Inoculation of GFP-siCCR6–transfected CTCL cells into NOG mice. (A) Expression of CCR6 in CTCL cell lines transiently transfected with siCCR6(705 or 1058). Left: relative expression of CCR6 in GFP-empty-CTCL (shown as “GFP-empty”) (My-La or HH), GFP-miR-150-CTCL cells transiently transfected with scrambled (shown as “+siSC”), or siCCR6 (shown as “+siCCR6”). Right: western blot analysis of CCR6 in GFP-empty (control) or GFP-miR-150 transduced CTCL cells transiently transduced with siCCR6(1058). Tubulin is the control for CCR6; 5S is the control for miR-150. (B) qPCR and western blot analyses of CCR6 in My-La and HH cells stably transfected with GFP-siCCR6. Relative expression of CCR6 in GFP-scrambled (control) CTCL (shown as “GFP-scrambled”), GFP-siCCR6 (705 or 1058)-CTCL, or GFP-miR-150-CTCL cells. GFP-siCCR6 expressing transfected cells were sorted and obtained with >99% purification. Northern blots against miR-150 are also shown below the western blot data. (C) Survival curves for NOG mice inoculated with My-La and HH transfected with GFP-scrambled (shown as “control”), GFP-siCCR6(1058), or GFP-miR-150 (n = 10 each). Asterisks (*) indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01. (D) Percentages of BM and spleen involvement in NOG mice inoculated CTCL cells transduced with GFP-control, GFP-siCCR6, or GFP-miR-150. NOG mice (n = 6 each) were sacrificed at day 31 (My-La) or day 55 (HH). P values were calculated using Student t test. Asterisks (*) indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001. Bars are means ± SEM of 6 independent experiments.