Figure 2. ABL kinase inhibition or depletion increases transcription-replication conflicts (TRCs) in SCLC cells.

(A) Schematic representation of TRCs.

(B) DMS53 cells were treated with vehicle (DMSO) or ABL001 (20 μM) for 16h, and proximity ligation assay (PLA) was performed using S9.6 (R-Loops) and anti-PCNA (replication forks) antibodies (scale bars, 10 μm). Quantification of PLA foci per nucleus, where each dot represents an individual nucleus; n = 95–230 nuclei per experiment with 3 biological replicates. Student’s t test ****p < 0.0001.

(C) DMS53 cells were treated for 24 h with vehicle, 20 μM ABL001, 20 μM DRB, or dual treatment with ABL001 and DRB. Representative images of PLA assay with S9.6 and anti-PCNA antibodies (594 nm) (scale bars, 10 μm). Quantification of PLA foci per nucleus, where each dot represents an individual nucleus; n = 71–204 nuclei per experiment with 2 biological replicates. One-way ANOVA with Tukey-Kramer test for multiple comparisons ****p < 0.0001.

(D) DMS53 cells were transduced with either shSCR or shAA for 5 days, then treated with or without 20 μM DRB for the final 24 h. Representative images of the PLA assay with S9.6 and anti-PCNA antibodies (594 nm) (scale bars, 10 μm). Quantification of PLA foci per nuclei, with the mean value representing PLA foci per nucleus; n = 50–100 nuclei per experiment with 2 biological replicates. One-way ANOVA with Tukey-Kramer test for multiple comparisons ****p < 0.0001.

Data in B, C, and D represented as mean +/− SEM.