Figure 5

GS-293 cells require cell-cell contact to detect cAMP from donor cells. (A) GS-293 cells were grown on transwell permeable supports and FSHR-293 cells were grown on the bottom of a separate well. Prior to experiment, the transwell chamber with the GS-293 cells was placed above the FSHR-293 cells and medium was replaced with assay medium (referred to as ′No cell-cell contact” configuration). GS-293 and FSHR-293 cells were also co-cultured in the transwell permeable support wells (referred to as “Cell-Cell contact” configuration). (B) In “No cell-cell contact” configuration, GS-293 cells were unable to detect cAMP production from FSHR-293 cells stimulated with rFSH (200 mIU/ml) and the luminescent signal detected was comparable to unstimulated GS-293 cells (control). The cell-cell contact configuration however yielded much higher luminescence, proving the detection of cAMP by GS-293 cells from donor cells requires cell-cell contact. (C) In cell-cell contact configuration, rFSH stimulation in the upper chamber yielded a higher response than rFSH stimulation in the bottom well due to higher effective concentration of rFSH in the former. (B and C) Data represented as mean of duplicates for one representative experiment (± SD; positive direction) with at least three independent repeats.