Skip to main content
. 2024 Oct 29;27(12):111283. doi: 10.1016/j.isci.2024.111283

Figure 2.

Figure 2

Effect of antibody drug conjugates (ADCs) on survival and viability in HGSOC cells

(A) Cell growth assays (XTT) were performed on TROP2-positive (PEO1 and PEO1-olaJR) HGSOC cells in triplicate wells, both in normal media or after pretreatment with a nontargeting antibody drug conjugate control (cADC) or SG (10 μg/mL, 30 min at 37°C and washed thrice in PBS), prior to adding ATRi berzosertib (0–2 μM) for 48 h. Graphs show mean absorbance values at 490 nm corrected for background ±SD for each treatment (n = 3 biological replicates) against a concentration gradient (Log2) of ATRi. For all treatments we replaced 0 values for ATRi with 0.01 to enable plotting of absorbance values on a logarithmic X axis. Statistical determinations were done with Student’s t test and shown as p-values. The p-values for SG treatment versus untreated are indicated by asterisks (∗) while SG versus cADC are indicated by hashes (#). Slopes for each curve are derived from linear regression formula “y = mx+b” where y = Y-intercept, x = ATRi (μM) and b = correlation coefficient, calculated from the linear part of the respective curves.

(B) Graphs show mean cell growth values ±SD of cells with cADC or SG alone relative to untreated, derived from panel A (n = 3 biological replicates). Statistical significance (p-values) was determined with Student’s t test.

(C) Clonogenic assays were performed over 7 days with increasing concentrations of berzosertib (ATRi). Graphs show mean ± SD of stained area (n = 3 biological replicates). Statistical significance (p-values) determined using Student’s t test.

(D) Representative images from Matrigel plates after crystal-violet staining are shown. Graphs below image panels show total number of crystal-violet-stained invaded cells from imaged panels ±SD (n = 3 biological replicates). Statistical significance (p-values) was determined using Student’s t test. Experiment was performed twice. Scale bar is 100 μm.

(E) Flow cytometric analysis was conducted to assess cell viability with AnnexinV-FITC and 7AAD reagents on cells pretreated with SG or ATRi berzosertib (1 μM) alone or SG pretreatment followed by ATRi treatment overnight. Percentage of cells positive for AnnexinV alone (early apoptosis) or both AnnexinV and 7AAD (dead) are shown within respective quadrants. Graphs below show percentage of live (gray) and dead cells (black), while numbers above bars indicate fold increase in dead cells for each treatment relative to untreated. Images are representative of 3 biologically independent experiments.

(F) Cells with or without SG pretreatment were treated with ATRi berzosertib (1 μM) for 48 h and then stained with propidium iodide for DNA quantification and cell cycle analysis. Cell populations as fractions of total cells are shown over appropriately marked G1, S, or G2/M phases. Images are representative of 2 experimental replicates. For all images, p-values are ∗, #p < 0.05, ∗∗, ##p < 0.01, and ∗∗∗, ###p < 0.001, ns, not significant.