Figure 5. Characterization of F9 cell lines overexpressing Hoxa1-399 and Hoxa1-993.

(A) Total RNA from F9 Wt and the stably transfected cell lines F9-399-83, F9-399-72, and F9-993-10 treated with 100 μM ZnCl₂ with or without 1 μM RA for the indicated times were subjected to Northern blot analysis and probed with various Hoxa1 and/or RA target genes. One representative is shown, although the experiments were performed three times (except where indicated) with different RNA preparations with similar results. Arrow, expression of the total endogenous Hoxa1. Arrowhead, expression of exogenous Hoxa1-399 mRNA. (B) Quantitation of the mRNA levels of Hoxa1 and/or RA targets by phosphorimager analysis from three independent experiments (except Pbx1 and Meis1 where n=2). Northern blots were normalized to GAPDH mRNA levels, and the data are shown as fold increase over the relative mRNA levels of F9 Wt zinc-treatment for 24 hrs. *** very significant; indicates a p value of <0.001, ** very significant; indicates a p value of <0.01,* significant; indicates a p value of <0.05 when comparing relative mRNA levels to F9 Wt under the same experimental condition. (C) RT-PCR analysis of the Hoxb1 transcript in F9 Wt, F9-399-83, and F9-399-72 cells after 72 hr treatment with 100 μM ZnCl₂ with or without 1 μM RA.