Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: (A) BF images five out of the seven cell lines, including telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive line VA13. (B) Quantification of hTAPAS and hTERT mRNA levels by qRT-PCR. (C) RT–PCR analysis of hTERT splice isoforms using primers spanning exons 2–3 and 5–9. An inverse relationship between hTAPAS and hTERT expression was observed in HFF-1 and BJ fibroblasts (low hTERT , detectable hTAPAS ) and in HEK293T and NALM6 cells (high hTERT , absent hTAPAS ), whereas intermediate patterns were detected in IMR90, VA13, and iPSCs, with iPSCs maintaining moderate hTERT expression despite high hTAPAS levels.

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: Targeted DNA methylation profiling was performed using CRISPR–Cas9 enrichment followed by Nanopore sequencing across a ∼9 kb region spanning hTAPAS through hTERT intron 2 (Chr. 5: 1,196,006–1,205,206) and a ∼6.5 kb region covering introns 6–8 (Chr. 5: 1,174,035–1,180,535). The analysis included telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive cell lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive cell line VA13. DNA methylation levels at individual CpG sites are depicted across the indicated genomic regions, including hTAPAS , the THOR region, the core promoter, exon1, intron1, exon 2 and intron 2. Methylation for each individual CpG is shown as a percentage, with unmethylated CpGs depicted in red and methylated CpGs in blue. Methylation across intron 2 was consistently high (80–100%) in all cell lines, whereas regions encompassing hTAPAS , the THOR region, exon 2, and introns 6–8 displayed marked variability between cell types. CpGs within the hTAPAS region were highly methylated in telomerase-positive cells and in the ALT-positive VA13 line, but largely unmethylated in fibroblasts, with partial methylation observed in IMR90. The core hTERT promoter and exon 2–proximal regions remained mostly unmethylated in all cell lines except VA13, which exhibited substantial hypermethylation

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: DNA Methylation Assay, CRISPR, Nanopore Sequencing, Methylation