Mild catalytic defects of tert rs61748181 polymorphism affect the clinical presentation of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a disorder of accelerated lung aging. Multiple pieces of evidence support that the aging biomarker short telomeres, which can be caused by mutations in telomerase reverse transcriptase (TERT), contribute to COPD pathogenesis. We hypothesized that short telomere risk-associated single nucleotide polymorphisms (SNPs) in TERT, while not able to drive COPD development, nonetheless modify the disease presentation. We set out to test the SNP carrying status in a longitudinal study of smokers with COPD and found that rapid decline of FEV1 in lung function was associated with the minor allele of rs61748181 (adjusted odds ratio 2.49, p = 0.038). Biochemical evaluation of ex vivo engineered human cell models revealed that primary cells expressing the minor allele of rs61748181 had suboptimal telomere length maintenance due to reduced telomerase catalytic activity, despite having comparable cell growth kinetics as WT-TERT expressing cells. This ex vivo observation translated clinically in that shorter telomeres were found in minor allele carriers in a sub-population of COPD patients with non-declining lung function, over the 5-year period of the longitudinal study. Collectively, our data suggest that functional TERT SNPs with mild catalytic defects are nonetheless implicated in the clinical presentation of COPD.

Telomere biology disorders consist of a spectrum of tissue degenerative disorders that have variable clinical manifestations and severities, but share the same disease etiology-short telomeres 1 . Telomeres are DNA-protein structures located at the ends of eukaryotic chromosomes 2 . Telomeres shorten with cell divisions and they limit the proliferation of human cells by inducing cellular senescence when a critical length is reached. Telomerase is a ribonucleoprotein complex that can copy the template within its RNA component, telomerase RNA (TER), to telomeric DNA by using its own polymerase, telomerase reverse transcriptase (TERT). De novo synthesis of telomeric repeats at chromosome ends by telomerase delays the depletion of telomeres and extends the proliferation lifespan 3 .
At the cellular level, mean telomere length is dynamically determined by the inherited starting telomere length, the rate of cell turnover, and the rate of telomere synthesis if telomerase is expressed in the cell. Individuals with the shortest telomeres (first percentile) in the general population are susceptible to a spectrum of premature aging disorders 1 , including idiopathic pulmonary fibrosis (IPF), even if they did not carry mutations in known telomere biology genes 4 . At the organismal level, telomere biology disorders, mostly caused by genetic deficiencies in telomerase function, affect tissue regenerative potential and present clinically as diverse disorders. Bone marrow is one tissue with a high proliferative demand that is particularly sensitive to short telomeres, as hematopoietic renewal failure occurs when the replicative potential and the stem cell pool of an affected individual is depleted. Consequently, bone marrow failure diseases, including dyskeratosis congenita and aplastic anemia, are frequently observed in patients with severe telomerase dysfunction.
On the other hand, the lung is a tissue with lower replicative demand but is also frequently affected by telomere dysfunction 1,5 . Patients with mutations in TERT suffer from IPF in an autosomal dominant pattern due to telomerase haploinsufficiency and the consequent short telomere-induced tissue renewal defects 6 . Furthermore, IPF is found in up to 20% of all patients with dyskeratosis congenita, the prototype and the first identified  7 . Disease presentation in the lung is driven by stem cell proliferative failure following telomere dysfunction in type 2 alveolar epithelial cells (AEC2) 8 . Chronic obstructive pulmonary disease (COPD) is a multi-factorial trait where inflammation of the peripheral airways and/or loss of elastic recoil due to the destruction of alveoli (emphysema) cause irreversible progressive airflow limitation 9 . Patients with COPD are known to have short telomere length in their peripheral blood leukocytes [10][11][12] . Notably, the discovery of rare telomerase mutations in emphysema patients was previously reported 13,14 . Stanley et al. sequenced the telomerase genes (TERT and TER) in COPD patients across two large cohorts (COPDgene and the Lung Health Study (LHS)) and discovered that 1% of patients with early-onset severe emphysema had deleterious TERT mutations 15 . The prevalence of TERT driver mutations in COPD is comparable to alpha-1 antitrypsin deficiency-the only well-characterized genetic cause of emphysema 15,16 . Based on the findings of telomere dysfunction in COPD/emphysema, it has been proposed that stem cells in the lung (i.e., AEC2) were exhausted following cellular senescence during the pathogenesis and progression of COPD 9 .
