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. 2012 Apr 15;125(8):1929–1944. doi: 10.1242/jcs.095067

Fig. 7.

Fig. 7.

Inhibition of ERK/MAPK signaling results in multiple lumen formation downstream of cytokine expression. (A) Glucocorticoids reduce ERK phosphorylation. Western blot from 3D culture grown for 7 days in BIE, BIE plus dexamethasone (DEX) or BIE plus hydrocortisone (HC) shows no modification of Akt phosphorylation, increased GR expression and reduction of ERK phosphorylation. Membranes were reproved with tubulin to show equal protein loading. (B) Inhibition of ERK/MAPK causes a dose-dependent increase in multiple-lumened glands. Endometrial epithelial glands were grown for 7 days in BIE medium alone or BIE plus TNFα and increasing doses of the ERK inhibitors UO126 or PD0325901. Quantification of single-lumened glands (top) and representative images (bottom) showing phalloidin immunostaining (red). Nuclei were evidenced by Hoechst 33342 staining (bue). (C) Inhibition of ERK/MAPK does not change ERα expression. Western blot from 3D cultures grown for 7 days in BIE plus increasing doses of UO126 shows an effective inhibition of ERK phosphorylation and no modification of ERα protein levels. Membranes were reproved with tubulin to show equal protein loading. (D) Inhibition of ERK/MAPK does not repress TNFα or IL1α expression. Endometrial epithelial glands were grown for 7 days in BIE plus increasing doses of UO126. mRNA was extracted and TNFα (left) and IL1α (right) expression was analyzed by real-time PCR. Results are expressed as relative mRNA levels. (E) TNFα, ICI182170 and shRNA targeting ERα restore ERK phosphorylation in glucocorticoid-treated cells. Top: Western blot shows ERK phosphorylation of cultures treated with BIE or BIE+DEX and 50 ng/ml TNFα, 10 nM ICI182170 (ICI) or no additives (−) for 7 days. Bottom: Western blot shows ERK phosphorylation in cultures infected with FSV vector or ERα shRNA1 and treated with BIE alone or BIE+DEX. Membranes were reproved with ERα antibodies to ensure correct inhibition of ERα. Membranes were also reproved with tubulin and total ERK to ensure equal loading. (F) Percentage of single- versus multiple-lumened glands grown for 7 days in BIE+DEX or BIE+DEX plus TNFα with (+) or without (−) 5 μM UO126. Scale bars: 50 μm. *P≤0.05.