Journal: Cancer Pathogenesis and Therapy

Article Title: Metabolic pathways and chemotherapy resistance in acute myeloid leukemia (AML): Insights into Enoyl-CoA hydratase domain-containing protein 3 ( ECHDC3 ) as a potential therapeutic target

doi: 10.1016/j.cpt.2025.08.002

Figure Lengend Snippet: Changes in mitochondrial function following ECHDC3 knockdown. (A) TMRE staining results based on ECHDC3 -knockdown cells. siNC cells emitted bright red-orange fluorescence. Cells treated with a mitochondrial membrane-potential disrupter, CCCP, showed very weak or complete absence of red-orange fluorescence. The average fluorescence intensity of the cells was calculated and quantitatively analyzed. (B–C) mtDNA copy number ( MT–CO1 and MT–CO2 ) was quantified via quantitative RT-PCR; (D) Quantitation of mitochondrial SOD activity, wherein SOD activity decreased in ECHDC3 -knockdown cells. (E) Mitophagy biomarkers were detected via western blotting. β-Actin was used as a control. (F–I) Quantitation of the mitophagy pathway protein. Values were presented as mean ± standard error. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. CCCP: Carbonyl cyanide m-chlorophenyl hydrazone; ECHDC3 : Enoyl-CoA hydratase domain-containing protein 3; mtDNA: Mitochondrial DNA; RT-PCR: Real-time polymerase chain reaction; SOD: Superoxide dismutase; TMRE: Tetramethyl rhodamine ethyl ester.

Article Snippet: Western blotting was performed to determine the expression of mitochondrial proteins, using the Mitophagy Antibody Sampler Kit (Cat# 43110, Cell Signaling Technology [CST], MA, USA) and an anti-β-actin mouse monoclonal antibody (Cat# 3700, CST, MA, USA).

Techniques: Knockdown, Staining, Fluorescence, Membrane, Quantitative RT-PCR, Quantitation Assay, Activity Assay, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: A mutualistic association of CDK5 and 18E6 proteins. (A) (i). The represented immunoblot image of CDK5 upregulates the level of 18E6 protein. HEK 293 cells were transfected with an increasing amount of pcDNA3.1: His-CDK5 (1, 2, and 4 μg) and a fixed amount of pcDNA3.1: HA-18E6 (2 μg). (ii). The represented immunoblot image of 18E6 upregulates the level of CDK5 protein. HEK 293 cells were transfected with an increasing amount of pcDNA3.1: HA-18E6 (1, 2, and 4 μg) and pcDNA3.1: His-CDK5 (2 μg). (B) (i). The represented immunoblot image of CDK5 upregulates the level of 18E6 protein in the HeLa cell lines. HeLa cells were transfected with an increasing amount of pcDNA3.1: His-CDK5 (1, 2, 3, and 4 μg). (ii). The represented immunoblot image of 18E6 upregulates the level of CDK5 protein in the SAS cell lines. SAS cells were transfected with an increasing amount of pcDNA3.1: HA-18E6 (1, 2, 3, and 4 μg). (C) (i). The represented immunoblot image of CDK5, E6AP, and p53 after silencing 18E6/E7 expression. Total protein lysates were extracted from HeLa cell lines transfected with siRNA against control (siCtrl) or 18 E6 and E7 (si18 E6/E7) for 72 h. (ii-v) The relative expression levels of 18E6, CDK5, p53, and E6AP relative to β-actin and analyzed using ImageJ and GraphPad Prism (n = 3). All data were presented as means ± standard error of the mean (SEM). (∗, P < 0.05; ∗∗, P < 0.01). (D) The represented immunoblot of CDK5, pCDK5 and HPV 18E6 proteins in the stable expression of HPV 18 in the HGK12 cell line (HPV-null primary keratinocytes cell line), HC: Treatment with 0.2 μM CP681301; LC: Treatment with 0.1 μM CP681301.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

Techniques: Western Blot, Transfection, Expressing, Control

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: CP681301 treatment destabilized E6, reduced E6AP and rescued p53. (A). HeLa and C33A cell lines were treated with CP681301 at the concentrations of 0.6 μM and 1.5 μM for 24 h. The protein extracts were subjected to Western blotting for the detection of CDK5, 18E6, E6AP, p53, and β-actin proteins. (B). CP681301 disrupted the 18E6-E6AP protein complex. The inhibition of CP681301 was detected using the GST-pull down assay, where CP681301 was incubated with the purified GST-18E6 protein and Flag-E6AP protein for 2h. After extensive washing, the bound E6AP protein was detected via Western blotting using an anti-Flag antibody. The immunoblot (IB) on the upper panel shows the interaction of E6AP with GST-18E6, while the lower panel shows the Ponceau S stained of the blot.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

Techniques: Western Blot, Inhibition, Pull Down Assay, Incubation, Purification, Staining

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: CP681301 inhibits CDK5 and 18E6 in HeLa cell line, and disrupted the CDK5-HPV18E6 association directly. (A) (i). CP681301 disrupted the CDK5-18E6 protein complex. The inhibition of CP681301 was detected using the GST-pull down assay, where CP681301 was incubated with the purified GST-18E6 protein and CDK5 protein for 2h. After extensive washing, the bound CDK5 protein was detected via Western blotting using an anti-CDK5 antibody. The immunoblot (IB) on the upper panel shows the interaction of CDK5 with GST-18E6, while the lower panel shows the Ponceau S stained of the blot. (ii). The bar graph shows the quantification of the relative level of CDK5 to GST-18E6 protein indicated from 3 independent experiments (n = 3). Quantitation was performed using ImageJ software, and the statistical analysis was performed using Prism. (B). The summarisation of the CP681301 binding affinity value with 16E6, 18E6 and CDK5. (C) (i). The immunoblot shows the protein levels of HPV18E6 and CDK5 at different concentrations of CP681301. HeLa cells were treated with different concentrations of CP681301 (0.2 to 3.2 μM) or DMSO as indicated. Total cell lysates were subjected to western blotting with anti-CDK5, anti-18E6, and anti-β-actin antibodies. (ii). The EC50 of 18E6 in the treatment of CP681301. (iii). The EC 50 of CDK5 in the treatment of CP681301. The percentage of CDK5 and 18E6 protein levels was analyzed using the nonl i near regression function in GraphPad Prism 8.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

Techniques: Inhibition, Pull Down Assay, Incubation, Purification, Western Blot, Staining, Quantitation Assay, Software, Binding Assay