ERα36 overexpression lowers MCF-10A cell proliferation rate.

<p>A. Quantification of MCF-10A/Zeo and MCF-10A/ERα36 cell viability by crystal violet assay after 48h culture. ERα36 overexpression triggers a 35% decrease of cell proliferation. Each bar represents mean ± S.E.M. N = 3. *: <i>P</i><0.05. B. Doubling time of MCF-10A/Zeo and MCF-10A/ERα36 cells was measured by crystal violet cell counting at t = 0 (immediately after seeding), t = 24h, 48h and 72h of culture. Given the measurements of living cells at t = 72h and t = 0, doubling time was calculated assuming a constant growth rate. Each bar represents mean ± S.E.M. N = 4. *: <i>P</i><0.05. C. Representative western blot analysis of Cyclin D1 expression in MCF-10A/Zeo and MCF-10A/ERα36 cells (left panel). α-Tubulin was used as a loading control. Quantification of corresponding band intensity (right panel) indicate a 60% decrease of cyclin D1 expression in MCF-10A/ERα36 compared to MCF-10A/Zeo cells. Each bar represents mean ± S.E.M. N = 3. **: <i>P</i><0.01. D. Relative Cyclin D1 expression in MCF-10A/Zeo and MCF-10A/ERα36 cells after 24h DMSO or 10μM 5,15 DPP exposure. Results depicted in the histogram are represented as 5,15 DPP versus DMSO ratio. In the presence of 5,15 DPP, cyclin D1 protein expression increases by 81% only in MCF-10A/ERα36 cells. </p

Whole mount mammary tree parameters analyses in wt and Tg mice at weaning (PND21).

<p>F2 or F4 mammary glands from wt or Tg mice where mounted. Computational analysis of mammary tree total extension (A), number of branching (B) or end buds (C) was performed using a dedicated software. No significant difference was observed between wt and Tg mice. F2wt: N = 4, F2 Tg: N = 6, F4wt: N = 6, F4Tg N = 5.</p

Histological analysis of F4 wt and Tg mice at weaning and adulthood.

<p>F₄ mammary gland slices from 3 week old (weaning) or 16 week old (adult) virgin female wt or Tg mice were stained with hematoxylin/eosin (A). S, stroma; E, epithelium; L, lumen; Ad, adipocyte. The epithelium thickness, the stromal thickness and lumen diameter of mammary ducts were measured in wt or Tg mice at weaning (B) and adulthood (C). Scale bar, 15 μm. Each bar represents mean ± S.E.M. N>5 per group. *: <i>P</i> <0.05, ***: <i>P</i> <0.001.</p

ERα36 overexpression enhances migratory potential.

<p>A. A wound was performed on a confluent monolayer culture of MCF-10A/Zeo and MCF-10A/ERα36 cells. Histogram depicts the wound healing when measured after a 6-hour culture (left panel). A representative picture of the migrating MCF-10A/ERα36 cells is presented in the right panel. Each bar represents mean ± S.E.M. N = 5. *: <i>P</i> <0.05. B-C. MCF-10A/Zeo and MCF-10A/ERα36 were cultured for 24 hours. B. CDH1 and CDH2 gene expression was measured by RT-PCR analysis. The housekeeping gene RPLPO was used as the reference gene. Each bar represents mean ± S.E.M. N = 3. *: <i>P</i> <0.05. **: <i>P</i> <0.01. C. Expression of cell-cell adhesion proteins was studied by immunofluorescence with specific antibodies: anti-β-catenin, anti-E-cadherin and anti-N-cadherin (AlexaFluor 555). Average fluorescent signal intensities are quantified from 5 cells from 5 fields per condition. Each bar represents mean ± S.E.M. N = 3. *: <i>P</i> <0.05, ** <i>P</i> <0.01. </p

ERα36 overexpression stimulates apoptosis resistance.

<p>MCF-10A/Zeo and MCF-10A/ERα36 cells were exposed to 0.25μM staurosporin (STS) or vehicle (Veh) for 6 hours. A. Cleavage of PARP1 (cPARP1), Caspase 7 (cCasp7) and Caspase 3 (cCasp3) were evaluated with specific antibodies (left panel). GAPDH was used as a loading control. Results depicted in the corresponding histogram are represented as STS versus Vehicle ratio (right panel). ERα36 overexpression triggered a significant 34%, 60% and 30% decrease of PARP1, Caspase 7 and Caspase 3 cleavage, respectively. Each bar represents mean ± S.E.M. N = 4. *: <i>P</i> <0.05. B. Cytochrome c (Cyt. C) release (red, AlexaFluor 555) and DNA fragmentation were respectively assessed by immunofluorescence and TUNEL assay after STS exposure in MCF-10A/Zeo and MCF-10A/ERα36 cells (left panel), then quantified as shown in the corresponding histogram (right panel). No cytochrome c release or DNA fragmentation can be detected in untreated cells (not shown). Each bar represents mean ± S.E.M. N = 3. *: <i>P</i> <0.05, ***: <i>P</i> <0.001.</p