal domain of PTCH1 in cancer promotes tumourigenesis by non-canonical activation of a GLI-PI3K loop

doi: 10.1038/s41388-026-03698-9

Figure Lengend Snippet: A Top: GLI1 protein levels in SCR, C9 and C15 cells. GAPDH is shown as loading control. Bottom: expression of PTCH1 , GLI1 and GLI 2 by qPCR. Expression levels were normalized to GAPDH mRNA and represented as fold change relative to control SCR cells ( n = 3). B Representative images for IHC staining of GLI1 and negative control (NoAb- no primary antibody) in sections of xenograft tumours generated with SCR and C9 cells. C Effect of increasing concentrations of GANT61 on viability of SCR, C9 and C15 cells after 72 h ( n = 3). The inset displays the calculated IC 50 in each cell line. D Effect of 0.5 μM KAAD-cyclopamine (KAAD-CP), 5 μM GANT61 or vehicle (DMSO) on the viability of SCR, C9 and C15 cells after 72 h ( n = 3). E Effect of 5 μM GANT61 or vehicle control (DMSO) on the colony formation ability of SCR, C9 and C15 cells upon culture for 10 days (representative images at the end of the experiment). F Number of colonies per 1000 plated cells at day 10 in the same conditions as ( E ) ( n = 3). G GLI1 protein levels in SCR, C9 and C15 cells transduced with lentiviruses encoding control scrambled (shScr) or GLI1 targeting (shGLI1) shRNAs. H Viability of SCR, C9 and C15 cells following 72 h of transduction with shScr-lenti or shGLI1-lenti in complete medium. Data are expressed as % of shScr ( n = 3). I Change in cell number of SCR cells in complete medium 48 h after transfection with empty vector, Gli1 K512R or Gli1(2-413)-VP16, compared to untransfected C9 and C15 cells ( n = 3). J Representative immunoblot depicting Gli1 protein levels following ectopic expression of Flag–Gli1-K518R and/or Gli1(2-413)–VP16 in SCR cells used in the proliferation assay shown in ( I ). The top blot was developed with a mix of anti-Gli1 and anti-VP16 antibodies. β-actin was used as loading control. All quantitative data represent the mean ± SEM of independent biological repeats (indicated n), performed in technical replicates. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following antibodies were used: EGFR (Cell Signaling Technology #2256, WB 1:1,000), AKT (Cell Signaling Technology #4691, WB 1:1,000), phospho- AKT (Ser473) (D9E) XP (Cell Signaling Technology #5012, WB 1:1,000), p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology #4695, WB 1:1,000), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology #4376, WB 1:1,000), Gli1 (Cell Signaling Technology #2643, WB 1:1,000), Phospho-PKA substrate (Cell Signaling Technology #9624, WB 1:1,000), GAPDH-HRP (Proteintech #HRP-60004, WB 1:3,000), Vinculin (Santa Cruz #sc-73614, WB 1:10,000), Poly-ADP ribose Polymerase (Cell Signaling Technology #9542, WB 1:2,000), anti VP16 (Santa Cruz #sc7545, WB 1:1,000),HA-Tag (Invitrogen #26183, WB 1:10,000), goat anti-rabbit IgG HRP (BioRad #172-1019, WB 1:3,000), goat anti-mouse IgG (H L)-HRP conjugate (Bethyl #A90-116P, WB 1:3,000).

Techniques: Control, Expressing, Immunohistochemistry, Negative Control, Generated, Transduction, Transfection, Plasmid Preparation, Western Blot, Proliferation Assay

Journal: Journal of Advanced Research

Article Title: Antimycin A inhibits alpha-herpesvirus replication by disrupting the formation of pyrimidinosomes

