Fig. 2. TIMELESS and oligomeric PRDX2 form a replisome-based redox
sensor. (A) Analysis of intracellular ROS (superoxide plus hydroxyl radicals)
by flow cytometry after the indicated treatments, and with or without
pretreatment with NAC. Aph, aphidicolin. (B) (Left) DNA fiber labeling protocol.
(Right) Replication speed in cells exposed to the indicated treatments with
or without NAC (n = 200 fibers for each condition). (C) Reciprocal FLAG
coimmunoprecipitation (FLAG-IP) followed by Western blotting of extracts
from U2OS cells or its derivatives stably expressing FLAG-tagged TIMELESS or
PRDX2 as indicated. (D) (Left) Representative images. (Right) Quantification
of the sum of PLA focus intensity per cell nucleus obtained with the indicated
antibodies. Scale bars, 10 mm. (E) Western blots of purified cellular fractions
under reducing (left) and nonreducing (right) conditions (Cyt, cytoplasm;
Nuc, nucleus; Chr, chromatin; n, number of PRDX2 monomers in a given
higher-order assembly). (F) (Top) Western blot of purified cellular fractions
under nonreducing conditions from cells exposed to the indicated treatments.
(Bottom) Bar graph representing the degree of chromatin-bound higher
oligomeric PRDX2 (n4) in the indicated conditions [mean ± SEM (error bars);
n = 4 technical replicates]. (G) Western blots of coimmunoprecipitated
FLAG-TIMELESS under nondenaturing conditions from whole-cell lysates from
cells exposed to the indicated treatments.