Fig 1.
Schematic pathway of polyunsaturated fatty acid (PUFA) synthesis.
The map shows the elongation and desaturation steps of omega 3 (n-3) and omega 6 (n-6) fatty acids connected to the major actions of ELOVL5, ELOVL2, FADS1 and FADS2.
Fig 2.
The effect of E2 treatment on the expression of PUFA synthesis enzymes in MCF7 cells.
MCF7 cells were treated with 10 nM E2 and/or 10 μM ICI182,780 compared or vehicle (ethanol) for 6 hours. Elovl2, Elovl5, Fads2 and Fads1 mRNA levels (A, B, C and D) were determined by quantitative RT-PCR and normalized to the reference gene 36B4. Results shown are means ± SE of two individual experiments in triplicate. Statistical significances are indicated as *P<0.05 and **P<0.01.
Fig 3.
E2 time-response of PUFA synthesizing enzyme expression in MCF7 cells.
MCF7 cells were treated with 10 nM E2 or vehicle (c) for 0, 6, 12 or 24 hours and Elovl2, Elovl5, Fads1 and Fads2 mRNA levels (A, B, C and D) were determined by quantitative RT-PCR and normalized to the reference gene 36B4. Results shown are means ± SE of two individual experiments in triplicate. Statistical significances are indicated as *P<0.05 and **P<0.01.
Fig 4.
The effect of tamoxifen on PUFA synthesizing enzyme expression in MCF7 cells.
MCF7 cells were treated with 5 μM or 10μM tamoxifen with and without 10 nM E2 or vehicle (c) for 24 hours and Elovl2, Elovl5, Fads1 and Fads2 mRNA levels (A, B, C and D) were determined by quantitative RT-PCR and normalized to the reference gene 36B4. Results shown are means ± SE of two individual experiments in triplicate. Statistical significances are indicated as *P<0.05, **P<0.01 and ***P<0.001.
Fig 5.
ERα overexpression does not modify the expression of PUFA elongases and desaturases in MCF7 cells.
MCF7 cells were transfected with different concentrations (50ng or 500ng) of ERα or empty plasmid (V) as indicated for 24 hours followed by incubation with 10 nM E2 or vehicle (c) for 6 hours. (A) ERα, (B) ERβ, (C) Elovl2, (D) Elovl5, (E) Fads1 and (F) Fads2 mRNA expression were determined by quantitative RT-PCR normalized to the reference gene 36B4. Results shown are means ± SE of three individual experiments in triplicate. Statistical significances are indicated as *P<0.05, **P<0.01 and ***P<0.001.
Fig 6.
ERβ overexpression did not influence the expression of PUFA elongases and desaturases in MCF7 cells.
A) MCF7 cells were transfected with different concentrations (50ng or 500ng) of ERβ or empty plasmid (V) as indicated for 24 hours followed by incubation with 10 nM E2 or vehicle (c) for 6 hours. (A) ERα, (B) ERβ, (C) Elovl2, (D) Elovl5, (E) Fads1 and (F) Fads2 mRNA expression were determined by quantitative RT-PCR normalized to the reference gene 36B4. Results shown are means ± SE of two individual experiments in triplicate. Statistical significances are indicated as *P<0.05, **P<0.01 and ***P<0.001, n.d = not detectable.
Fig 7.
ERα knock-down reduces Elovl2 expresion in MCF7 cells.
MCF7 cells were transiently transfected with 50 nM ERα siRNA or siRNA control for 48 hours followed by incubation with 10 nM E2 (black bars) or vehicle (white bars) for 4 hours. (A) Western blot analysis using total protein extracts was performed to assess ERα and β-actin (control) protein levels. (B) Quantification of ERα protein levels, and (C) Elovl2 and (D) Elovl5 mRNA expression levels determined by quantitative RT-PCR normalized to the reference gene 36B4. Results shown are means ± SE of two individual experiments in duplicate. Statistical significances are indicated as *P<0.05, **P<0.01 and ***P<0.001.
Fig 8.
Binding of ERα to ChIP analysis of the Elovl2 enhancer in MCF7 cells.
(A) Schematic representation of two putative estrogen response elements, ERE1 and ERE2, within the Elovl2 enhancer with the primer pairs used for ChIP assays illustrated as black arrows denoted a, b and c with the “a” primer pair positioned 5’ adjacent to the ERE1, the “b” primer pair covering the ERE1, the “c” primer pair 3’ of the ERE1 and the “d” primer pair covering the ERE2 site. (B) Fold difference for ERα and IgG (control) binding for E2 and vehicle treated MCF7 cells using primer pair a, primer pair b, primer pair c and primer pair d. Statistical significances are indicated as *P<0.05, **P<0.01 and ***P<0.001.