Single nucleotide polymorphisms (SNPs) in the TERT region have been linked to telomere biology disorders, including, but not limited to, IPF 17 and bone marrow failure 18,19 . Notably, a few non-coding (intronic) SNPs in the TERT gene were recently reported to associate with increased risks of COPD in Chinese and Japanese populations 20,21 . While rare, disease-causing TERT mutations precipitate the development of tissue regenerative deficiency such as emphysema 22 , in this study, we were interested in understanding whether common TERT SNP associated with mild telomere maintenance defects could modify the risk and clinical presentation of COPD.
The non-synonymous TERT SNP rs61748181 features a C > T nucleotide substitution, resulting in an amino acid change from alanine to threonine (TERT p.A279T). It is located at a linker domain of the N-terminus of TERT. Targeted deep sequencing has linked this SNP to bone marrow failure 18 , lung cancer 23 and esophageal cancer 24 . Compared to other disease-associated TERT SNPs, it has a relatively high minor allele frequency (MAF) of 3.58% 25 (Supplementary Table 1). Here, using a combination of human cell models, genetic analysis, and clinical data mining of the LHS cohort, we set out to investigate the effects of TERT SNP rs61748181 in the progression of COPD. Results rs61748181 carrying status does not affect the overall clinical progression of patients in the LHS cohort. The MAF of rs61748181 in the Caucasians from the LHS population was 3.29% (Table 1).
The locus showed a slight deviation from Hardy-Weinberg equilibrium (p = 0.040), with a higher number of homozygotes for the minor allele than expected. The deviation is in agreement with a recent report on the excess of homozygotes for rs61748181 minor T allele in COPD patients than in control subjects 26 . 500 out of 3596 samples (13.90%) from Caucasian subjects in the LHS cohort failed the genotyping assay, likely due to DNA sample degradation, as we noted that there were unremarkable differences in baseline characteristics of the demographic profile between the typed and undetermined participants (Supplementary Table 2).
We sorted the Lung Health Study participants by annualized change (i.e., the slope) of FEV1 over 5 years (ΔFEV1 5yrs ) and tested the association between rs61748181 and disease progression by a multivariate linear regression model. While age and smoking (both smoking history in pack-years and smoking status during the 5 years of the LHS study) affected disease progression, we did not observe any significant difference in ΔFEV1 5yrs between the rs61748181 minor allele carriers (CT and TT) (n = 197) and those with the CC genotype (n = 2899) (Supplementary Table 3). The lack of correlation between rs61748181 carrying status and ΔFEV1 5yrs suggested that COPD disease progression is not likely to be primarily driven by the carrier status of rs61748181. Table 1. The demographic profile of the LHS population*. *Caucasian only. ^Sustained quitters are defined as abstinence from all smoked tobacco products at all 5 years of the LHS annual follow up visits (Y1-Y5), confirmed by salivary cotinine and expired carbon monoxide data. # Intermittent quitters consist of self-report smoking history in any annual visits (Y1-Y5); and participants who failed any validation tests. Participants who did not attend a given annual visit were counted as smokers in that visit, and an intermittent quitter. † % of predicted value. www.nature.com/scientificreports/ Higher prevalence of rs61748181 carriers among rapid decliners of lung function than non-decliners in the LHS cohort. The lack of direct clinical correlation with telomerase genetic defects is not unexpected in telomere biology diseases, as physiological function and tissue renewal are protected by residual telomere length until a critical threshold for telomere dysfunction (unprotection) is reached. We reasoned that when carriers of TERT SNPs are subjected to an increased demand for telomere replenishment to maintain tissue integrity, the possible mild defects in telomere maintenance would be exacerbated. Over time, carriers of TERT SNPs should experience accelerated deterioration of existing pathological conditions, as a function of the sum total of all etiologic