Figure 3. Transient and constitutive MCF7 overexpression of Y537S or D538G mutated ERα (mutER) induces ligand-independent tumorigenic growth with pS294 formation, prevented by the CDK2 inhibitor Dinaciclib but not by the CDK4/6 inhibitor Palbociclib.

A. Following transient transfection of MCF7 cells with control (C or wt = pSG5-HEGO) or mutER expression vectors (#37 = Y537S, #38 = D538G), transfer to E2-free media (x 24-48 h), and subsequent culture treatment (0.5 μM x 60 min) with either Dinaciclib (DIA) or Palbociclib (PD), cells were harvested, proteins extracted, ERα immunoprecipitated and immunoblotted for pS294, pS118, pS167, and total ERα. B. Young nude mice were bilaterally implanted with knock-in MCF7 sublines (8 mice per subline), ERmut(Y537S) or ERcontrol(AAV), and observed for tumorigenic growth in the absence of exogenous E2 supplementation, with only the ERmut expressing cells showing E2-independent tumor growth. At 21 days, tumors were excised, protein extracted, immunoprecipitated first for pS294 (1^st IP) and then for remaining ERα (2^nd IP). Both IPs were immunoblotted for pS294, pS118, and total ERα. C. Knock-in MCF7 sublines, MCF7(Y537S), MCF7(D538G) and control MCF7(AAV), were serially passaged in E2 free media (C), transiently stimulated with E2 (10 nM x 20 min) +/- pretreatment (0.5 μM x 60 min) with Dinaciclib (D) or Palbociclib (PD) before cell harvest, protein extraction, ERα immunoprecipitation and immunoblotting for pS294, pS118, and total ERα as shown.