Supplementary Figure 6: MCM2 binding is not required for incorporation of H3.1–H4 into chromatin but is important for stability of H3.1–H4.

(a) Representative images of newly synthesized TMR* labelled SNAP-HA-H3.1 wt and R63A K64A as described in Fig. 3b. Scale bar, 50 μm. (b) Analysis of total chromatin-bound and soluble SNAP-HA-H3.1 in stable cell lines used in Fig. 3b&c. Total SNAP-HA-H3.1 wt or R63A K64A were measured by TMR* labelling and direct fixation. The dot-plot showed median TMR* intensities from 3 independent experiments each including 5 technical replicates of total > 5,000 TMR* positive cells. Error bars, s.d. n=3. Unpaired t-test: *, P < 0.05. (c) SNAP-HA-H3.1 mRNA levels in stable cell lines used to measure the histone stability in Fig. 3c. Expression of e-H3 mRNA levels was measured by qPCR using two different primer sets, normalized to GAPDH and shown relative to wt. Error bars, s.d n=3 (for primer-set 2 wt, n=2). (d , e) Immunofluorescence of U-2-OS cells conditionally expressing Flag-HA-MCM2 WT or Y81A Y90A mutant. Cells were induced by tetracyclin (tet) for 24 hours and fixed directly (d) or pre-extracted (e) by CSK-T. HA staining confirmed induction of FLAG-HA-MCM2 in non-extracted cells (d) and showed typical cell cycle dependent MCM2 staining patterns in pre-extracted cells (e). Representative images of typical G1, early S and mid S phase were shown. Note: because MCM2-7 is unloaded as S phase progresses, late S and G2 cells are negative for MCM2. Flag-HA-MCM2 patterns were scored in 3 independent experiments (n=3), error-bars represent standard deviation, > 200 HA positive cells were scored per experiment. Scale bars, 20 μm (d) and 5 μm (e).