Fig. 2.

Long-term reduction of NOVA1 progressively shortens telomeres. a Terminal restriction fragment length (TRF-Southern blot) analysis of control shRNA or NOVA1 shRNA at two population doublings (PD). b Rescue of shRNA knockdown of NOVA1 with a shRNA mutant cDNA in H1299 cells (stable cell lines were measured a minimum of six times over several passages). hTERT splicing was determined with RT-ddPCR assays. c Western blot of NOVA1 shRNA rescue in H1299 cells (representative image of stable cell lines, measured three times over three passages in culture). d hTERT expression in rescue H1299 cells as determined by RT-PCR of exons 5–9 (representative image; n = 3). e Rescue of shRNA knockdown of NOVA1 with a shRNA mutant cDNA partially restores telomerase activity in H1299 cells (n = 6). Telomerase activity was determined with droplet digital TRAP (ddTRAP with 50 cell equivalents added to the assay). Student’s t test set at *p < 0.05 for significance. f Western blot of V5-tagged NOVA1 expression in Calu6 cells (representative image of stable cell lines). g hTERT splicing profile in Calu6 cells with and without NOVA1 (n = 6). hTERT splicing was determined with RT-ddPCR assays. h Telomerase enzyme activity (ddTRAP with 50 cell equivalents added to the assay) in Calu6 cells with and without NOVA1 (n = 3). Student’s t test set at *p < 0.05 for significance). Data are expressed as means and standard error of the mean where applicable. *p < 0.05. RT: reverse transcription, ddPCR: droplet digital PCR. + indicates presence of shRNA or cDNA construct. − indicates absence of shRNA or cDNA construct (b, d, e). Supplementary data associated with this figure can be found in Supplementary Figure 2