FIG. 5.

Induction of Dmrt1 by 8-bromo-cAMP requires ongoing transcription but not translation. A, Sertoli cells were treated with the protein synthesis inhibitor cycloheximide in the presence and absence of 8-bromo-cAMP and RNA levels for Dmrt1 and actin were measured by RPA as described in the legend for Fig. 2. Where indicated Sertoli cells were pretreated with cylcohexamide (CHX) for 30 min. The CHX treated cells were either left to incubate an additional 4 h (+CHX) or treated with 8-bromo-cAMP (1 mm) for the same time period (+cAMP & CHX). The other samples included cells cultured in the absence of addition treatments (no treatment) for the same period of time or ones treated with 8-bromo-cAMP alone for 4 h (+cAMP). B, Sertoli cells were treated with the transcriptional inhibitor actinomycin D in the presence and absence of 8-bromo-cAMP and RNA levels for Dmrt1 and actin were measured by RPA. Where indicated Sertoli cells were pretreated with actinomycin D (ActD) for 30 min. The Act D treated cells were either left to incubate an addition 4 h (+ActD) or treated with 8-bromo-cAMP (1 mm) for the same time period (+cAMP & ActD). The other samples included cells cultured in the absence of addition treatments (no treatment) for the same period of time or ones treated with 8-bromo-cAMP alone for 4 h (+cAMP). Arrows mark the protected fragments and tRNA and RNA from freshly isolated Sertoli cells were added as negative and positive controls, respectively.