Fig 2: CNTF and CNTFRa immunofluorescence in HTR-8/SVneo (a–f) and BeWo (g–l) cells. CNTF immunostaining (green) is localized in the cytoplasm and nucleoplasm of both HTR-8/SVneo (b–c) and BeWo (h–i) cells, whereas the nucleoli are CNTF-negative. CNTFRa immunostaining (red) is localized in the cytoplasm and is especially intense in the perinuclear region of both HTR-8/SVneo (e–f) and BeWo (k–l) cells. The nuclei are stained blue. a–c, g–l Bars = 100 mm; d–f bars = 50 mm. Negative control: c’, f’, i’, l’ bars = 100 mm (HTR-8/SVneo) and 50 mm (BeWo)

Fig 3: Wound closure experiments. CNTF did not affect HTR-8/SVneo cell mobility. a–d, a’–d’ Representative photographs show the effect of CNTF treatment on cell migration at different time points (0, 4, 8, 24 h). e The graph, representing the cell-free area, shows that CNTF treatment did not affect wound closure. In treated and untreated cells, wound closure was completed in 24 h. Results are expressed in arbitrary units (A.U.) and reported as histogram bars. Bars = 100 mm. Data are mean ± SD (n = 3)

Fig 4: Immunohistochemistry of CNTF (a–d) and CNTFRa (e–h) in normal first-trimester (a, b, e, f) and third-trimester (c, d, g, h) placentas. a In first-trimester placental villi, CNTF is expressed in villous cytotrophoblast cells (arrows) whereas the syncytiotrophoblast is negative. b The extravillous cytotrophoblast cells are CNTF-positive, showing intense staining in the proximal portion of cell columns, near the villus (*). In third-trimester placental villi, only the syncytiotrophoblast (arrowheads) shows immunostaining (c, d). In (d), a terminal villus shows that the syncytiotrophoblast is CNTF-positive. In first-trimester placental villi, CNTFRa (e, f) is expressed in villous cytotrophoblast cells (arrows) whereas the syncytiotrophoblast is negative. Foetal vessels are CNTFRa-positive (e: V). g, h In third-trimester placental villi, only the syncytiotrophoblast (arrowheads) shows CNTFRa immunostaining. i, j Negative controls. b Bar = 50 mm; c, e, g, i, j bars = 100 mm; a, f, h bars = 30 mm; d bar = 25 mm

Fig 5: In HTR-8/SVneo and BeWo cells, CNTF induced pSTAT3 upregulation and pERK downregulation. Left, Western blots (a–f); right, histograms (a’–f’) showing band quantification. a, a’, b, b’, c, c’ HTR-8/SVneo cells and d, d’, e, e’, f, f’ BeWo cells after treatment with 20, 50 or 100 ng/ml rhCNTF. a’, d’ significant increase in pSTAT3 protein levels; b’, e’ unchanged pAKT protein levels; c’, f’ significant reduction in pERK protein levels. Results are expressed in arbitrary units (A.U.) and reported as histogram bars. pSTAT3, pAKT and pERK1/2 quantities were normalized using total STAT3, AKT and ERK1/2, respectively. Data are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001