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. 2021 Apr 6;49(8):4350–4370. doi: 10.1093/nar/gkab180

Figure 3.

Figure 3.

DDX19A is involved in R-loop homeostasis. (A) Representative immunofluorescence microscopy images of NIH/3T3 cells expressing the indicated shRNAs for 10 days and stained for R-loops with the S9.6 antibody. Transfection with RNAseH1 24 h before fixation serves as a control for the S9.6 antibody specificity. Images are maximum intensity projections of a Z-stack covering the whole nucleus. Scale bars are 10 μm. (B) Box and whisker plot showing the quantification of the R-loop spot count per nucleus in images from (A). Images were analysed using CellProfiler™ software. Box-and-Whisker plots indicate the median and the 10–90 percentile from three independent experiments (**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, Student's t-test). (C) DRIP-qPCR analysis of RNA:DNA hybrid structures at the synP element upon recruitment of rTetR-LSD1 and under suppression of Ddx19a. Total nucleic acids were extracted from NIH/3T3 cells expressing the synP-mCherry reporter, the rTetR-LSD1 fusion protein and the indicated shRNAs at day 14 with and without DOX treatment and used as input for the IP with the S9.6 antibody. qPCR signals are shown relative to -DOX. Circles represent independent replicates (n = 4, mean ± s.e.m.; **P ≤ 0.01, ***P ≤ 0.001, n.s. = non-significant, Student's t-test). Pre-treatment of the input samples with RNAseH1 before DRIP was used as a negative control. (D) DRIP-qPCR analysis of RNA:DNA hybrid structures at selected endogenous loci under suppression of Ddx19a expression. Total nucleic acids were extracted from NIH/3T3 cells expressing the synP-mCherry reporter, the rTetR-LSD1 fusion protein and the indicated shRNAs for 10 days and used as input for the IP with the S9.6 antibody. qPCR signals are shown relative to shControl. Circles represent independent replicates (n = 3, mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.01, n.s. = non-significant, Student's t-test). (E) Representative result of an in vitro RNA:DNA hybrid unwinding assay using recombinant DDX19A. On the right of the image, the Cy5-labeled single strand DNA (ssDNA) and the RNA:DNA hybrid substrate are shown schematically. The RNA is colored blue, the DNA black and the Cy5 label is indicated with a red star.