Article Figures & Data
Figures
- Figure 1.
Quantitative detection of sgHPRT-induced mutation by HRM analysis. A, Graphical representation of the targeting of HPRT locus using CRISPR/Cas9 system. B, HEK293T cells were transfected with sgHPRT plasmid, and genomic DNA was extracted at different time points. T7E1 cleavage assay was performed to evaluate the targeted mutation. C, Melting data were collected and normalized as described in Materials and Methods (top), and a subtractive plot showed the relative fluorescence difference (ΔF) to the control curve (bottom). D, Different mutation efficiencies were induced by transfection of proportional sgHPRT and control plasmid. The data were presented in a three-dimension plot where x-axis showed the temperature, y-axis showed the ΔF, and z-axis showed the mutation efficiency. E, Correlation of the mutation efficiency with the maximum value of ΔF (ΔFmax).
- Figure 2.
Design and validation of the screening for NHEJ inhibitors. A, Schematic depicting the screening procedures. HEK293T cells were transfected with sgHPRT plasmid for 5 hours, and subcultured into 96-well plates in the presence of the screening compounds. Twenty-four hours later, genomic DNA was extracted and subjected to HRM analysis. Two DNA-PKcs inhibitors, NU7441 and KU-0060648, were incubated with cells in 96-well plates. Three-dimensional plots show the HRM data of different concentrations of NU7441 (B) and KU-0060648 (D). Linear correlation between the concentrations and ΔFmax for NU7441 (C) and KU-0060648 (E).
- Figure 3.
Results of the screening of an approved drug library. A, The drugs were classified according to their pharmacologic action. The composition of the 10 categories was shown in the circle chart. B, Scatter plots of the screening results of 1,540 compounds. Tables of top hits of Category no. 7 (cardiovascular system; C) and Category no. 8 (nervous system; D).
- Figure 4.
Dose–response analysis of the screening hits. The chemical structures, and the cytotoxic and NHEJ-inhibiting effects of ouabain (A) and penfluridol (B) were shown in a dose-dependent manner. HEK293T cells were treated with different concentration of ouabain or penfluridol for 24 hours. C, Measurement of NHEJ and HR activities by using the NHEJ and HR reporter plasmids. HEK293T cells were transfected with I-SceI–linearized plasmids and incubated with ouabain or penfluridol for 24 hours. A DsRed-expressing plasmid was cotransfected as transfection control. Recircularized plasmids by NHEJ or HR have the intact GFP gene that can be assayed by flow cytometry. *, P < 0.05; **, P < 0.01 versus DMSO-treated group, n = 4.
- Figure 5.
Radiosensitizing effects of ouabain and penfluridol. A, HeLa cells were incubated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then exposed to 8 Gy of X-ray, and further incubated with these drugs for 3 hours. Neutral comet assay was performed 12 hours postirradiation. B, The percentage of DNA content in the tails were analyzed; *, P < 0.05 versus DMSO-treated group, n = 3. Representative images of the colony formation assay of ouabain (C) and penfluridol (E) were shown. Treatment of ouabain (D) and penfluridol (F) increased the radiosensitivity of HeLa cells. *, P < 0.05 versus DMSO-treated group, n = 3.
- Figure 6.
Impairment of DSB repair by ouabain and penfluridol. A, HeLa cells were incubated with 0.1 μmol/L ouabain or 5 μmol/L penfluridol for 2 hours, then exposed to 8 Gy of X-ray, and further incubated with these drugs for 3 hours. Twelve hours postirradiation, γH2AX and 53BP1 foci formations were visualized by immunofluorescent staining. B, Four hours postirradiation, the expressions of phosphorylated DNA-PKcs, ATM, and Chk2 were analyzed by Western blotting. *, P < 0.05 versus DMSO-treated group, n = 3.
Additional Files
Supplementary Data
- Supplementary Figures S1-S4 and Supplementary Methods - Figure S1: HRM analysis of the sgHBEGF-induced mutation; Figure S2: Figure S3: Selected hits of NHEJ inhibitors; Figure S4: The direct effects of ouabain and penfluridol on DNA-PK were examined by using DNA-PK activity assayin vitro.
- Table S1 - Full table of the screening results.
Volume 17, Issue 2
