Fig 1: PFN1 and ALOX5 immunolocalization in endometrium. (a–d) Representative photomicrographs of endometrial biopsies showing localization of PFN1 protein in the stroma (s), luminal (le) and glandular (ge) epithelium in the proliferative (a,b) and late secretory (c,d) phases. (e–g) PFN1 immunostaining intensity scoring in the luminal epithelium (e, n = 3), glandular epithelium (f, n = 3) and stroma (g, n = 3). (h–k) Representative photomicrographs of endometrial biopsies showing localization of ALOX5 protein in the stroma, luminal and glandular epithelium in the proliferative (h–i) and late secretory (j–k) phases. (l–n) ALOX5 immunostaining intensity scoring in the luminal epithelium (l, n = 4), glandular epithelium (m, n = 4) and stroma (n, n = 4). Inserts show negative staining. Data are mean ± SEM, *p < 0.05, significant difference from proliferative phase; e-g;l-n: Mann Whitney t-test.
Fig 2: EVT CM enhanced HESC decidualization via profilin 1 (PFN1). (a) HESCs (n = 4) were treated with E for 14 days and CM treatments (Hek293, HTR8[/SVneo], EVT) included from day 7. PRL secretion was measured on day 14. PRL secretion was significantly elevated by factors in EVT CM compared to media alone or cell line control CMs. (b) HESCs (n = 4) were treated with E+MPA for 14 days with CM treatments (Hek293, HTR8, EVT) included from day 7. PRL secretion was measured on day 14. PRL secretion was elevated in HESC treated with EVT CM compared to Hek293 control CM but not significantly changed compared to media alone or HTR8/SVneo CM. (c) Only EVT CM contained progesterone (n = 5). (d) PRL secretion by HESC was significantly higher when treated with EVT CM compared to a range (0–300 nmol/L) of MPA concentrations (n = 3/group). (e–k) HESCs were decidualized with E+MPA for 12 days before being subjected to functional assays with CM or PFN1 treatments. (e–g) Decidualized HESC adhesion (e, n = 3), proliferation (f, n = 4) and migration (g, n = 6) were significantly enhanced by treatment with EVT CM. (h) PFN1 treatment during decidualization (days 7–14 of E+MPA treatment) significantly increased HESC PRL secretion on day 14 (h, n = 4). i. PFN1 treatment significantly enhanced proliferation of decidualized HESC (n = 3). (j,k) PFN1 treatment had no effect on decidualized HESC adhesion (j; n = 3) or migration (k; n = 4). Data are mean ± SEM, *p < 0.05, significant difference from control; ^p < 0.05 significant difference between treatments labelled with ^. a,b,d: Friedman test; d,e,f,g,h: paired t-test; i, j,k: repeated measures one-way ANOVA.
Fig 3: Exogenous profilin 1 decreased HESC ALOX5 expression. (a) PFN1 significantly decreased ALOX5 mRNA in minimally decidualized (MD) HESC (decidualized for 2 days, do not secrete detectable PRL; n = 4–6/group) but had no significant effect on decidualized (D) HESC (decidualized for 10–12 days, secrete PRL). (b) ALOX5 mRNA expression declined during in vitro HESC decidualization (n = 4–10/group). (c) Prolactin confirmation of decidualization (n = 4–10/group). mRNA expression was determined by semi-quantitative PCR normalized to 18S. Data are mean ± SEM, *p < 0.05, significant difference from control. a: paired t-test; b,c: one-way ANOVA.
Fig 4: Exogenous profilin 1 decreased THP-1 ALOX5 expression. (a) ALOX5 mRNA was decreased after 24 hr treatment with PFN1 (100 μM) in THP-1 cells (n = 4). ALOX5 protein was decreased after (b) 48 h (n = 3) and (c) 72 hr (n = 3) of PFN1 (100 µM) treatment on THP-1 cells determined by Western blot normalized to GAPDH. mRNA expression was determined by semi-quantitative PCR normalized to 18S. Data are mean ± SEM, *p < 0.05, significant difference from control. a-c: student’s t-test.
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