Fig 1: Analysis of hTR, hTERT and telomerase activity in the hTERT IP samples. (a) hTERT was immunoprecipitated from HEK 293T cell lysate and eluted from beads with excess amounts of the corresponding peptide antigen as described in ‘Materials and Methods’ section. hTR in the input (0.67%), flow through (0.67%) and elution (40%) fractions was examined by northern blot, with H1 RNA serving as an internal control. Western blot with Abcam ab32020 was performed on the input (0.1%), flow through (0.1%) and elution (6%) fractions, with ß-actin serving as an internal control. (b) Telomerase activity in the input (1%), flow through (1%) and elution (7.5%) fractions was examined by the direct enzyme assay as described in ‘Materials and Methods’ section. (1, 2) Duplicate repeats of experiment. +2 and +4, size markers made by extending the DNA primer by two or four nucleotides. LC, labeled unextended primer, serving as a loading control. (c) Summary of hTR levels, Abcam ab32020 western signals and telomerase activity levels present in flow through and elution fractions, as normalized to input levels, in the specific experiment shown in (a) and (b). Note that the telomerase activity in the elution (~69% input) is higher than that lost from input to flow through (~35% input), probably because cellular factors present in input/flow through but not elution are inhibiting telomerase activity. Replicates of this experiment in HEK 293T and HeLa cells are summarized in Table 2.
Fig 2: TERT promoter hypermethylation and elevated expression correlate with aggressive clinical phenotypes in TNBC. (A, B). TERT expression in different TNM stages and pathological N stages among patients from the TCGA cohort. (C, D). The relative methylation levels of cg26006951 and cg07380026 in different pathological N and T stages in patients in the TCGA cohort.
Fig 3: TERT expression and promoter CpGs were associated with the expression of key immunomodulators in TNBC. (A). Correlation heatmap of TERT expression and two differentially methylated CpG sites (cg26006951 and cg07380026) with 74 key immunomodulators in the TCGA cohort. (B–D). The correlation of TERT expression, cg26006951, and cg07380026 methylation level with the immune checkpoint molecules LAG-3, PD-1, PD-L1, CTLA4, TIGIT, or PD-L2, respectively. * P < 0.05; ** P < 0.01.
Fig 4: NXF1-exported TERC facilitates the cytoplasmic assembly of the telomerase complex and its subsequent nuclear localization(A) Interaction between NXF1 and TERC. RIP assay was performed with NXF1 or XPO1 antibody on A549 cell lysates as previously described.37 RIP with IgG served as control. RT-qPCR was performed to measure the amount of TERC in the precipitates. Data were presented as mean ± SD (n ≥ 3), and p value was calculated by Student’s t-test in comparison with the IgG control.(B) Measurement of nuclear TERC enrichment by RT-qPCR. Whole-cell (wce) and nuclear (nuc) RNA extracts were prepared from A549 cells without (control) or with NXF1 knockdown (NXF1-si1, NXF1-si2). RT-qPCR was performed for each extract to measure the level of TERC, using U6 (known to remain in the nucleus after transcription by Pol III) as an internal control. Relative nuclear enrichment of TERC was determined by its nuclear/total expression normalized to the same ratio within the control. Data were presented as mean ± SD (n ≥ 2), and p values were determined by Student’s t-test in comparison with the control.(C) Representative image of western blotting to detect TERT in A549 cells without (ctrl) or with NXF1 knocked down (Nsi1 and Nsi2). Numbers indicate the relative ratios of TERT/histone H3.(D) Quantification of the blots in (C). Data were presented as mean ± SD (n ≥ 3), and p values were analyzed by Student’s t-test in comparison with the control.(E) Relative telomere repeat number in NSCLC cells with NXF1 knockdown. Cellular DNA was extracted for qPCR to detect the telomere repeats, and 36B4 served as the single-copy gene control. Data are presented as mean ± SD (n ≥ 2). p values were determined by Student’s t-test.(F) A model for the trafficking and assembly of the telomerase components. The mature TERC is exported to the cytoplasm by NXF1, where it mediates the assembly of the telomerase ribonucleoprotein complex. The fully assembled telomerase holoenzyme is imported into the nucleus for telomere maintenance and transcriptional regulation.
Fig 5: Knockdown of TERC blocks the nuclear localization of TERT(A) Top and middle: Representative images of western blotting to detect the level of TERT in whole-cell extracts (wce) and nuclear extracts (nuc) of A549 and H460 cells without (ctrl) or with TERC knocked down (si1 and si2). β-tubulin served as whole cell and cytoplasmic loading control, while histone H3 served as whole cell and nuclear loading control. Numbers indicate the ratios of TERT/histone H3. Bottom: Quantification of the blots. Data were presented as mean ± SD (n ≥ 3).(B) Top: Representative images of the subcellular localization of TERT detected by immunofluorescence in A549 cells. Nuclei were stained with DAPI. Scale bar, 10 μm. Bottom: Quantification by calculating the ratio of nuclear/cytoplasmic GFP signal intensity in each cell.(C) Left and middle: Representative IHC staining of TERT in xenograft tumors from A549 cells without (control) or with TERC stably knocked down by its specific shRNA (TERC-sh) (400× magnification). Scale bar, 50 μm. Right: Quantification by calculating the percentage of the cells with nuclear staining of TERT. The numbers in the brackets in (B) and (C) denote the number of cells examined; and p values were determined by Student’s t-test in comparison with the control.
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