Supplementary Data

Figure S1. Validation of the hTR and hTERT RT-qPCR primers. (a) Examination of the efficiencies of RT-qPCR primer pairs hTR 1-4. Each standard curve was generated by plotting the Ct values of the PCR reactions against the logarithm of the mass of Std hTR added (unit: fg) to base 10. The slopes of the standard curves are all close to-3.3, which is the theoretical value for a perfectly efficient primer pair. The function and R 2 value of each curve are shown. We also performed the template-titration experiment on cDNA prepared from HEK 293T total RNA, and found the four primer pairs were equally efficient in that context as well (data not shown). (b) Examination of the specificities of hTR RT-qPCR primer pairs 1-4. PCR products obtained with each primer pair, using cDNA prepared from HEK 293T total RNA as the template, were visualized by gel electrophoresis. Only a single band of the expected size was obtained with each primer pair tested. Lanes 1-4: RT-qPCR products obtained with primer pairs hTR 1-4; Lane 5: 50 bp DNA ladder (Promega). (c) Examination of the efficiencies of RT-qPCR primer pairs hTERT 1 and 2. Each standard curve was generated by plotting the Ct values of the PCR reactions against the logarithm of the mass of total cDNA added (unit: ng) to base 10. The slopes of the standard curves are both close to-3.3. The function and R 2 value of each curve are shown. (d) Examination of the specificities of hTERT RT-qPCR primer pairs 1 and 2. PCR products obtained with each primer pair, using cDNA prepared from HEK 293T total RNA as the template, were visualized by gel electrophoresis. Only a single band of the expected size was obtained with each primer pair tested. Lanes 1 and 4: RT-qPCR products obtained with primer pairs hTERT 1 and 2, respectively; Lane 2 and 3: 50 bp DNA ladder (Promega). Figure S2. Analysis of hTERT and telomerase activity in the hTERT IP samples HEK 293T and HeLa cell lysates were prepared in CHAPS lysis buffer as described in Materials and Methods. After the first round of hTERT IP, the flow through sample was used as the input sample for the second round of hTERT IP. (a) Upper panels: Telomerase activity in the original input, first round flow through (second round input) and second round flow through samples was examined by a direct assay …

. Analysis of hTERT and telomerase activity in the hTERT IP samples HEK 293T and HeLa cell lysates were prepared in CHAPS lysis buffer as described in Materials and Methods. After the first round of hTERT IP, the flow through sample was used as the input sample for the second round of hTERT IP. (a) Upper panels: Telomerase activity in the original input, first round flow through (second round input) and second round flow through samples was examined by a direct assay (1 and 2, Duplicate repeats of experiment. +2 and +4, size markers made by extending the DNA primer by two or four nucleotides. LC, labelled unextended primer, serving as a loading control). Lower panels: Western blot with Abcam ab32020 was performed on these samples with β-actin as an internal control. (b) Summary of Abcam ab32020 western signals and telomerase activity levels present in the two rounds of flow through fractions, as normalized to input levels. Note that Abcam ab32020 western signal is not depleted although more than 70% of the telomerase activity is depleted after the two rounds of IP (compare 2nd round flow through with input). S-methionine counts per unit of hTERT was plotted against the amount of cold methionine added in Lane 4-7. As shown in Lane 1, if hTERT plasmid was omitted, little radioactivity was detected by SDS-PAGE; the same result was obtained from liquid scintillation counting (data not shown). Lanes 2-7 suggest that the RRLs contained pre-existing methionine. However, the pre-existing methionine concentration is negligible compared to the exogenously supplemented concentration, since the ensuing ratio of 35 S signal present in the Std hTERT protein (measured by SDS-PAGE) to the western blot signal of hTERT was inversely proportional to the amount of cold methionine added. (b) Assuming all the western signals detected by Abcam ab32020 in the cell lysate samples are from endogenous hTERT, hTERT protein levels in HEK 293T and HeLa cells were quantified by western blot. The number of hTERT molecules in the lysates was calculated by comparing the western blot signals obtained from these samples to that from a known number of Std hTERT molecules.

