Thanasoula et al. - Fig. S1

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1 S HK1si G1 Thanasoula et al. - Fig. S1 G2/M HK2si hours after double thymidine block release Figure S1. U2OS synchronous cell cycle progression. U2OS cells transfected with, POT1, HK1, HK2 or control GFP sirns were grown for 48 hours before synchronization by double thymidine block and release. ells collected at the indicated times after release were processed by FS analyses of N content. Similar patterns of cell cycle distribution were obtained in at least two independent experiments.

2 hanges in POT1 mrn relative to 18S mrn P<0.001 Thanasoula et al. - Fig. S2 sirn: GFP POT1 GFP POT1 * S317 HK1 T68 HK2 S15 p53 +MSO +caffeine +caffeine +MSO fraction of phospho H3-positive cells relative to control Figure S2.. Real-time RT-PR analysis of sirn-mediated POT1 depletion. U2OS cells transfected with POT1 or control GFP sirn were grown for 24 hours. mrn was isolated and the levels of POT1 transcript were determined using real-time RT-PR. The P value was calculated using an unpaired two-tailed t test.. affeine treatment abrogates the G2/M checkpoint response to telomeres uncapped through sirn-mediated inhibition of or POT1. U2OS cells transfected with, POT1 or control GFP sirns were grown for 48 hours before synchronization by double thymidine block and release in fresh media for 10 hours. affeine or solvent (MSO) were added to the media 4 hours prior to collection. ell extracts were prepared and immunoblotted as shown.. U2OS cells treated as in () were stained with propidium iodide and an antibody against phosphorylated histone H3-Ser 10 and analysed by flow cytometry. n=10,000 cells were analyzed for each sample. Error bars represent S of two independent experiments. P values were calculated using an unpaired two-tailed t test.

3 WI38 (V13), colcemid-treated Thanasoula et al. - Fig. S3 p53 S20 p53 S15 NS NS MSO UN-01 Go p53 wt vector P< fraction of phospho H3-positive cells relative to control U2OS+ Go6976 UN-01 P<0.001 MSO sirn: U2OS, hours after thymidine block release 5 h (S) GFP POT1 10 h (G2/M) GFP POT1 fraction of phospho H3-positive cells relative to control Figure S3.. Ser15 residue of p53, a target for TM/TR-dependent phosphorylation, is required to prevent mitotic entry of WI38(V13) human cells with uncapped telomeres. WI38(V13) cells were transfected with p53-encoding constructs or vector alone, 24h after treatment with, POT1 or control GFP sirns. Twenty four hours later cells were treated with colcemid for 3 hours, stained with propidium iodide and an antibody against phosphorylated histone H3-Ser 10 and analysed by flow cytometry. N=10,000 cells were analyzed for each sample. Error bars represent S of at least two independent experiments. P values were calculated using an unpaired two-tailed t test.. HK1 chemical inhibition rescues the G2/M arrest in POT1-depleted cells. U2OS cells transfected with POT1 or control GFP sirns were grown for 48 hours before synchronization by double thymidine block and release in fresh media for 10 hours. UN-01 and Go6976 inhibitors, or MSO control were added to the media 4 hours before collection. ells were stained with propidium iodide and an antibody against phosphorylated histone H3-Ser 10 and analysed by flow cytometry. N=10,000 cells were analyzed for each sample. Error bars represent S of two independent experiments. P values were calculated using an unpaired two-tailed t test.. U2OS cells were synchronized by double thymidine block and release in fresh media for 10 hours. UN-01 and Go6976 inhibitors, or MSO control were added to the media 4 hours before collection. ell extracts were immunoblotted as shown.. Telomere damage induced by depletion leads to 25 reduction in G2/M and 25/ reduction in S. 25 is not downregulated by exposure to IR either in S or G2/M. U2OS cells transfected with control GFP, or POT1 sirns were grown for 48 hours before synchronization by double thymidine block and release. Extracts prepared at the indicated times after release were immunoblotted as shown. Tubulin was used as a loading control. s controls, U2OS cells at 5 or 10 hours after release from double thymidine block were exposed to 10 Gy of IR, allowed to recover for 2 hours and collected for cell extract preparation.

4 HT116, 10 hours after thymidine block release MG132: sirn: GFP POT1 GFP POT1 * 25 - Thanasoula et al. - Fig. S4 HT116+Myc-Ub, 10 hours after thymidine block release, + MG132 Input sirn: GFP POT1 25 IP: IgG anti-25 sirn:gfp POT1GFP POT1 72 k fraction of phospho H3-positive cells relative to control MG132: esirn: control control anti-myc 55 k 25 anti-25 Figure S4.. Proteasome-mediated destruction of the 25 prevents mitotic entry in the presence of uncapped telomeres in p53-proficient HT116 cells. HT116 cells transfected with, POT1 or control GFP sirns were grown for 48 hours before synchronization by double thymidine block and release in fresh media for 10 hours. Proteasome inhibitor (MG132) was added to the media 3 hours prior to collection. ell extracts were prepared and immunoblotted as shown. Extracts from cells exposed to 10 Gy of IR were used as a control. *, non-specific band.. HT116 cells treated as in. were stained with propidium iodide and an antibody against phosphorylated histone H3-Ser 10 and analysed by flow cytometry. N=10,000 cells were analyzed for each sample. Error bars represent S of three independent experiments. P values were calculated using an unpaired two-tailed t test.. HT116 cells transfected with esirn for (Sigma) or control were processed as in.. or POT1 inhibition triggers 25 ubiquitylation in HT116 cells. HT116 cells were treated with, POT1 or control GFP sirns for 24 hours before transfection with Myc-ubiquitin expressing construct. Twenty four hours later cells were synchronized by double thymidine block and release in fresh media for 10 hours. Proteasome inhibitor (MG132) was added to the media 3 hours prior to collection. ell extracts used as input and proteins immunoprecipitated with IgG control or anti-25 antibody were immunoblotted as shown.

5 sirn: GFP POT1 R51 GFP POT1 R51 R51si Thanasoula et al. - Fig. S5 R k R51si POT1+HK2si +HK2si POT1+HK1si +HK1si HK2si HK1si +colcemid +colcemid Figure S5.. hronic N damage induced by R51 depletion does not lead to 25 degradation in G2/M and the ensuing mitotic entry block is not rescued by proteasome inhibition. U2OS cells transfected with indicated sirns for 48 hours were synchronized by double-thymidine block and released in fresh media for 10 hours. Proteasome inhibitor (MG132) was added to the media 3 hours prior to collection. s controls, U2OS cells at 10 hours after release from double thymidine block were exposed to 10 Gy of IR, allowed to recover for 2 hours and collected for cell extract preparation.. U2OS cells treated as in. were stained with propidium iodide and an antibody against phosphorylated histone H3-Ser 10 and analysed by flow cytometry. N=10,000 cells were analyzed for each sample. Error bars represent S of three independent experiments.. bsolute values for the flow cytometry analyses of percentages of phosphorylated histone H3-Ser 10 stained cells shown in Fig. 4 and Fig. 5.