factors that contribute to the loss of tissue integrity, leading to accelerated progression of COPD pathologies 9,27 . Based on this model, we explored the effect of this TERT SNP in patients with extremely rapid disease progression or no progression ( Table 2). In a subset of 464 individuals (15% of the LHS population), carriers of rs61748181 TT or CT genotypes were more prevalent among rapid decliners (7.76%) than non-decliners (3.02%), with an adjusted OR of 2.49 (Table 3). In agreement with our model, restricting the comparator populations to patients with extremely rapid disease progression and no progression allowed us to tease out the accumulating biological effect of this TERT SNP. rs61748181 in TERT can lead to compromised telomere length maintenance secondary to reduced telomerase activity. Next, we applied an ex vivo human cell model to assess the telomere maintenance capacity of the minor alleles of rs61748181 and two other TERT variants 28 . In addition to rs61748181, we include the telomere biology disorder-associated TERT SNP rs377639087, which features the deletion of codon 441 (TERT p.∆E441) and serves as a known telomerase activity defective control. rs112614087 (TERT p.A615T), which has a heterozygosity of 0.500 29 and therefore is deemed unlikely to change TERT function, was chosen as the variant with normal telomerase activity control. The details of the TERT SNPs involved in the biochemical study are summarized in Supplementary Table 1.
We used site-directed mutagenesis to introduce the sequences encoded by the minor alleles of the SNPs into the TERT open reading frame (Supplementary Table 4). Like other human fibroblasts, the human foreskin fibroblast BJ cell line silenced its endogenous TERT expression, whereas its TER expression is intact. Stable genomic integration of the coding sequences of TERT (WT and the SNP variants) through infection with retrovirus vectors (pBabe-Hygro-flag3B-TERT) in BJ cells restored telomerase activity. An examination of protein expression revealed that TERT protein levels in these reconstituted cell lines were comparable to each other (Fig. 1A). The population growth kinetics of the TERT-expressing BJ cell lines followed typical logarithmic curves (Fig. 1B). While the BJ vector control cell line (BJ-vector), which does not express telomerase activity, entered replicative senescence following 10 PDLs post-antibiotic selection, all BJ fibroblasts expressing recombinant telomerase (WT/SNP variants) were proliferating continuously in our ex vivo cell model system. Using this ex vivo system, we found the minor allele of SNP rs61748181, when translated into a variant version of TERT (expressed as TERT variant hereafter), was deficient in telomere length maintenance (Fig. 1C,D) while its TERT protein expressions were comparable (Fig. 1A) and growth kinetics remained the same as WT-TERT up to 50-60 PDLs (Fig. 1B). Table 2. Characteristics of the study participants in the rapid and non-decliner groups. *Rapid vs Nondecliners (Fisher's exact test and two-tailed t test). Statistical analyses were done using the JMP Statistics software package version 13 (SAS Institute Inc.) and R version 3.5.1.

Parameters
Rapid decliners (N = 232) Non-decliners (N = 232) p value* www.nature.com/scientificreports/ To understand the mechanism behind defective telomere length maintenance by TERT variants, we applied the primer extension assay to check single nucleotide incorporation (nucleotide addition processivity, NAP) and repeat synthesis (repeat addition processivity, RAP) in WT-TERT and TERT SNP variant cell lines. The RAP activity of the rs61748181-TERT was 94.4 ± 5.5% compared to the WT-TERT (n = 6, p = 0.3313) and its NAP activity was 81.1 ± 7.3% of WT (n = 6, p = 0.0323) ( Supplementary Fig. 1). The RAP and NAP activity of the telomerase activity defective control rs377639087-TERT was 99.41 ± 6.48% (p = 0.929) and 71.16 ± 11.22% (p = 0.022), respectively. We concluded that the TERT protein encoded by rs61748181 had similar repeat addition capacity as WT-TERT, but reduced nucleotide addition processivity. This minor difference in the polymerase's processivity translated to the accumulated defects in telomere repeat synthesis overtime, as observed in the cell culture experiments.

