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. 2022 Jan 28;5(5):e202101088. doi: 10.26508/lsa.202101088

Figure S10. Analysis of several stable U2OS clones expressing Treslin/TICRR-ΔCIT or various C-terminal truncation mutants.

Figure S10.

(A) Schematic giving an overview over the Treslin/TICRR mutants used in this figure. (B, C, D) Immunoblots (i) to assess transgene expression levels and BrdU-flow cytometry (ii) and propidium iodide profiles (iii) to determine overall DNA replication of the indicated Treslin/TICRR mutants shown in (A). The following U2OS clones were used for main Fig 4: Treslin/TICRR-ΔCIT-5, ΔC853-5. Immunoblots of whole cell lysates used mouse anti-GFP and Ponceau staining (as a loading control). Flow cytometry was done after replacing endogenous Treslin/TICRR against the indicated siRNA-resistant transgenes using RNAi. Density plots are shown. Parental U2OS cells and a line expressing Treslin/TICRR-WT served to control the experiment. Dashed lines show BrdU peak level of the respective control siRNA-treated cell line in the same experiment. Clones were picked that expressed the Treslin/TICRR transgenes at similar or higher levels than Treslin/TICRR-WT to avoid under-estimating the capability of the mutants to support replication. For Treslin/TICRR-ΔC651, only low-expressing clones were found. The results are still conclusive, though, because all clones were capable to support replication. (E) Quantification of overall replication in mutant Treslin/TICRR U2OS cell lines described in (A, B, C, D), based on BrdU-propidium iodide flow cytometry experiments as described in (B, C, D). For comparison, the Treslin/TICRR-core clones are shown in addition to the usual control lines. The quantifications indicate that ΔCIT, ΔC651, and ΔC853 mutants were active. It also shows the clonal variability that did not clearly correlate with expression levels, as indicated by the Treslin/TICRR-ΔC651 clones 1–3. Error bars: SEM; sample numbers (n): 8 (none; WT), 5 (ΔCIT-5; ΔC853-5), 4(ΔCIT-7); 3 (ΔC651-2; ΔC651-3; ΔC651-38; ΔC853-29, core-35; core-41); 2 (ΔC853-13); significance tests: parametric, unpaired, two tailed t test, *P ≤ 0.05.