Fig. 4
From: JMJD1B, a novel player in histone H3 and H4 processing to ensure genome stability
Histone deposition defects, G2/M cell cycle arrest, and DNA damage are induced upon siJMJD1B. a Scheme illustrating the H3.1- and H3.3-SNAP for in vivo labeling assays with red fluorescent TMR-Star in quench-chase-pulse experiments to label newly H3-SNAP synthesized during the 2 h chase. b Western blot of crude cell extracts from siLuciferase (siLuc), siJMJD1B, and siHIRA treated cells. c Box-and-whisker plot of the TMR intensity in the nucleus of HeLa cells for H3.1-SNAP (left) and H3.3-SNAP (right) histones. Whiskers indicate the range of the intensities measured. A Mann–Whitney statistical test established the significance of the difference between siLuciferase and siJMJD1B for H3.1 (p < 2.6 × 10−11) and H3.3 (p < 2.2 × 10−16), and siLuciferase and siHIRA for H3.3 (p < 2.2 × 10−16). d Cell cycle profiles of HeLa cells treated with siControl or siJMJD1B synchronized with a double 2 mM Thymidine block. The graphs show DNA profiles of cells released from the Thymidine block after 0, 2, 4, 6, 8, and 10 h. The standard deviation was obtained from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, Student’s t-test. e Immunodetection of γH2AX from siControl and siJMJD1B treated HeLa cells after 72 h. The arrow points to micronuclei. On the right, quantitation of micronuclei observed in siControl and siJMJD1B, expressed as the mean of micronuclei per cell upon counting 100 cells of each condition. The standard deviation was obtained from 4 independent countings **p < 0.01, Student’s t-test