N(%)
Differences in median telomere length between CC homozygotes and rs61748181 minor allele carriers are evident in subgroups of LHS patients with extreme telomere synthesis demands. Considering the telomere maintenance defects observed in fibroblasts expressing the minor T allele of rs61748181 over the long term in our cell model, patients carrying the minor allele of rs61748181 should be at risk of increased telomere loss regardless of their COPD progression. We reasoned that we should be able to observe telomere length differences between CC homozygotes and rs61748181 minor allele carriers (genotypes CT and TT) in the LHS population. To test this, we investigated the association between rs61748181 minor www.nature.com/scientificreports/ allele carrying status and telomere lengths in 3050 genotyped Caucasian patients from the LHS with measurable telomeres. Our analyses did not reveal any statistical differences in median telomere length between rs61748181 minor allele carriers and CC homozygotes ( Fig. 2A). Notably, the LHS cohort showed non-normal distribution of telomere length with several participants harboring leukocytes telomere measurements up to forty times longer than the median (Fig. 2B); perhaps not surprisingly, as it is well-known that telomere length regulation is multi-factorial, based on genetic, lifestyle and environmental influences. We reasoned that the minor telomerase catalysis defects conferred by this SNP would be most evident in the subpopulation of patients with the highest demand for optimal telomere synthesis. Next, we accessed the leukocyte telomere length after dividing the cohort into four groups based on changes in their lung function over 5 years (ΔFEV1 5yrs ). In each group, the leukocyte telomere length for the rs61748181 minor allele carriers was compared to their CC counterparts. In the fourth quartile (Fig. 2C), consisting of patients with no observable decline in their lung function over 5 years monitoring period (positive ΔFEV1 5yrs ), carriers of rs6178181 minor allele (n = 43) had shorter leukocyte telomere lengths than CC patients (n = 719). This difference in telomere lengths based on SNP carrying status was statistically significant (p = 0.03). Similarly, rs6178181 minor allele carriers (n = 45) in the first quartile (where patients had the fastest disease progression and most negative ΔFEV1 5yr values) tended to have shorter leukocyte telomere lengths than CC patients (n = 717), though statistical significance was not reached. The difference in telomere length between rs6178181 minor allele carriers and CC individuals diminished in the middle subgroups (Fig. 2C), which may explain the lack of correlation between rs61748181 carrier status with telomere length, when the entire LHS cohort was considered ( Fig. 2A).

Discussion
We investigated the biological and clinical effects of a TERT SNP rs61748181 which has previously been linked to telomere biology disorders 18 . With clinical samples collected in the LHS, we revealed that carrier status of the SNP rs61748181 aggravated existing disease conditions in COPD patients. Using a combination of molecular and cell biology tools in the laboratory, we further demonstrated that the non-synonymous SNP rs61748181 could lead to defective telomerase catalytic activity and deficient telomere length maintenance. Regression analysis showed that the association was independent of known risk factors such as aging and smoking history. These results implicated TERT as a biologically plausible candidate gene for lung function decline and COPD pathogenesis. Not only did our results agree with the previous observation that deleterious mutations in TERT contribute to COPD pathogenesis 15 , we also extended this observation to demonstrate that common variants in TERT with low penetrance may nonetheless modify disease presentations. Our ex vivo results demonstrated in a laboratory setting that, when other confounding factors for telomere attrition were fixed, fibroblasts with recombinant expression of the disease-associating TERT variants were predetermined to suboptimal telomere maintenance. However, deficient telomere length maintenance in this context did not translate to a loss of proliferative capacity, as BJ cells expressing the minor allele of rs61748181 were kept in culture for up to 50-60 PDLs, the limit of fibroblast divisions during a lifetime 30 , and demonstrated the same growth rate as WT-TERT expressing cells. To our knowledge, this is the first study to investigate the long-term effects of common TERT variants on telomeres in primary cell lines. Our results confirmed that the short telomere risk-associated SNP rs61748181 encoded a TERT variant that was deficient in telomere length maintenance. We provided strong experimental evidence to support the previous clinical observations that in subjects with bone marrow failure, carriers (heterozygous and homozygous) of this SNP had shorter telomeres than their age-matched peers 18 . These biochemical results are also supported by reports in different cellular and experimental systems 24,31 .
