Fig. 3. Preventing TIMELESS dissociation suppresses the slow fork
response and leads to genomic instability. (A) Western blots of iPOND
samples prepared as in Fig. 1A from cells treated with siRNAs and HU as
indicated. (B) QIBC of TIMELESS chromatin loading in cells treated with
siRNAs and HU as indicated. (C) (Top) DNA fiber labeling protocol. (Bottom)
Replication speed in cells treated as indicated (n = 200 fibers for each
condition). (D) Quantification of g-H2AX in PCNA-positive cells. Data were
derived from QIBC analysis of cells treated with siRNAs as indicated; exposed to
HU for 30 min; and stained for DAPI, g-H2AX, and PCNA. (E) (Top) Examples
of ultrafine anaphase bridges (UFBs) marked by Bloom’s syndrome helicase
(BLM) (red) between anaphase chromosomes counterstained by DAPI (blue).
(Bottom) Quantification of UFBs in cells treated with the indicated siRNAs
(mean ± range; n = 2 technical replicates). (F) (Left) QIBC of 53BP1 nuclear
bodies (NBs) in cells treated with the indicated siRNA and counterstained for
cyclin A and DAPI to stratify cell cycle progression (n = 10,000 cells for each
condition; colors indicate the number of 53BP1 nuclear bodies per nucleus).
(Right) Quantification of 53BP1 nuclear bodies in cells treated as indicated;
inset shows examples of G1 cells (cyclin A negative) with 53BP1 nuclear bodies.