INM localized Scs2-Siz2 is required for telomere tethering during mitosis and G1-phase of the cell cycle. (A–C) NE tethering of Tel6R in WT and siz2^S522A-V5₃ cells (A), as well as NE tethering of Tel14L in WT (B and C), ulp1^K352E/Y583H-V5₃, ulp1^K352E/Y583H-V5₃ siz2^S522A-V5₃ (B), yku70Δ, yku80Δ, and sir4Δ cells (C) were examined as described in Fig. 6. (D) Shown are epifluorescence images of WT cells producing Sir4-GFP and NE/ER–localized Sur4-mCherry. Merged images were used to assess the relative position of Sir4-GFP foci with respect to the NE (identified by Sur4-mCherry). Images were rendered using the unsharp mask filter in ImageJ. Bar, 2 µm. Bar graphs show the percentage of total Sir4-GFP foci at the NE (middle panel) and the average number of Sir4-GFP foci per nucleus (right panel) in WT and siz2Δ cells at the indicated cell cycle stage. Note, cells in anaphase/telophase show double the number of Sir4-GFP foci as seen in G1- and S-phase cells. Graphs in A–D represent data from at least three biological replicates. n = 50 cells/replicate/cell cycle stage. Error bars represent SD. Asterisks: significant difference relative to WT using a two-tailed Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (E) Sir4-PrA binding to chromatin adjacent to Tel6R was assessed by ChIP and qPCR analysis using asynchronous cultures of WT, ulp1^K352E/Y583H-V5₃, siz2^S522A-V5₃, and ulp1^K352E/Y583H-V5₃ siz2^S522A-V5₃ cells. Graphs represent at least three biological replicates. Error bars = SEM. Asterisks: significant change relative to WT using a two-tailed Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01. Ana/Telo, Anaphase/Telophase; Phos, phosphorylation.