This is the first study both to show the increased risk of rapid lung function decline in COPD patients carrying rs61748181 minor allele and to investigate the association of this SNP with short leukocyte telomeres in patient subsets from in a large cohort study of COPD. The current model of cellular senescence-based COPD pathogenicity 9 stipulates that the carrier's stem cell compartments would remain proliferative until their telomeres were too short and trigger stem cell exhaustion, which finally presented as rapid decline and poor lung performance at the end stage of the study. Telomeres shorten at similar rates in blood and somatic tissues 32 . Despite variation, telomere lengths correlate among most human tissue types, including lung tissues 33,34 . Notably, it is recently reported that carriers with rare variants in TERT, though not showing telomere biology disorders, have shorter telomere length across all tissues 34 . Therefore, our measurement of leukocyte telomere lengths in COPD patients should be representative of the lengths in their lung tissues.
We also considered the potential influence from a collider effect in our observed correlation between the rs61748181 minor allele carrier status with short leukocyte telomere length. For the collider effect to be true, two variables (telomere length and carrier status of rs61748181) are independently associated with an observation (COPD disease progression, in this case), but they should not be associated. Given that we have biological and empirical reasons to believe short telomere lengths are predicted by the rs61748181 minor allele, and we did not see an association between telomere length and COPD disease progression, we concluded that the collider effect is not relevant here. Finally, by stratifying our analyses to disease progression subgroups, the collider effect interference, if there is one, should be minimized.
An important limitation of our study is a lack of a validation cohort to confirm the clinical effects of rs61748181 carrying status. To reliably determine lung disease progression, longitudinal follow-up and repeated observations are necessary. The impediments from these resource-intensive follow-up procedures are compounded by the low allelic frequencies of rs61748181, requiring a large sample size to enroll in the study. While we do not have access to clinical samples from such a clinical study in lung function decline, we contend that the effects of rs61748181 should apply to other chronic tissue degenerative disorders. Our data should provide the impetus to include TERT non-synonymous SNPs in disease progression assessments.
An individual's telomere length is determined by multiple factors, including genetics, inherited telomere length, lifestyle and environmental factors 35 . While the effect of environmental insults, such as smoking or infection, was highly variable and subject to huge inter-individual and temporal variations, the effects represented by the inherited telomere length and from the genotype of telomere-related genes are fixed. The comprehension of these variable and fixed effects could facilitate understanding of the pathogenesis and progression in complex disorders. Carriers with genetic variants of low penetrance in telomere biology genes could remain asymptomatic, while the telomere length maintenance defects keep accumulating as long as telomere length does not reach the threshold level. When these individuals encountered other fixed and/or variable factors that accelerate cellular turnover and telomere loss, such as exposure to cell death stimulants including smoking or recurrent infections, the defects in telomerase catalysis would be exacerbated. Over time, carriers of TERT SNPs should experience accelerated worsening of existing pathological conditions, as a function of the sum total of all etiologic factors that contributed to the loss of tissue integrity, leading to earlier onset or progression of multiple aging-related diseases, such as cancer 27 , IPF 9 , or COPD 9 (Fig. 3).
