Supplemental Fig. S1
The distribution of oxytocin (OXT) receptor expression according to the lobe of normal mouse pituitary gland. Immunohistochemical staining shows the different expression of OXT receptor according the lobe of normal mouse pituitary gland (n=2, ×100). AL, anterior lobe; IL, intermediate lobe; NL, neural lobe.
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Supplemental Fig. S2
Effects of oxytocin (OXT) and OXT receptor antagonist on cell viability. AtT20 and GH3 cells were treated with various concentrations of OXT (0 to 500 nM) for 24 and 48 hours, respectively. (A, C) These panels demonstrate the effects of OXT on cell viability in AtT20 and GH3 cell lines, respectively. (B, D) These panels show the effects of OXT with or without OXT receptor antagonist L-368,899 (0 to 500 nM) on proliferation of AtT20 and GH3 cells, respectively. Data are presented as mean±standard error of the mean (n=3 to 4). aP<0.05 vs. OXT 0 nM.
enm-34-302-s002.pdf
Fig. 1Expression of oxytocin (OXT) receptor in normal mouse pituitary gland and immunofluorescence staining of OXT receptor in AtT20 and GH3 cells. (A, B) Immunohistochemical staining shows that normal mouse pituitary gland was stained positively for OXT receptor (n=2, ×40 and ×200, respectively). (C) It indicates positive control for OXT receptor staining in uterine endometrial tissue (n=3, ×200). (D) It indicates immunofluorescence co-staining of OXT receptor (red color) and corticotropin-releasing hormone receptor (green color) in AtT20 cells (magnification, ×630). (E, F) These panels show the staining for OXT receptor (green color) in AtT20 and GH3 cells (×400), respectively. AL, anterior lobe.
Fig. 2Cell cycle analysis in AtT20 cells. AtT20 cells were treated with oxytocin (OXT) and/or OXT receptor antagonist L-368,899 for 6 or 24 hours, and cells were stained with propidium iodide. DNA contents were analyzed by flow cytometer to assess cell cycle phases (n=3). (A) Flow cytometry of AtT20 cells treated with OXT and/or OXT receptor antagonist for 6 or 24 hours. (B, C) Representative histogram data of the cell cycle analysis of AtT20 cells treated with OXT and/or OXT receptor antagonist for 6 or 24 hours. PI-A, Propidium iodide-Area. aP<0.05 vs. control group; bP<0.05 vs. L-368,899 treatment group.
Fig. 3Effects of oxytocin (OXT) on gene expression of (A) OXT receptors and (B) pro-opiomelanocortin (POMC) in AtT20 cells. AtT20 cells were exposed to OXT (50 nM) for 30 minutes, 1, 3, 6, 12, and 24 hours. mRNA levels of POMC and OXT receptor genes were estimated at each time point using quantitative real-time polymerase chain reaction. Data are presented as mean±standard deviation (n=4 to 6). aP<0.05 vs. control group.
Fig. 4Effects of oxytocin (OXT) treatment on adrenocorticotropic hormone (ACTH) secretion in AtT20 cells. To determine ACTH level, conditioned medium was harvested and analyzed by enzyme-linked immunosorbent assay. X-axis indicates control and OXT treatment groups according to time point. Y-axis indicates ACTH level per 105 cells. Data are presented as mean±standard error of the mean (n=3). aP<0.05 vs. control group.
Fig. 5Effects of oxytocin (OXT) on proliferating cell nuclear antigen (PCNA) and extracellular-signal-regulated kinase 1/2 (ERK1/2) in AtT20 cells. AtT20 cells were incubated with (A) 50 nM OXT and/or (B) 10 nM OXT receptor antagonist L-368,899 for 24 hours, and protein levels of PCNA and ERK1/2 in cells were examined by Western blot analysis. Data are presented as mean±standard error of the mean (n=3). P-ERK, phosphorylated extracellular-signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05 vs. control group.
Fig. 6Effects of oxytocin (OXT) on proliferating cell nuclear antigen (PCNA) and extracellular-signal-regulated kinase 1/2 (ERK1/2) in GH3 cells. GH3 cells were incubated with (A) 50 nM OXT and/or (B) 10 nM OXT receptor antagonist L-368,899 for 24 hours, and protein levels of PCNA and ERK1/2 in cells were examined by Western blot analysis. Data are presented as mean±standard error of the mean (n=3). P-ERK, phosphorylated extracellular-signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. aP<0.05 vs. control group.