Overexpression of miR-124a in ATL cells inhibits tumor growth in vivo. (A) Mature miRNA detection of miR-124a in ATL patient samples (n = 17) vs healthy noninfected donor. miR-24 served as an internal control. (B-C) Induction of miR-124a in ED-40515(–) cells inhibits cell proliferation. Pre–miR-124a expression was detected by real-time PCR (B) after doxycycline (Dox) addition. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. Cells were counted every 48 hours after receiving 2 μg/mL Dox (every 48 h) (C). Results represent at least 2 independent experiments. (D) Loss of proliferation/cell cycle markers (cyclins A, B1, and E, and survivin) in miR-124a–expressing ATL cells, 6 days after receiving 2 μg/mL Dox every other day. Tubulin was used as a loading control. (E) ED-40515(–) cells expressing miR-124a enter cell cycle arrest after induction with 2 μg/mL Dox every day for 5 days. (F) Cell survival markers (Bax, Bcl-xl, and Bcl-2) are altered in ED-40515(–)–miR-124a expressing cells 5 days after receiving 2 μg/mL Dox every day. Tubulin served as a loading control. (G) ED-40515(–)–pTRIPZ (n = 12) or miR-124a (n = 12) TET-On cells were injected into the right or left flank of NOG mice. Pictures are representative of 2 mice simultaneously receiving pTRIPZ (left) or miR-124a (right) TET-On ED-40515(–) cells. (H) Real-time PCR detection of pre–miR-124a in a tumor model of ATL. GAPDH served as a control. (I) In vivo growth of NOG mice engrafted with ED-40515(–) pTRIPZ (left) or miR-124a (right) cells (plotted as the average tumor volume (mm³) (calculated as the [width²  × length]/2) per every 7 days). (J) The percentage of tumors with volumes of 200 m³ or larger was plotted against the days after implantation into mice. Volumes were calculated every 7 days, up to 21 days. Statistics are calculated using the N-1 2-proportion test, with the number of tumors in each group (n = 12) 200 m³ or larger, considered positive.