In contrast with our ex vivo model of rs61748181, where we observed telomere maintenance defects along the entire time course of the study, shorter telomeres were only observed in carriers of rs61748181 with COPD progression in the fourth quartile (non-decline in lung function group). We contend that while telomerase catalysis defects and accelerated telomere shortening should be consistent in carriers of rs61748181 within the LHS population, each individual's variable factors (pathological factors and disease progression rate) could mask this direct correlation. Accordingly, we did not observe any cohort-wide association between rs61748181 with shorter telomere length. The moderate differences in telomere catalysis between CC homozygotes and rs6178181 minor Scientific Reports | (2021) 11:4333 | https://doi.org/10.1038/s41598-021-83686-z www.nature.com/scientificreports/ allele carriers in a population could only be unmasked when the effects of these variable factors are removed, or approaching a fixed rate. We reasoned that maximum telomere attrition can be evident in the patient subgroup with the fastest disease progression profile. When we examined the subgroup of patients with the most negative ΔFEV1 5yrs , rapid lung tissue loss due to the end stage of the pathological processes indeed translated to a trend of shorter telomeres in carriers of rs61748181 minor allele than in CC patients, although statistical significance was not reached. Paradoxically, we demonstrated statistically significant difference in median telomere lengths between carriers of rs61748181 minor allele and CC-individuals, in a subgroup of patients with the most positive ΔFEV1 5yrs . Higher prevalence of smoking cessation ( Supplementary Fig. 2) in this sub-group contributed to the stabilization of lung functions and reduced the lung tissue renewal demand that were associated with rapid telomere attrition. As well, reduction of direct oxidative damage on telomeres from cigarette smoke may also mitigate a significant environment variable in telomere length maintenance. We contend that the reduction in both (pathological and environmental) variables in telomere length regulation provided the opportunity to unmask the catalytic defects of rs61748181 minor allele at a population level. Variants associated with mild penetrance in telomerase deficiency did not lead to cellular senescence in the absence of other confounding factors, and will not, on their own, drive the onset of chronic degenerative disorders. However, in the presence of accelerated cellular turnover, resulting from exposure to chronic environmental irritants and/or other genetic perturbations that affect tissue-specific functions, the presence of these genetic variants in the telomere biology pathway would be combined, resulting in faster telomere attrition. The effect of mildly deficient genetic polymorphisms in telomere biology genes can be seen as modifiers in accelerated COPD disease progression, when the pathogenetic process leads to excessive demand in lung tissue regeneration. Although the understanding of genetic profile in telomere biology genes does not unequivocally predict one's disease susceptibility, we contend that a thorough understanding of all the determinants that affect cellular Figure 3. The impact of genetic defects in telomere maintenance on lung function deterioration. Telomere Biology Diseases (TBD, right panel) are caused by rare genetic mutations that severely compromise the synthesis and/or the structural protection of the telomeres. TBD patients display extremely short telomeres at a young age, precipitating the onset of clinical symptoms. In contrast, carriers of common TERT genetic variants (SNPs) (middle panel) are not expected to display pathogenesis associated with short telomeres in the absence of additional factors (environmental, pathological or in combination with other genetic determinants) that accelerate telomere attritions. Even so, these carriers are still genetically predetermined to a decreased capacity to maintain their telomeres, when compared with noncarriers (left panel). These deficiencies, while silent phenotypically within the general population, could be unmasked by the massive increase in regenerative demand associated with tissue degenerative conditions, as seen in the case of COPD. Our work provides a proof-of-principle for novel epidemiological and clinical evaluations of the common SNPs in telomere biology genes on their effects as genetic modifiers rather than drivers of chronic