Figure 2.
Progesterone mediates its effects via non-nuclear receptors on LLC1 tumor cells
(A) Expression of the mRNA for nuclear progesterone receptor (Pgr) in LLC1 tumor cells. 1 μg of total RNA was reverse transcribed to cDNA, and PCR was carried out. mRNA from BALB/c ovaries was employed as a positive control (PC). The primer employed (sequence provided in Table S2) amplifies both isoforms (A and B) of Pgr. The vertical gap between the bands denotes missing wells in the gel (Data S1). Gapdh controls are shown.
(B) LLC1 tumor cells were incubated with mifepristone (500 nM) for 12 h followed by incubation with Prog (50 μM) for 48 h. Vehicle (Veh) for mifepristone: DMSO; Veh for mifepristone + Prog: DMSO + ethanol. Cell viability was assessed by MTT. Data are plotted as percentage viability over untreated cells. Values represent arithmetic mean ± SD (n = 3). ∗∗∗p < 0.001 by the unpaired t-test. ns: non-significant.
(C) Experimental set-up similar to Figure 2B. Proliferation was assessed by measuring the incorporation of 3H-thymidine. Values represent arithmetic mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01 by the unpaired t-test.
(D) Expression of mRNA for non-nuclear progesterone receptors in LLC1 tumor cells. mRNA from BALB/c ovaries was employed as a positive control (PC). 1 μg of total RNA was reverse transcribed to cDNA, and PCR was carried out. The vertical gap between the bands denotes missing wells in the gels (Data S1). Gapdh controls are shown.
(E) LLC1 tumor cells were incubated with phosphodiesterase inhibitors IBMX and Ro-20-1724 for 30 min before incubation with forskolin (100 μM/10 min) or Prog (100 μM/30 min). Vehicle (Veh) for forskolin: DMSO. A luminescence-based assay for cAMP was then performed. Each luminescence (RLU) value was normalized to blank. Concentrations were calculated using four-parameter logistic curve regression model. Values represent arithmetic mean ± SD (n = 2). Value compared to respective vehicles. ∗∗∗p < 0.001 by the unpaired t-test.