Figure 7
The p8 protein increases TNT formation. (A) Orf-I amino acid sequences of HTLV-1 positive cell lines. (B) Transduction efficiency determined by flow cytometry of percentage GFP positive cells in Jurkat-memCherry cells transduced with pSDM, pSDM-N26(p8)-HA or pSDM-G29S(p12)-HA. (C) Immunoblotting of Jurkat-memCherry cells (ctr), Jurkat-memCherry; pSDM, pSDM-G29S-HA and pSDM-N26-HA incubated with anti-HA antibody. β-actin was used as loading control. (D) TNT quantification of Jurkat-memCherry; pSDM, pSDM-G29S-HA and pSDM-N26-HA. Results are presented as mean from three independent experiments performed in duplicates. (E) TNT verification in Jurkat-memCherry-pSDM-N26-HA cells. The cells were fixed and stained with AF350 phalloidin to visualize F-actin (blue) and anti-α-tubulin to visualize tubulin (cyan, AF633), transduced cells are GFP positive, plasma membrane memCherry (red). Scale bar: 10 µm. Result is representative of three independent experiments. (F) Jurkat-memCherry-pSDM-N26-HA cells were untreated or treated with 1 µM cytarabine (AraC) for 24 h and TNTs were quantified in live cells. Results are shown as mean from three independent experiments performed in duplicates. (G) Immunoblotting of Jurkat-memCherry-pSDM-N26-HA cells not treated or treated with 1 µM AraC incubated with anti-HA antibody. β-tubulin was used as loading control. Representative blot of three independent experiments is shown. Unpaired t-test was performed to evaluate statistic significance. F-test was performed for individual variation. GraphPad Prism (Version 6.03) was used (**p < 0.01, ***p ≤ 0.001). Full-length immunoblots are presented in Supplementary Fig. 5. Adobe photoshop CS6 was used to prepare the images. The contrast was enhanced on the whole image to better represent and visualize the thin TNT structures.
