MiR-150 and siCCR6 collaborate to inhibit CTCL cell migration. (A) Migration of My-La (top) and HH (bottom) cells in the presence of different extracellular stimuli; 5 × 10⁵ cells in 100 μL of medium without HS and CCL20 in the upper chamber through a coated basement membrane toward different concentrations of CCL20 (0.1∼10 ng/mL), with or without 2% HS at lower chamber (500 μL). The lower chamber contained HS free or CCL20, HS, CCL20, or CCL20 + HS. 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. (B) A schematic illustration of the migration assay described above. (C) ELISA of CCL20 in upper and lower chambers (transwell assays) by use of GFP-empty (shown as “control”), GFP-siCCR6 (shown as “siCCR6”), or GFP-miR-150 (shown as “miR-150”) transduced CTCL cells in indicated condition in the lower chamber. (D) A schematic illustration of the ELISA assay (HS + CCL20 condition in the lower chamber). (E) Migration of CTCL cells [GFP-empty+siSC (control), GFP-empty+siCCR6(1058) (transient), GFP-siCCR6(1058)+siSC, GFP-miR-150+siSC, and GFP-miR-150 +siCCR6(1058)] in the presence or absence of CCL20 (0 or 0.5 ng/mL), or HS (2%) in the lower chamber. 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 3 independent experiments. (F) A schematic diagram of the relationship between IL-22, CCL20, CCR6, and miR-150 in CTCL. NS: not significant.