MOESM2 of HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo

Additional file 2: Fig. S2. Ablation of the HTLV-1 CTCF-binding site significantly decreases HTLV-1 p30 expression in vitro in HTLV-1∆CTCF immortalized PBLs when compared to HTLV-1p12Stop, but not when compared to HTLV-1. p30 gene expression was assessed via qPCR. RNA was isolated from HTLV-1-immortalized (PBL.HTLV-1), HTLV-1∆CTCF-immortalized (PBL.HTLV-1∆CTCF, and HTLV-1p12Stop-immortalized (PBL.HTLV-1p12Stop) PBLs. cDNA was synthesized from 1 µg of RNA, then a 45-cycle qPCR was performed in duplicate using 2 µL of cDNA per reaction and a p30-specific primer/probe set. Total copy number was determined using a standard curve generated by duplicate samples of log10 dilutions of p30 standard (primer/probe set and standard described in materials and methods). Copy numbers were normalized to 106 human GAPDH (hGAPDH). Bars represent mean log10 p30 copy normalized to hGAPDH. Error bars indicated standard deviation. PBL.HTLV-1∆CTCF showed a significant decrease in p30 gene expression when compared to PBL.HTLV-1p12Stop (p 0.025). While subjectively decreased, the difference in p30 expression between PBL.HTLV-1∆CTCF and PBL.HTLV-1 was not significant (p 0.175). One way ANOVA with multiple comparisons was used for statistical analysis with significance denoted by values with p < 0.05.