Table 1.

Result highlights of the study published by Li et al. [18] showing tumorigenic ID8 ovary cancer surface epithelial cells are affected by FSH via FSHR-3

Experiments	Results highlights
In vitro studies	ID8, a tumorigenic cell line derived from mouse ovarian surface epithelial cells when exposed to EGF (10 ng/ml) or FSH (20 ng/ml), showed greater proliferation response after FSH treatment compared to EGF in vitro.
Associated signaling	The canonical FSHR-1 is associated with stimulation of adenylate cyclase whereas the growth factor variant FSHR-3 signals via ERK1/2 activation and calcium influx in the absence of increased intracellular concentrations of cAMP. In mammalian cells transfected with FSH-R3, FSH has been shown not only to increase the influx of extracellular calcium, but also to stimulate cell proliferation and ERK phosphorylation in a calcium-dependent fashion FSH failed to stimulate cAMP accumulation in ID8 cells despite the presence of functional adenylate cyclase. Rather, FSH stimulated ERK phosphorylation. After FSH addition, the phosphorylated forms of ERK1 and ERK2 were evident at 10 min, elevated after 30 min and declined to basal levels after 60 min. Pretreatment of cells with MEK inhibitor [PD98059, 100 μM] abolished FSH mediated proliferation as well as cell signaling Effects of calcium channel antagonist SNX-482 were assessed on ID8 MOSEC to determine FSH effect on influx of calcium via voltage-gated channels. SNX-482 prevented FSH from stimulating both MOSEC growth and ERK activation
Southern Blotting	Oligonucleotide probes specific for Exons 7 & 11 were used for Southern blot analysis of genomic DNA isolated from ID8 cells. Hybridization analysis with both the probes recognized 11.6 and 4.3 kb fragments
Northern Blotting	RNA blot using the same probes as above identified 1.9 Kb band
Western Blotting	Commercial antibody against FSHR (Santacruz) directed against an N-terminal sequence of the FSH-R recommended to detect FSHR-1 was found to detect two bands of 75 and approximately 50 kDA which corresponded to FSHR-1 and FSHR-3 respectively
Immunocytochemistry	Surface expression of FSHR-3 and SNX-482 sensitive Cav2.3 channel in MOSEC was shown by immunocytochemistry