Fig 2.

αBE4 enhancer activity in response to En-1 treatment. (A) Serum-starved HL-1 cells were treated with either different concentrations of En-1 (25, 50, 100 nM) for 12, 24 and 48 hrs before collection or vehicle (water). Total RNA levels were examined by Northern blotting with a CryAB cDNA probe. 18S ribosomal RNA was used as a loading control, (lower panel). Quantification of time and concentration dependent effect of En-1 on CryAB mRNA levels in HL-1 cells (n= 2). (B) HL-1 cells were cotransfected with an expression plasmid encoding Nished or parental vector where indicated along with CryAB-Luc, or mutBE4-Luc plasmids. After preincubation in serum-free medium cells were treated with 50 nM En-1. Luciferase activities in cells treated with En-1 are expressed to the mean value obtained from cells exposed to vehicle (water). In cells co-transfected with pcDNAV5-Nished (Nished) treated with En-1, luciferase activities are expressed relative to the mean value derived from cells cotransfected with pcDNAV5 (parental vector) and CryAB-Luc or mutBE4-luc plasmids and treated with En-1. Renilla luciferase expression was used for normalization. Experiments have been repeated at least three times. Data are mean ± SE. **P < 0.01.