Figure 2

GLuc-miR-122 reporter knockdown by inducible short hairpin expression is reversible and RSL1 concentration-dependent. a. Gaussia luciferase (GLuc) reporters used in transfections: pTKGLuc: G aussia luciferase under control of the constitutive HSV-TK promoter; pTKGLuc-miR122 carries two tandem miR-122 targets (arrows) inserted in the 3' untranslated region of GLuc. b. Left panel: NIH3T3-47/X1-miR122 cells were transfected with pTKGLuc-miR122 or pTKGLuc and treated with DMSO (white bars) or 0.5 μM RSL1 (gray bars) for 48 h, or 0.5 μM RSL1 for 24 h followed by DMSO (RSL1/DMSO, black bars) for 24 h. GLuc reporter activity was assayed from transfected cell culture supernatants and RNA was prepared from the same cells. Values are expressed as a percent of the mean GLuc activity of non-induced cells (+/- 1SD). Right panel: Northern blot analysis of RNA prepared from the cells transfected and treated as shown in Left panel. Probes: miR-122 guide strand (top), U6 loading control (bottom). c. NIH3T3-47/X1-miR122 cells were transfected with pTKGLuc-miR122 or control pTKGLuc plasmid, and treated with DMSO or increasing concentrations of RSL1. Mean GLuc activity of induced cells is expressed relative to mean GLuc activity of non-induced (DMSO-treated) cells (+/- 1SD). Western blot: cell culture supernatants from c, detected with anti-GLuc antibody. Lane 1: DMSO; lanes 2-4: 0.05 μM, 0.5 μM and 5.0 μM RSL1 respectively.