FIGURE 7.
Gαs-dependent gonadotropin mRNA regulation is mediated by released autocrine/paracrine peptide(s). A–C, LβT2 gonadotrope cells were transfected with control or Gαs siRNAs via nucleofection. Two days after siRNA transfection, medium was replaced with fresh medium. At day 3, overnight conditioned medium (CM) was collected from siRNA-transfected cells, and it was added to non-siRNA-treated recipient cells. A, conditioned medium from control and Gαs knockdown cells was added to the non-siRNA-treated recipient cells for 6 h, and the effect on FSHβ and Gαs transcription was monitored by real-time PCR analysis. B, conditioned medium from control and Gαs knockdown cells passed through 3-kDa molecular mass cutoff filter. Concentrate fraction (enriched with more than 3-kDa proteins) and filtrate fraction (depleted from more than 3-kDa proteins) were added to non-siRNA-treated recipient cells for 6 h. C, conditioned medium from control and Gαs knockdown cells was treated with proteinase K and then heat-denatured. Both control and proteinase K-treated conditioned media were added to the recipient cells, and the effect on FSHβ transcription was monitored by real-time PCR. D, conditioned medium from control and CTX-pretreated cells was collected and was added to non-CTX-treated recipient cells for 6 h. FSHβ and LHβ mRNA expression was monitored by real-time PCR. For statistical analysis, two-tailed t test was used. **, p < 0.01; *, p < 0.05. Error bars, S.E.