HTLV-1 Tax induces Fascin in T cells. (A) Quantitative PCR of Fascin mRNA in the presence (Tesi) and absence (Tesi/Tet) of Tax; for complete Tax repression, cells were grown in medium containing 1 μg/mL tetracycline for 10 days (Tesi/Tet). Relative copy number was determined by normalizing Fascin and Tax transcripts to those of β-actin (ACTB). Mean values ± SE were compared using a paired t test (n = 4). *P < .05. (B) Representative diagram of Fascin (quantitative PCR) and Tax (RT-PCR) mRNA in Tax-inducible JPX9 cells (wt-Tax) and control cells JPX9M (inactive Tax mutant) after incubation with 20μM CdCl₂ for 0, 6, 12, 24, and 48 hours. (C) Test of Fascin and Tax copy numbers (quantitative PCR) for correlation using Pearson test after transfection of Jurkat T cells with increasing amounts of pcTax (0, 5, 10, 15, 20, 25, 30, and 35 μg) replenished with pcDNA3.1 to 40 μg. (D) Quantitative PCR of Fascin mRNA in freshly isolated CD4⁺ T lymphocytes from ATLL patients LFA, LFK, and LFN directly after isolation (t = 0 hours) and 18 hours after cultivation. (E) Quantitative PCR and immunoblot of Fascin. Relative copy number was determined by normalizing Fascin transcripts to those of ACTB. The means of 3 independent experiments ± SE are shown. ivt indicates in vitro-transformed; ATLL, derived from adult T-cell leukemia/lymphoma; ALL, derived from acute lymphoblastic leukemia; and HD, healthy donors. In immunoblots, detection of Hsp90α/β served as loading control. (F) Comparison of Fascin copies between HTLV-1/Tax-positive cells shown in panel E and uninfected controls (ALL, CD4⁺, PBMCs) using a 2-tailed Mann-Whitney test (n = 3). **P < .01. Horizontal bars represent the median; ■, HTLV-1-transformed cells; ●, HTLV-1-negative ALL cells; ▴, CD4⁺ T lymphocytes from HD; and ▵, PBMCs from HD. (G) Immunofluorescence of C8166 (HTLV/Tax+) and Tesi (Tax+) cells using Phalloidin X-Texas Red for detection of actin and anti-Fascin and secondary anti–mouse Alexa Flour 488 antibodies. Images were acquired using a Leica LAS AF DMI 6000 fluorescence microscope equipped with a 63× ACX PL APO oil immersion lens and a Leica DFC 360 FX camera. Leica Application Suite 2.0.2 software was used. (H) Comparison of invasion of ATLL-derived, serum-starved HuT-102 cells, transduced with lentiviral vectors expressing either a nonsense shRNA (ctrl.) or shFascin, into extracellular matrix in transwell assays performed in quadruplicate. In immunoblots, protein expression of Fascin, Tax, and Hsp90α/β was controlled. One representative experiment is shown.