Journal: Cell Reports Methods

Article Title: Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

doi: 10.1016/j.crmeth.2025.101270

Figure Lengend Snippet: Induction of resistance in vitro induces a reduction in PARP trapping as assessed by the EGFP-PARP1-mCherryFP FRET biosensor (A–C) (A) Schematic of the treatment protocol to induce in vitro resistance to olaparib or rucaparib. OVCAR4 EGFP-PARP1-mCherryFP cells were treated daily with 20 μM olaparib or rucaparib for 8 weeks. Resistance was assessed by SRB assay to (B) rucaparib or (C) olaparib and compared to vehicle-treated parental OVCAR4 EGFP-PARP1-mCherryFP cells. t tests were performed to confirm IC 50 values, n = 3 biological repeats. (D and E) (D) Live-cell FLIM-FRET HCA assays were performed to assess changes in donor lifetime of the EGFP-PARP1-mCherryFP FRET biosensor upon 1 h treatment with rucaparib or (E) olaparib. Single-cell FLIM data show population-level distributions of mean-weighted fluorescence lifetime. Data are shown as mean ± SEM, n = 3 biological repeats, and statistical significance was assessed using one-way ANOVA. (F) Change in FRET biosensor donor fluorescence lifetime of vehicle-exposed (left) or olaparib-exposed (right) cells upon treatment with rucaparib at 0, 10, 30, or 50 μM for 1 h. One-way ANOVA (Kruskal-Wallis) with Dunn’s multiple comparison test was used to assess statistical significance between treatment conditions. (G and H) (G) RT-qPCR analysis of PARG and (H) TP53BP1 relative mRNA expression normalized to GAPDH expression. n = 3 biological repeats. (I) Representative images of rucaparib and EGFP fluorescence in OVCAR4 EGFP-PARP1-mCherryFP cells following vehicle, rucaparib, or olaparib exposure in vitro for 9 weeks. (J) Quantification of average intracellular rucaparib intensity of images in (I), each point is a single cell average and error bars represent population median. n = 3 biological repeats.

Article Snippet: TaqMan primer probes ( GAPDH (Hs02786624_g1), PARG ( Hs00608254_m1 ), TP53BP1 (Hs00996827_m1), ABCB1 (Hs00184500_m1), Thermo Fisher Scientific).

Techniques: In Vitro, Sulforhodamine B Assay, Fluorescence, Comparison, Quantitative RT-PCR, Expressing

Journal: Cell Reports Methods

Article Title: Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

doi: 10.1016/j.crmeth.2025.101270

Figure Lengend Snippet: In vivo resistance generation to olaparib leads to a reduction in PARP trapping (A) OVCAR4 EGFP-PARP1-mCherryFP cells were injected intraperitoneally (I.P.) into female CD1 nude mice and allowed to grow for 21 days before mice were treated with vehicle or olaparib (5 mg/kg) daily for 14 days. Mice were culled upon reaching humane endpoint, and omental tumors and ascites were harvested. (B) Representative images showing OVCAR4 EGFP-PARP1-mCherryFP cells grown in 2D monolayers before injecting into CD1 nude mice (left) and the ascites harvested from the peritoneal cavity of these mice after 90 or 92 days for vehicle- and olaparib-treated groups, respectively. Images were acquired using the Incucyte S3 with 10× objective. (C) Quantification of average spheroid area per condition. (D) Viability assays were performed on the ascites cells grown in 2D, treated with olaparib for 72 h. Survival scores are normalized to vehicle-treated controls. Error bars represent SEM, n = 3 biological replicates. (E) Representative IHC images of MDR1 (brown stain) from OVCAR4 EGFP-PARP1-mCherryFP tumors, scale bars: 1 mm. (F) OVCAR4 EGFP-PARP1-mCherryFP omental tumors harvested at day 90 (vehicle) or 92 (olaparib) were stained for MDR1 by IHC. The number of MDR1-positive tumor cells was quantified using QuPath. Statistical significance was tested using an unpaired t test. (G and H) (G) RT-qPCR analysis of PARG and (H) TP53BP1 relative mRNA expression normalized to GAPDH expression. n = 3 biological repeats. (I) Representative images of rucaparib intrinsic fluorescence (left), EGFP-PARP1 (middle), and merged image (right) in the vehicle- or olaparib-exposed populations, scale bars: 25 μm. (J) Quantification of median rucaparib signal per cell. n = 3 biological repeats. (K) Fluorescence lifetimes of EGFP measured in FLIM assays of OVCAR4 EGFP-PARP1-mCherryFP cells that had been harvested from mice ascites of each group. Cells retrieved from vehicle- and olaparib-treated mice were incubated with olaparib (0–50 μM) for 1 hour prior to fluorescence microscopy. Error bars show mean and SEM value, n = 3 biological replicates. One-way ANOVA (Kruskal-Wallis) with Dunn’s multiple comparison test was used to assess statistical significance between treatment conditions.

Article Snippet: TaqMan primer probes ( GAPDH (Hs02786624_g1), PARG ( Hs00608254_m1 ), TP53BP1 (Hs00996827_m1), ABCB1 (Hs00184500_m1), Thermo Fisher Scientific).

Techniques: In Vivo, Injection, Staining, Quantitative RT-PCR, Expressing, Fluorescence, Incubation, Microscopy, Comparison

Journal: Journal of medicinal chemistry

Article Title: Discovery of Potent Isoquinolinequinone N -Oxides to Overcome Cancer Multidrug Resistance.

doi: 10.1021/acs.jmedchem.4c00705

Figure Lengend Snippet: Figure 10. Effect of compound 25 on the NCI-H460 cellular expression of proteins involved in proliferation, cell death and DNA damage, analyzed by Western Blotting. (A) Representative blots of TP53BP1, PARP-1, cyclin D1, p21, cyclin E, p-histone H2A.X, p53, and procaspase-3, after cells treatment for 48 h with medium, DMSO (vehicle), doxorubicin (positive control), GI50 concentration (0.35 μM), or 2 × GI50 concentration (0.7 μM) of compound 25. V1, % of vehicle used at the GI50 concentration of compound; V2, % of vehicle used at 2 × GI50 concentration of compound. Doxo: doxorubicin at 50 nM. (B) Graphical representation of the expression of proteins analyzed. β-actin was used as loading control. Results are the mean ± SEM from 3 independent experiments. * p ≤0.05, ** p ≤0.01 when comparing DMSO vs compound treatment.

Article Snippet: The membranes were incubated for 90 min at RT with the following primary antibodies: caspase-3 (1:200; sc-56053), cyclin D1 (1:200; sc-8396), cyclin E (1:100; sc-377100), p21 (1:200; sc-6246), p53 (1:200; sc-126), PARP-1 (1:200; sc-8007), p-Histone H2A.X (1:200; sc-517348), TP53BP1 (1:100; sc-515841), and β-actin (1:200; sc13118) from Santa Cruz Biotechnology, Dallas, Texas, USA.

Techniques: Expressing, Western Blot, Positive Control, Concentration Assay, Control