Fig 2.

Incubation with Mithramycin A and siRNA knockdown of Sp1 and Sp3 inhibit basal RIIα protein expression in human myometrial cells. (A) Decrease in RIIα protein levels monitored by Western blotting after 6 hrs treatment of human myometrial cells with 2 μM Mithramycin. Data are expressed as mean ± S.E.M. Data were obtained from six myometrial cell preparations. *P, 0.05 Student’s t-test (n 5 6) treated versus untreated cells. (B) Mithramycin A at 200 νM also decreased RIIα protein levels as monitored by Western blotting after 24 hrs treatment of human myometrial cells indicating further specificity of the effect. Data are expressed as mean ± S.E.M. *P < 0.05 Student’s t-test (n = 4) treated versus untreated cells. Equal loading in each case (A and B) was confirmed using a β-actin antibody. (C) Effect of Sp1/Sp3 siRNA transfection of human myometrial cells on RIIα protein expression. Myometrial cells were transfected with siRNAs directed against both Sp1 and Sp3 as well as control non-target siRNAs. Expression of Sp1, Sp3 and RIIα protein levels after 48 hrs transfection were monitored by Western blotting. A substantial decrease in Sp1, Sp3 and RIIα protein levels was observed with Sp1/Sp3 siRNAs compared to control (no siRNAs) or non-target siRNAs. A GAPDH antibody was used to confirm specificity of the siRNA transfection. Data are means ± S.E.M. (n 5 5) from two myometrial cell preparations.