Figure S3. Treslin/TICRR-ΔSTD and Treslin/TICRR-core expressing cells progress slower through S-phase.

(A) Stable U2OS cell lines expressing no transgene or siTreslin-resistant GFP-Flag-Treslin/TICRR-WT, 2PM, ΔSTD, or core were arrested in a double thymidine block and treated with siTreslin or siCtr 8 h after release from the first thymidine arrest, so that the siRNA would take effect only after the genome had been replicated. After release for 0, 6, or 10 h from the second thymidine block, cells were analysed by propidium iodide flow cytometry. Histograms show overlays between the samples for the three time points and the gates used to calculate amount of cells in the early S phase and the late S phase, respectively. Propidium iodide histograms show relative cell count. Clones Treslin/TICRR-ΔSTD-11 and Treslin/TICRR-core-35 were used. (B) Quantification of number of cells in early S-phase or late S-phase, 0 and 10 h after a double thymidine release of samples described in (A), using the gates show in (A). At 0 h after release, all samples show around 70% of cells in early the S phase, consistent with similar synchronisation by the double thymidine arrest. 10 h after release, U2OS cells treated with siCtr and Treslin/TICRR-WT expressing cells treated with siTreslin show almost 60% of cells had progressed to late S-phase, with 10–20% remaining in the early S phase. In contrast, only around 30% U2OS cells treated with siTreslin and cells expressing Treslin/TICRR-2PM, ΔSTD, or core had progressed to the late S phase by 10 h. This shows that Treslin/TICRR-ΔSTD or core expressing cells replicated at similar rates as cells expressing the inactive Treslin/TICRR-2PM mutant or cells lacking Treslin/TICRR, indicating that Treslin/TICRR-ΔSTD and core do not support normal S-phase replication. (C, E) Chromatin of cells shown in (A) was isolated for immunoblotting with goat anti-Mcm2 and mouse anti-PCNA antibodies. Coomassie (Coom.) staining of low molecular weight part including histones controlled for loading. Samples shown in (C, E) are the same shown in (A) and were processed in parallel. 10 h after double thymidine release, U2OS cells treated with siCtr and Treslin/TICRR-WT expressing cells treated with siTreslin show that pre-RCs became largely cleared from chromatin, and replisomes (PCNA on chromatin) were also severely decreased, consistent with genome replication being nearly complete at 10 h. In contrast, in U2OS cells treated with siTreslin and in cells expressing Treslin/TICRR-2PM, ΔSTD, or core, pre-RCs and replisomes were cleared from chromatin at much slower rates, consistent with slow replication. (D) Quantification of Mcm2 (i) and PCNA (ii) signals of immunoblots shown in (C). (F) Quantification of Mcm2 (i) and PCNA (ii) signals of immunoblots shown in (E).