Journal: Oncotarget

Article Title: Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

doi: 10.18632/oncotarget.6490

Figure Lengend Snippet: A. Evaluation of ACF stability using increasing concentrations of ZnPC-containing cell phantoms (ZnPC-CPs) with or without irradiation. ACF degradation was monitored using fluorescence spectroscopy ( n = 4 per concentration). B. Cells were incubated with ACF for 24 hours, after which the uptake of ACF was determined using fluorescence spectroscopy. Data were normalized to protein content ( n = 4 per concentration). C. ACF toxicity was determined after 24-hour incubation under either normoxic (red line) or hypoxic (blue line) conditions using the WST-1 method ( n = 4 per group). Treatment efficacy of ACF and ACF + PDT was tested in SK-ChA-1 cells after 4 hours at D. normoxic and E. hypoxic culture conditions ( n = 6 per group). (F, G) Relative caspase 3/7 activity was determined 4 hours after PDT at incubation at F. normoxic or G. hypoxic culture conditions ( n = 6 per group). H. Lactate production by SK-ChA-1 cells treated with ACF and ACF + PDT was evaluated after 24 hours at normoxic (red bars) or hypoxic (blue bars) culture conditions ( n = 6 per group). I–P. Analysis of DNA damage after control (CTRL), ACF, PDT, and ACF + PDT treatment. Cells were kept for 4 hours under normoxic (I-L) or hypoxic conditions (M-P) post-treatment. Cells were stained with DAPI (nuclei, blue) and phospho-H2AX (DNA double-strand breaks, red). The arrowhead in panel P indicates apoptosis. Readers are referred to the experimental section for the significance of the statistical symbols.

Article Snippet: For the assessment of DNA damage, cells were fixed with a mixture of 4% paraformaldehyde and 0.2% sucrose for 5 min and permeabilized in 0.1% TX-100 (in PBS) for 5 min. Next, cells were washed with 1 mL of PBS and incubated for 16 hours with mouse anti-human phospho-H2AX-AlexaFluor647 (Cell Signaling Technology, Danvers, MA) at a 1:100 dilution in 0.5% BSA and 0.15% glycine (in PBS, staining buffer) at 4°C.

Techniques: Irradiation, Fluorescence, Spectroscopy, Concentration Assay, Incubation, Activity Assay, Staining

Journal: Oncotarget

Article Title: Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

doi: 10.18632/oncotarget.6490

Figure Lengend Snippet: A–D. SK-ChA-1 cells were incubated with ACF for either (A, B) 24 hours or (C, D) 48 hours, after which the cell cycle profile was analyzed with flow cytrometry using propidium iodide staining ( n = 3 per group). E. Flow cytometric analysis of SK-ChA-1 cells that were incubated with ACF for either 24 hours (in grey) or 48 hours (in white), after which the fraction of apoptotic (annexin V-positive) and F. necrotic (TO-PRO-3-positive) cells was determined ( n = 3 per group). (G, H) SK-ChA-1 cells were exposed to ACF for G. 24 hours or H. 48 hours and intracellular DCF fluorescence was determined as a measure of ROS production. I–P. Analysis of DNA damage after control (CTRL) or ACF treatment. SK-ChA-1 cells received ACF or CTRL treatment for (I-L) 24 hours or (M-P) 48 hours, and were subsequently stained with DAPI (nuclei, blue) and phospho-H2AX (DNA double-strand breaks, red).

Article Snippet: For the assessment of DNA damage, cells were fixed with a mixture of 4% paraformaldehyde and 0.2% sucrose for 5 min and permeabilized in 0.1% TX-100 (in PBS) for 5 min. Next, cells were washed with 1 mL of PBS and incubated for 16 hours with mouse anti-human phospho-H2AX-AlexaFluor647 (Cell Signaling Technology, Danvers, MA) at a 1:100 dilution in 0.5% BSA and 0.15% glycine (in PBS, staining buffer) at 4°C.

Techniques: Incubation, Staining, Fluorescence