doi: 10.1016/j.jare.2025.05.016

Figure Lengend Snippet: Antimycin A exhibits extensive antiviral activity against alpha-herpesvirus. Antimycin A effectively inhibited HSV-1 (A-C) and HSV-2 (G-H) infections in Vero E6 cells, PRV (D-E) infection in PK-15 cells, and EHV-1 (J-K) infection in RK13 cells. Vero-E6 cells, PK-15 cells, and RK13 cells were pretreated for 12 h with increasing concentrations of Antimycin A and then infected with HSV-1 (A-C), PRV (D-E), HSV-2 (G-I), and EHV-1 (J-K) at MOIs of 0.5, 0.1, 0.5, and 0.5, respectively. At 24 hpi, cells were fixed and analyzed by fluorescence imaging. (A, D, G and J) Infection levels were quantified using a fluorescent microplate reader (black curve), while cell viability was measured using the CCK-8 Assay (orange curve). The CC50 for each compound was calculated via a four-parameter logistic nonlinear regression model in GraphPad Prism. Dotted lines indicate 50 % inhibition. Data represent the means ± SEM from n = 3 independent experiments of infectious virions, normalized to DMSO-treated wells. The IC50 values for HSV-1, PRV, HSV-2, and EHV-1 were determined by nonlinear regression analysis. (B, E, H and K) eGFP expression in infected cells, either untreated (0 μM) or treated with various concentrations (0.0015–5 μM) of Antimycin A, was visualized by fluorescence microscopy at the same time point. Representative images are shown. Bars, 300 µm. Magnification, ×10. (C, F and I) Western blot analysis was performed to quantify infection in cells infected with HSV-1, PRV, or HSV-2. For HSV-1, infection was assessed using ICP4, VP16, and gD as markers. For PRV, infection levels were quantified by immunoblotting for UL54. For HSV-2, infection was quantified by immunoblotting for VP16. β-actin was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in this study included goat BHV-1 antisera, mouse anti-PRV UL47 monoclonal antibody (MAb), mouse anti-HSV-1 ICP4 monoclonal antibody (Santa Cruz, sc69809), mouse anti-HSV-1 VP16 monoclonal antibody (Santa Cruz, sc7545), mouse anti-HSV-1 gD monoclonal antibody (Santa Cruz, sc21719), and mouse anti-β-actin MAb (Proteintech, 66009–1-lg).

Techniques: Activity Assay, Infection, Fluorescence, Imaging, CCK-8 Assay, Inhibition, Expressing, Microscopy, Western Blot, Control

Journal: Journal of Advanced Research

Article Title: Antimycin A inhibits alpha-herpesvirus replication by disrupting the formation of pyrimidinosomes

doi: 10.1016/j.jare.2025.05.016

Figure Lengend Snippet: Antimycin A exhibits extensive antiviral activity against alpha-herpesvirus. Antimycin A effectively inhibited HSV-1 (A-C) and HSV-2 (G-H) infections in Vero E6 cells, PRV (D-E) infection in PK-15 cells, and EHV-1 (J-K) infection in RK13 cells. Vero-E6 cells, PK-15 cells, and RK13 cells were pretreated for 12 h with increasing concentrations of Antimycin A and then infected with HSV-1 (A-C), PRV (D-E), HSV-2 (G-I), and EHV-1 (J-K) at MOIs of 0.5, 0.1, 0.5, and 0.5, respectively. At 24 hpi, cells were fixed and analyzed by fluorescence imaging. (A, D, G and J) Infection levels were quantified using a fluorescent microplate reader (black curve), while cell viability was measured using the CCK-8 Assay (orange curve). The CC50 for each compound was calculated via a four-parameter logistic nonlinear regression model in GraphPad Prism. Dotted lines indicate 50 % inhibition. Data represent the means ± SEM from n = 3 independent experiments of infectious virions, normalized to DMSO-treated wells. The IC50 values for HSV-1, PRV, HSV-2, and EHV-1 were determined by nonlinear regression analysis. (B, E, H and K) eGFP expression in infected cells, either untreated (0 μM) or treated with various concentrations (0.0015–5 μM) of Antimycin A, was visualized by fluorescence microscopy at the same time point. Representative images are shown. Bars, 300 µm. Magnification, ×10. (C, F and I) Western blot analysis was performed to quantify infection in cells infected with HSV-1, PRV, or HSV-2. For HSV-1, infection was assessed using ICP4, VP16, and gD as markers. For PRV, infection levels were quantified by immunoblotting for UL54. For HSV-2, infection was quantified by immunoblotting for VP16. β-actin was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used in this study included goat BHV-1 antisera, mouse anti-PRV UL47 monoclonal antibody (MAb), mouse anti-HSV-1 ICP4 monoclonal antibody (Santa Cruz, sc69809), mouse anti-HSV-1 VP16 monoclonal antibody (Santa Cruz, sc7545), mouse anti-HSV-1 gD monoclonal antibody (Santa Cruz, sc21719), and mouse anti-β-actin MAb (Proteintech, 66009–1-lg).

Techniques: Activity Assay, Infection, Fluorescence, Imaging, CCK-8 Assay, Inhibition, Expressing, Microscopy, Western Blot, Control