Article Figures & Data
Figures
- Figure 1.
Effects of si53BP1-transfection on the cytotoxicity of anticancer drugs administered with or without olaparib to KYSE70 cells. (A) Two and 6 days after transfection, total protein was extracted from whole-cell lysates, and western blotting was performed for 53BP1 and β-actin (reference protein) detection. (B) siRNA-transfected cells were seeded onto 96-well plates. After culturing for 24 h, cells were exposed continuously to the indicated drug concentrations for 1 week with or without olaparib (5 μM). Cell viability was determined using the CellQuanti-Blue™ cell viability assay kit. Each point represents the mean±standard error (n=4). siRNA, Small interfering RNA; si53BP1, siRNA against 53BP1; siNC, negative control siRNA; 5-FU, 5-fluorouracil; CDDP, cisplatin; SN-38, 7-ethyl-10-hydroxy-camptothecin.
- Figure 2.
Effects of olaparib on the nuclear accumulation of γH2AX and 53BP1 induced by anticancer drugs in KYSE70 cells. Cells were seeded onto black 96-well plates. After culturing for 24 h, cells were exposed continuously to the indicated anticancer drug with or without olaparib (5 μM) for 24 h. The concentrations of anticancer drugs are as follows: (A) 5-FU, 1 μM; (B) CDDP, 1 μM; (C) docetaxel, 1 nM; (D) SN-38, 10 nM; (E) doxorubicin, 100 nM; and (F) temozolomide, 100 μM. Cells were immunostained for γH2AX or 53BP1, and their accumulation in the nucleus was examined by immunofluorescence microscopy after counterstaining with DAPI. Each set of panels shows the numbers of γH2AX foci in the nucleus (left) and the numbers of 53BP1 foci co-localised with γH2AX (right). Each bar represents the mean±standard error (n=3). Significant differences were determined by an analysis of variance followed by Tukey's test (**p<0.01 vs. control †p<0.05, ††p<0.01, N.S., not significant). 5-FU, 5-Fluorouracil; CDDP, cisplatin; SN-38, 7-ethyl-10-hydroxy-camptothecin.
- Figure 3.
Effects of olaparib on the nuclear accumulation of 53BP1 and REV7 caused by anticancer drugs in KYSE70 cells. Cells were seeded onto black 96-well plates. After culturing for 24 h, cells were exposed continuously to the indicated anticancer drug with or without olaparib (5 μM) for 24 h. The concentrations of anticancer drugs are as follows: (A) CDDP, 1 μM; (B) SN-38, 10 nM; (C) doxorubicin, 100 nM; and (D) temozolomide, 100 μM. Cells were immunostained for 53BP1 or REV7, and their accumulations in the nucleus were examined by immunofluorescence microscopy after counterstaining with DAPI. Each set of panels shows the number of 53BP1 foci in nucleus (left) and the number of REV7 foci co-localised with 53BP1 (right). Each bar represents the mean±standard error (n=3). Significant differences were determined by an analysis of variance followed by Tukey's test (*p<0.05; **p<0.01 vs. control; †p<0.05; ††p<0.01; N.S., not significant). 5-FU, 5-Fluorouracil; CDDP, cisplatin; SN-38, 7-ethyl-10-hydroxy-camptothecin.
- Figure 4.
Effects of siREV7-transfection on the cytotoxicity of anticancer drug with or without olaparib in KYSE70 cells. (A) After transfection for 2 and 6 days, total protein was extracted from whole-cell lysates, and western blotting was performed for REV7 and β-actin (reference protein) detection. (B) siRNA-transfected cells were seeded onto 96-well plates. After culturing for 24 h, cells were exposed continuously to the indicated drug concentrations with or without olaparib (5 μM) for 1 week. Cell viability was determined using the CellQuanti-Blue™ cell viability assay kit. Each point represents the mean±standard error (n=4). siRNA, Small interfering RNA; siREV7, siRNA against REV7; siNC, negative control siRNA; 5-FU, 5-fluorouracil; CDDP, cisplatin; SN-38, 7-ethyl-10-hydroxy-camptothecin.
- Figure 5.
Effects of si53BP1 transfection on KYSE70 cell apoptosis caused by anticancer drugs with and without olaparib. siRNA-transfected cells were seeded onto 6-well plates. After culturing for 24 h, cells were exposed continuously to the indicated anticancer drug with or without olaparib (5 μM) for 48 h. The concentrations of anticancer drugs were as follows: (A) CDDP, 1 μM; (B) SN-38, 10nM; (C) doxorubicin, 100 nM; and (D) temozolomide, 100 μM. Next, cells were harvested and stained with annexin V-fluorescein isothiocyanate and propidium iodide, and analysed using the Becton Dickinson LSRFortessa™ instrument. Each bar represents the mean±standard error (n=3). Significant differences were determined via analysis of variance followed by Tukey's test (*p<0.05; **p<0.01 vs. control of siNC; †p<0.05; and ††p<0.01). siRNA, Small interfering RNA; si53BP1, siRNA against 53BP1; siNC, negative control siRNA; CDDP, cisplatin; SN-38, 7-ethyl-10-hydroxy-camptotheci.
