Fig. 1: RS-35d, S-35d and R-35d differential effects on HR, RAD51 nuclear activity and DNA damage. | Cell Death Discovery

Fig. 1: RS-35d, S-35d and R-35d differential effects on HR, RAD51 nuclear activity and DNA damage.

From: Investigating synthetic lethality and PARP inhibitor resistance in pancreatic cancer through enantiomer differential activity

Fig. 1

a Effect on HR caused by RS-35d, S-35d and R-35d in BxPC-3 cells. Results are expressed as mean ± SD (n = 3). b mClover-Lamin assay for DNA repair by HR performed in HEK293 cells transfected with Lamin A sgRNA and mClover-Lamin A donor and treated with RS-35d, S-35d and R-35d. c Representative microscopic merged images of mClover-Lamin A fusion protein and DNA (DAPI staining) fluorescence after exposure to increasing doses of RS-35d, S-35d or R-35d (scale bar, 30 μm). d Analysis of HR-positive (mClover-Lamin A-positive) cells after administration of increasing doses of RS-35d, S-35d or R-35d (the three compounds were tested in independent experiments and their effects were compared to their respective vehicle control). Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with p < 0.01 or p < 0.001 vs CTRL. e Immunofluorescence detection of nuclear RAD51 in BxPC-3 exposed to 40 µM RS-35d, 20 µM S-35d or 20 µM R-35d alone or after pre-treatment with 50 µM cisplatin (CDDP). Representative images of DAPI-stained nuclei and the corresponding nuclear localisation of RAD51 immune-labelling (scale bar, 30 μm) and analysis of RAD51-positive nuclei (%). Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Tuckey’s multiple comparison test, with §§§p < 0.001 vs CTRL and ##p < 0.01 vs 50 µM CDDP. f Evaluation of DNA damage via nuclear γH2AX foci immune detection in BxPC-3 cells treated for 48 h with 40 µM RS-35d, 20 µM S-35d or 20 µM R-35d alone or in combination with 10 µM olaparib (OLA). Representative images of DAPI-stained nuclei and the corresponding γH2AX immune-labelling (scale bar, 50 μm) and analysis of γH2AX-positive nuclei (%). g Micronuclei evaluation in BxPC-3 cells treated for 72 h with 40 µM RS-35d, 20 µM S-35d or 20 µM R-35d alone or in combination with 10 µM OLA. Representative images of DAPI-stained nuclei (white asterisks indicate micronuclei presence) (scale bar, 15 μm) and analysis of micronuclei-bearing cells (%). Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Tuckey’s multiple comparison test, with §p < 0.05 or §§p < 0.01 vs CTRL; #p < 0.05, ##p < 0.01 or ###p < 0.001 vs 10 µM OLA; *p < 0.05, **p < 0.01 or ***p < 0.001 vs RAD51-BRCA2 inhibitor alone.

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