Fig 2: PES1 forms an active complex with telomerase.(A) MCF7 cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag. The immune complexes were eluted with Flag peptide and re-IP using anti-Myc or normal IgG. The resulting precipitates were used for detection of Flag-hTERT and Myc-PES1 expression by immunoblot. hTR levels were determined by RT-PCR. (B) MCF7 cell extracts were fractionated on Superose 6 size exclusion columns. Fast protein liquid chromatography (FPLC) chromatographic elution profiles are indicated. The elution positions of calibration proteins with known molecular masses (kDa) are shown by arrows, and an equal volume from each chromatographic fraction was used for determination of protein expression by immunoblot with the indicated antibodies. To increase the specificity of hTERT detection, cells were immunoprecipitated with anti-hTERT from Abbexa, followed by immunoblot with anti-hTERT from Abcam (hTERT after pre-purification). hTR levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and telomerase activity by telomerase repeat amplification protocol (TRAP). CHAPS buffer was used as a negative control for TRAP assay. IC, internal control; mAu, milli-Absorbance unit. Red frame indicates a potential complex with hTERT/hTR/DKC1/PES1. (C) MCF7 and U2OS cells were transfected with Flag-tagged PES1 or hTERT or empty vector, and immunoprecipitated with anti-Flag agarose. The resulting precipitates were used for assessment of Flag-PES1 or Flag-hTERT expression by immunoblot. hTR levels were determined by RT-PCR. β-Actin was used as a negative control for hTR determination. (D) MCF7-Flag-PES1+shPES1 cells or control (MCF7-Flag+shCon) cells were transfected with control small interfering RNA (siRNA) or hTERT siRNA as indicated and were analyzed as in (C). (E) Biotinylated hTR was incubated with purified GST-hTERT, GST-PES1, or GST-hTERT plus GST-PES1. The resulting complexes were subject to RNA pull-down assay. The amount of biotin-hTR was detected by the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).

Fig 3: PES1 correlates with telomerase activity and cellular senescence in breast cancer samples.(A) Representative immunohistochemical staining of PES1, telomerase activity, and telomere length in 43 human breast cancer samples. Scale bars, 100 μm. Correlation of PES1 levels with relative telomerase activity and telomere length was generated by Mann-Whitney test. (B) Representative immunohistochemical staining of PES1 and SA-β-gal in frozen breast cancer tissue sections from (A). Scale bars, 100 μm. Correlation between PES1 expression and SA-β-gal staining was generated by Fisher’s exact test. (C) A proposed model underlying the role of PES1 in regulation of telomerase and senescence. Telomerase minimally comprises TERT and TR. When PES1 expression levels are elevated in some cases (e.g., cancer), it facilitates assembly of TERT and TR by forming a complex with TERT and TR, resulting in high telomerase activity, long telomere, and inhibition of senescence. PES1 depletion has opposite effects.

Fig 4: PES1 enhances assembly of TERT and TR.(A) PES1 WT or KO MCF7 cells (left) and MEFs (right) were transiently transfected with the indicated plasmids and immunoprecipitated with anti-Flag. The precipitates were used for assessment of Flag-h/mTERT, PES1, and β-actin expression by immunoblot. TR levels were determined by RT-PCR. Flag-h/mTERT, Flag-tagged human or mouse TERT. Flag-hTERT was used for MCF7 cells, and Flag-mTERT was used for MEFs. (B) siRNA-mediated PES1 KD MCF7 or HepG2 cells were transfected with Flag-hTERT and siRNA-resistant Myc-tagged PES1, PES1 (ΔBRCT), or PES1 (W397R) as indicated and were analyzed as in (A). (C) siRNA-mediated PES1 KD MEFs were transfected with Flag-mTERT and siRNA-resistant Myc-tagged PES1, PES1 (ΔBRCT), or PES1 (W397R) as indicated and were analyzed as in (A). Data shown are mean ± SD of three independent experiments (A to C). **P < 0.01. (D) Biotinylated hTR was incubated with purified GST-hTERT and WT or mutant His-PES1. The resulting complexes were subject to RNA pull-down assay. Unlabeled hTR was used as a negative control. Asterisks indicate the positions of the expected full-length fusion proteins.

Fig 5: PES1 directly interacts with hTERT.(A) Cell extracts from MCF7 cells stably expressing Flag (control) or Flag-tagged hTERT (Flag-hTERT) were immunopurified with anti-Flag affinity gel, and the purified complex was subject to SDS–polyacrylamide gel electrophoresis (PAGE) and silver-stained. The differential protein bands were retrieved and analyzed by MS. n, number of peptides identified by MS analysis; MM, molecular mass. (B) HEK293T cells transiently transfected with Myc-tagged hTERT (Myc-hTERT) and Flag-PES1 or Flag-DKC1 were treated with or without RNase A (0.1 mg/ml), and immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with the indicated antibodies. (C) MCF7 cell lysates were immunoprecipitated with anti-hTERT from Abbexa and immunoblotted with anti-hTERT from Abcam and anti-PES1. IgG, immunoglobulin G. (D) Glutathione-Sepharose beads bound with GST-hTERT were incubated with purified His-tagged wild-type (WT) or mutated PES1. After washing the beads, the bound proteins were subjected to SDS-PAGE and immunoblotted with anti-GST antibody. Also shown on top of the graph is a schematic diagram of the PES1 protein, illustrating the location of the BRCT domain. Asterisks indicate the positions of the expected full-length fusion proteins. (E) Glutathione-Sepharose beads bound with WT or truncated hTERT were incubated with purified His-tagged PES1. After washing the beads, the bound proteins were examined by immunoblot with anti-His. Schematic diagram of the hTERT deletion constructs used is shown at the top. Asterisks indicate the positions of the expected full-length fusion proteins. TEN, telomerase N-terminal; TRBD, TR-binding domain; RT, reverse transcriptase; CTE, C-terminal extension.