Quantification of endogenous hTR levels through RT-qPCR
Take the quantification of the hTR level in HEK 293T cells using RT-qPCR with primer pair hTR 1 ( Figure 1a) as an example. Total RNA extracted from ~2140 HEK 293T cells was mixed with 0, 1, 2, 3, 4, 5 or 6 million Std hTR molecules. RT-qPCR was performed on cDNA samples prepared from these mixtures with the primer pair hTR 1 as well as an internal control primer pair for GAPDH. Based on the Ct values, the ratio of Std hTR/endogenous hTR in each sample was calculated with the following formula: In sample n, to which n million Std hTR molecules were added,

Std hTR
Endogenous hTR Ct0hTR 1: Ct value of the PCR reaction with the primer pair hTR 1 and cDNA from the sample 0; Ct0GAPDH: Ct value of the PCR reaction with the primer pair GAPDH and cDNA from the sample 0; CtnhTR 1: Ct value of the PCR reaction with the primer pair hTR 1 and cDNA from the sample n; CtnGAPDH: Ct value of the PCR reaction with the primer pair GAPDH and cDNA from the sample n.
Then the calculated ratio of Std hTR to endogenous hTR was plotted against the amount of Std hTR added to make a standard curve, as shown in Figure 1a. In this particular case, the function of the standard curve is y = 0.6336x + 0.0051. When y = 1, x is calculated to be 1.57, which means the amount of endogenous hTR molecules present in each sample is ~1.57 million. Dividing this number by the number of cells from which the total RNA was extracted (2140 in this case), the average hTR copy number per cell is estimated to be ~730 in this experiment. By liquid scintillation counting, we measured the 35 S signal in the original reaction system and in the beads slurry to be 464373 cpm/μl and 14985 cpm/μl, respectively. Therefore, the ratio of methionine present in each μl of the beads slurry to that added to the reaction system at the beginning was calculated:
The total amount of methionine added to the system is calculated by adding the amount of cold methionine and the amount of hot methionine together.
As SDS-PAGE analysis indicates ~93% of the 35 S signal in the beads slurry is present in the protein band (Figure 3a), and there are 13 methionine amino acids in each 3×FLAG-hTERT protein, the molecule number of hTERT protein present in each μl of the beads slurry should be: (3.9×10 11 ) × 93%/13 = 2.8×10 10 .
3. Quantification of the specific activity of telomerase First, we determined the 32 P signal present in the band of the 18-mer LC. By liquid scintillation counting, we measured that 3063 cpm radioactivity was present in the LC sample we added to each reaction system. Examination of the LC sample on the 10% polyacrylamide/7 M urea/1× TBE denaturing gel ( Figure S5a) indicated that ~90% of the 32 P signal was in the band of the 18-mer, which corresponded to 2746 cpm. We then determined the ratio of the 32 P signal present in the extension products to the 32 P signal present in the 18-mer band on the polyacrylamide gel. Taking replicate 1 of the endogenous telomerase elution in Figure 5b as an example, the ratio was ~1.7, so the 32 P signal present in the extension products was calculated: 2746 cpm × 1.7 = 4668 cpm.
The total 32 P signal in the [α-32 P]-dGTP added to the assay was measured by liquid scintillation counting, giving ~8.9×10 7 cpm. Thus, the ratio of dGTP incorporated in the extension products to the total dGTP added should be: 4668 cpm / 8.9×10 7 cpm = 5.2×10 -5 .
As the sequence of the extension products is the repetition of "GGGTTA", only half of which is G, the amount of total deoxyribonucleotides incorporated equals: 2.7×10 -14 mol × 2 = 5.4×10 -14 mol.
By northern blot, the amount of hTR in the elution sample added to this direct assay is 2.29×10 -17 mol ( Figure 5a), which is also the amount of telomerase monomers added to this assay. Therefore, the specific activity of telomerase quantified by this experiment is: 5.4×10 -14 mol nucleotides / (2.29×10 -17 mol telomerase monomers × 120 min) = 19.3 nucleotides incorporated per telomerase monomer per min.