Fig. 5: R-loop-dependent fork stalling is induced by partial inhibition of DNA synthesis with aphidicolin.
From: Excessive reactive oxygen species induce transcription-dependent replication stress
a–c RNase H1 overexpression rescues replication fork stalling induced by a low dose of aphidicolin (APH). a Experimental workflow of DNA fiber assays with U2OS T-REx [RNH1(WT)-GFP] cells. Doxycycline (Dox; 1 ng/ml) was added to induce the expression of RNase H1 (RNH1). b Scatter plot of the values of sister fork ratio obtained from three independent experiments for indicated conditions (n ≥ 153). c Scatter plot of the values of IdU/CldU tract length ratio obtained from three independent experiments for indicated conditions (n ≥ 402). d, e Co-localization of PCNA and elongating form of RNAPII in S-phase nuclei of U2OS cells after 1 h treatment with 0.2 μM APH, as revealed by proximity ligation assay (PLA) and EdU-pulse labeling. d Top: Experimental workflow. EdU, 5-ethynyl-2′-deoxyuridine (10 μM). Bottom: Representative galleries of PLA signal in cell nuclei exported from ScanR analysis program. Scale bar, 10 μm. e Top: Scatter plot of the number of PLA foci in individual cells for indicated conditions (n ≥ 2064). Gray bars indicate the median; p values were calculated by Mann-Whitney test. Bottom: Scatter plot of total DAPI (x-axis) and mean EdU (y-axis) intensities in individual cells. Colors indicate the number of PLA foci and correspond to the upper plot. Representative plots from three independent experiments yielding similar results are shown. NT non-treated. f Low but not high doses of APH induce nascent DNA degradation in BRCA2-depleted U2OS cells. Top: Experimental workflow of DNA fiber assays. A thymidine (400 μM) chase was included for all conditions to stop IdU incorporation. Bottom: Scatter plot of the values of IdU/CldU tract length ratio obtained from two independent experiments for indicated conditions (n ≥ 332). b, c, f Red horizontal lines indicate the median; p values were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Source data are provided as a Source Data file.
