Journal: International Journal of Molecular Sciences

Article Title: Polyamine Oxidase Expression Is Downregulated by 17β-Estradiol via Estrogen Receptor 2 in Human MCF-7 Breast Cancer Cells

doi: 10.3390/ijms23147521

Figure Lengend Snippet: E2-dependent PAOX downregulation is mediated by ESR2. MCF-7 cells were incubated in 10% CS-FBS in PRF-DMEM in the presence of DMSO (-; 100 µM), ESR1 antagonist MPP (M; 100 µM), ESR2 antagonist PHTPP (P; 100 µM), G protein-coupled ER antagonist G-15 (G; 100 µM), or non-specific ESR antagonist ICI 182,780 (I; 100 µM) for 5 h. MCF-7 cells were then incubated in 10% charcoal-stripped FBS in PRF-DMEM, with or without E2. Protein levels of PAOX, GREB1, and GAPDH were evaluated using Western blotting ( A ). The mRNA levels of PAOX , GREB1 , and GAPDH were evaluated by conventional (top) and quantitative (bottom) PCR ( B ). MCF-7 cells were transfected with siRNA for Con (scrambled), ESR1 , or ESR2 knockdown for 5 h. Protein levels of PAOX, ESR1, ESR2, GREB1, and GAPDH were evaluated using Western blotting ( C ). The mRNA expression levels of PAOX , GREB1 , and GAPDH were evaluated by conventional (top) and quantitative (bottom) PCR ( D ). Data are shown as the mean ± S.D. ( n = 3), normalized to GAPDH expression. * p < 0.05; ** p < 0.01; and *** p < 0.001 versus control.

Article Snippet: The following antibodies were used: anti-PAOX (MBS3211539), obtained from MyBioSource (San Diego, CA, USA); anti-ESR1 (sc-8002) and anti-ESR2 (sc-373885), obtained from Santa Cruz Biotechnology (Cambridge, UK); anti-GREB1 (ab-72997), obtained from Cell Signaling (Danvers, MA, USA); anti-c-JUN (2250S) and anti-c-FOS (9165S), obtained from Abcam (Cambridge, UK); anti-GAPDH (AbC-2003), obtained from AbClone (Seoul, Korea); and the horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG described previously [ ].

Techniques: Incubation, Western Blot, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Polyamine Oxidase Expression Is Downregulated by 17β-Estradiol via Estrogen Receptor 2 in Human MCF-7 Breast Cancer Cells

doi: 10.3390/ijms23147521

Figure Lengend Snippet: ESR2 interacts with AP-1, which binds to the AP-1 binding sites in the PAOX promoter in an E2-dependent manner. Primer pairs for PCR amplification of the AP-1 binding site and the half-ERE binding site in the PAOX promoter are indicated by arrows ( A ). The regions for PCR were −2896 to −2710 for the distal AP-1 site, −1271 to −1080 for the proximal AP-1 site, −2730 to −2477 for the distal putative half-ERE site, and −1100 to −1003 for the proximal putative half-ERE sites. Chromatin extracts were prepared from MCF-7 cells treated with or without E2, and immunoprecipitation of the extracts was performed using the indicated antibodies for ChIP assays ( B ). Chromatin extracts were immunoprecipitated using an anti-ESR1 antibody or anti-ESR2 antibody and then with control anti-IgG, anti-c-JUN, or anti-c-FOS antibody for Re-IP assays ( C ). PCR was performed with the above primer pairs, using DNA extracted from the immunoprecipitates as templates. PCR products were analyzed using 5% PAGE gels.

Article Snippet: The following antibodies were used: anti-PAOX (MBS3211539), obtained from MyBioSource (San Diego, CA, USA); anti-ESR1 (sc-8002) and anti-ESR2 (sc-373885), obtained from Santa Cruz Biotechnology (Cambridge, UK); anti-GREB1 (ab-72997), obtained from Cell Signaling (Danvers, MA, USA); anti-c-JUN (2250S) and anti-c-FOS (9165S), obtained from Abcam (Cambridge, UK); anti-GAPDH (AbC-2003), obtained from AbClone (Seoul, Korea); and the horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG described previously [ ].

Techniques: Binding Assay, Amplification, Immunoprecipitation