GPER triggers JKT-1 cell proliferation in vitro.

<p>JKT-1 cells were seeded in six-well plates (0,6×10⁶ cells/well). After 48 h, the JKT-1 cells were washed and oestrogen starved overnight in phenol red-free DMEM supplemented with 1% charcoal-stripped FBS. Serum-deprived JKT-1 cells were then incubated for 24 h with E2-BSA (1 nM), an impermeable E2-conjugate, which cannot thus interact with classical nuclear or cytoplasmic estrogen receptors, after a pre-treatment with G15 (1 nM), a GPER antagonist, or ICI-182,780 (1 µM), a pure ER antagonist, or <i>pertussis</i> toxin (100 ng/mL), a GPCR protein inhibitor. G1 (1 nM), a GPER-specific agonist, was used as a positive control. E2 at the same concentration (1 nM), which induces an inhibitory effect on cell proliferation neutralized by ER antagonist ICI (1 µM), was used for comparison. Histograms represent percentages of variation in the JKT-1 cell number compared with the control (0%), which was measured at 1,2×10⁶ cells/well after 24 hours of incubation in the steroid-free medium with ethanol (10% of variation represent an increase or a decrease of 60 000 cells). All results are expressed as means ± SEM of three independents experiments. * P<0.01.</p

Effects of GPER knockdown on E2 and E2-BSA-induced JKT-1 cell proliferation.

<p>(A) JKT-1 cells were transfected with 50 nM of siRNA designed to knockdown GPER or with control siRNA. After 72-h incubation, proteins were extracted and subjected to Western blotting to confirm the specific inhibitory activity of GPER siRNA after normalization with β-actin, which was evaluated as a house-keeping gene. (B) After 72 h transfection with 50 nM of GPER siRNA or control siRNA, JKT-1 cells were incubated for 24 h with E2 (1 nM) or E2-BSA (1 nM). Histograms represent the percentages of variation of the JKT-1 cell number compared to control without estrogens. Cell number was measured at 1,5×10⁶ cells/well after 24 hours of incubation in the steroid-free medium with ethanol (10% of variation representing an increase or a decrease of 90 000 cells). All results are expressed as means ± SEM of three independents experiments (*P<0.01).</p

Immunolocalization of GPER in JKT-1 seminoma cells.

<p>In JKT-1 cells, GPER (red fluorescence) had both a membranous and intracytoplasmic perinuclear localization (A), whereas the classical oestrogen receptor ERβ had an intracytoplasmic perinuclear and nuclear localization (B, blue fluorescence). E2-BSA, an impermeable E2 conjugate that does not cross the membrane, stained the cell membrane when coupled to FITC (C, green fluorescence). By double staining (D), E2-BSA (green) and GPER (red) co-localized at the membrane (yellow [merge]); GPER was also expressed in the cytoplasm (red). (A, B, C, D: magnification, ×63).</p

Dnmt2-deficiency affects the P-TEFb complex.

<p>(A) Cdk9 expression in Dnmt2-deficient and control ES cells was analyzed by quantitative RT-PCR (left) and Western blot assays (right). (B) Cdk9 expression in Dnmt2-deficient and control hearts was investigated by quantitative RT-PCR (left) (<i>n</i> = 5) and Western blot assays (right). (C) Quantitative RT-PCR for Ctip expression in the hearts of Dnmt2^-/- and Dnmt2^+/+ mice (<i>n</i> = 6). Northern blot assay (D) and quantitative RT-PCR (E) for Rn7sk (7SK) expression in the hearts of Dnmt2^-/- and Dnmt2^+/+ mice. (F) RNA immunoprecipitation using a 5-methyl Cytidine antibody with RNA extracted from Dnmt2^-/- and Dnmt2^+/+ hearts, followed by RT-PCR for Rn7sk (<i>n</i> = 6). Note that Rn7sk is significantly less methylated in Dnmt2^-/- hearts. (G) PTEF-b immunoprecipitation using an antibody against Cdk9 on lysates from Dnmt2- mutant and control ES cells followed by RT-PCR for Rn7sk (<i>n</i> = 3) and subsequent quantification. An anti Cyp2 antibody served as negative control. Note that less Rn7sk is associated to the P-TEFb complex in Dnmt2-deficient cells.</p

Primers for quantitative RT-PCR analysis.

<p>Primers for quantitative RT-PCR analysis.</p

Dnmt2-deficiency is associated with increased phosphorylation of RNA polymerase II.

<p>Representative Western Blots for RNA polymerase II (RNA Pol II, upper panels) and quantification of the ratio of phosphorylated to non-phosphorylated RNA Pol II (lower panels) in mouse hearts (A) and ES cells (B) with knockout of Dnmt2 and controls. β-actin was used as a standard. Data are mean ± SEM. *p < 0.05.</p

Schematic representation of Dnmt2-mediated RNA polymerase II transcriptional activity in cardiac growth.

<p>Storage of inactive protein in a complex with 7SK RNA and inhibitory proteins, release of active kinase (P-TEFb) in Dnmt2 knock out conditions. The phosphorylation of a C-terminal domain of the polymerase by P-TEFb allows elongation of the transcripts. RNAPII CTD phosphorylation increases mRNA and protein expression, which mediates cardiac growth in Dnmt2-deficient mice.</p