tissue-degenerative disorders. Methods COPD patients. This study used clinical data and biological materials from the LHS. The LHS cohort initially recruited 5887 smokers, aged 35-60, with mild airway obstruction across 10 clinical centers in North America and collected their written informed consents 36 . Spirometry was performed annually over a period of 5 years for all study participants and in a subset after 11 years of study initiation as previously described 37,38 . Lung function was assessed as forced expiratory volume in one second (FEV1) % of predicted, i.e., FEV1 adjusted for age, height and sex 39 . We focused our data analyses on self-identified Caucasian patients in the LHS collection (n = 3596), as they constituted the major ethnicity group in the patient collection, and to avoid the confounding effects from haplotype differences in our analyses. In our study, FEV1 measurements over the 5-year follow-up period (reported as the slope from linear regression of FEV1 values plotted against time, ΔFEV1 5yrs ) was used as a readout for changes in lung function and a measurement of COPD progression. For subgroup analyses based on disease progression, we restricted our study to 15% of the Caucasian subjects with confirmed genotype data, and included equal numbers of rapid-and slow decliners from each end of the linear regression of the rate of decline. This cutoff was used to capture extreme phenotypes that would be likely to be influenced by genetic factors, as we have done in previous studies. Using this cut-off, the rapid decliner group is restricted to LHS participants with ΔFEV1 lower than − 3.5, the characteristics of these selected subjects are shown in Table 2 Telomere length measurement. The Southern blot-based terminal restriction fragment analysis (TRF) was applied to determine average telomere lengths in cell populations 41 . Data from TRF blots were analyzed by ImageQuant Software (GE Healthcare, Piscataway, NJ, USA) using weighted average analyses 41 . Data were reported as the nearest kilobase using the 1 kb DNA ladder (New England Biolabs, Beverly, MA, USA) as a reference. Telomeres in peripheral blood leukocytes in the LHS cohort were measured by qPCR as previously described 11 .
Scientific Reports | (2021) 11:4333 | https://doi.org/10.1038/s41598-021-83686-z www.nature.com/scientificreports/ Protein extraction. Cells were removed from culture flasks with trypsin/EDTA. Cell numbers in 100 μL of suspension were counted by Coulter Counter (Beckman Coulter, Brea, CA, USA) according to the manufacturer's instruction and total cell numbers were calculated accordingly. Following the 2 × wash with ice-cold PBS, cells were resuspended in hypotonic lysis buffer (20 mM HEPES pH 8.0, 2 mM MgCl 2 , 0.2 mM EGTA, 10% glycerol, 1 mM DTT, and 0.1 mM PMSF). Cell suspensions were subjected to four consecutive freeze-thaw cycles from liquid nitrogen to a 37 °C water bath. NaCl was then added to the lysate in two parts to a final concentration of 400 mM. Cell suspensions were incubated on ice for 15 min before clearing the lysate by centrifugation at 13,200 rpm for 15 min. The supernatant (whole cell lysate, WCL) was transferred to a fresh tube, aliquoted, and stored in − 80 °C for further analysis. Protein concentrations were measured by Bradford protein assay.
Telomerase activity. Telomerase activity was measured by the classical radioactive primer extension assay 42 using protein extracts from primary BJ cell lines expressing recombinant TERT isoforms. The quantifications of telomerase catalysis and repeat addition processivity were previously described 43 . Immunoblot. 100 μg WCL were resolved in 5% SDS-PAGE gel. A standard western blotting protocol was applied for the detection of TERT expression in the cell lysates. A sheep anti-human TERT polyclonal antibody (0.5 μg/ml, Abbexa, Cambridge, UK) and a Donkey anti-sheep horseradish peroxidase-conjugated antibody (0.2 μg/ml, Jackson ImmunoResearch, West Grove, PA, USA) were sequentially applied before detection by ECL (PerkinElmer, Waltham, MA, USA) on Kodak X-ray films. The cytoskeletal protein vinculin was used as a normalization control. It has a molecular weight of 124 kDa, which is similar to TERT (127 kDa). Rabbit anti-human vinculin monoclonal antibody (0.1 μg/ml, Cell Signaling, Danvers, MA, USA) and Alexa Fluor 680 Dye (ThermoFisher Scientific, Waltham, MA, USA) were applied before detection by Licor Odyssey CLx Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA) and quantification by ImageJ software (NIH, Bethesda, MD, USA).