Decreased expression of FOXA2 promotes eutopic endometrial cell proliferation and migration in patients with endometriosis Anping Lin, Juan Yin, Chao.

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Decreased expression of FOXA2 promotes eutopic endometrial cell proliferation and migration in patients with endometriosis Anping Lin, Juan Yin, Chao Cheng, Zhu Yang, Huan Yang Reproductive BioMedicine Online Volume 36, Issue 2, Pages 181-187 (February 2018) DOI: 10.1016/j.rbmo.2017.11.001 Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Expression of FOXA2 in eutopic endometrium tissues from endometriosis patients and controls. Upper panel shows average expression levels of FOXA2 protein in different samples measured by Western blot analysis (lower panel). GAPDH was used as loading control. Samples were from 14 patients with endometriosis (patient) and 16 healthy women (ctrl). All samples were assayed in triplicate. Values are expressed as mean ± SEM. ctrl = control; # indicates statistical significance (P < 0.05). Reproductive BioMedicine Online 2018 36, 181-187DOI: (10.1016/j.rbmo.2017.11.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Knockdown of FOXA2 by siRNA in ESC. ESC from 16 healthy women were transfected with FOXA2 siRNA. An example of FOXA2 expression analysed by Western blotting in shown in the upper panel, while the average protein level of FOXA2 is shown in the lower panel. GAPDH was used as loading control. ESC = endometrial stromal cells. ctrl = control, indicates ESC from healthy women were treated with culture medium; vector: ESC were transfected with vector; FOXA2 siRNA: ESC were transfected with FOXA2 siRNA. Values expressed as mean ± SEM. All samples were assayed in triplicate. # indicates statistical significance (P < 0.05). Reproductive BioMedicine Online 2018 36, 181-187DOI: (10.1016/j.rbmo.2017.11.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Effect of FOXA2 on cell viability. Cell viability assays were performed using the MTT method on ESC transfected with FOXA2 siRNA. All samples were assayed in triplicate. ctrl = control, ESC were treated with culture medium; negative control: ESC were transfected with negative siRNA; FOXA2 siRNA, ESC were transfected with FOXA2 siRNA; ESC = endometrial stromal cells. # indicates statistical significance (P < 0.05). Reproductive BioMedicine Online 2018 36, 181-187DOI: (10.1016/j.rbmo.2017.11.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Effect of FOXA2 on cell apoptosis. ESC were transfected with FOXA2 siRNA, then the rate of apoptotic cells was detected by flow cytometry as described in Materials and methods. ESC = endometrial stromal cells; A: ctrl = control, ESC were treated with culture medium; B: vector, ESC were transfected with vector; C: negative ctrl, ESC were transfected with negative siRNA; D: FOXA2 siRNA, ESC were transfected with FOXA2 siRNA; E: data were expressed as mean ± SEM, # P < 0.05 indicates compared with ctrl. All samples were assayed in triplicate. Reproductive BioMedicine Online 2018 36, 181-187DOI: (10.1016/j.rbmo.2017.11.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Effect of FOXA2 on cell migration. Confluent of FOXA2 siRNA transfected ESC monolayers were wounded with a 10 µl pipette tip and cultured for 24 h. The black line indicates the wound edge, cell migration to the centre of the wound was observed using an inverted microscope, as shown on the left-hand panel (×100 magnification). Then, migrated cells were counted in the wounding area by a predefined frame. Data were analysed and are shown in the right-hand panel. All samples were assayed in triplicate. ESC: endometrial stromal cells; A: control, ESC were treated with culture medium; B: vector, ESC were transfected with vector; C: negative ctrl, ESC were transfected with negative siRNA; D: ESC were transfected with FOXA2 siRNA. Reproductive BioMedicine Online 2018 36, 181-187DOI: (10.1016/j.rbmo.2017.11.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Effect of progesterone on FOXA2 expression. ESC were treated with P4 (0, 10−7 to 10−5 M) for 24 h, respectively, mRNA level of FOXA2 was determined by real-time PCR. GAPDH was used as an endogenous control to normalize for differences. P4 at 0 M are the ctrl. All samples were assayed in triplicate. ctrl = control; ESC = endometrial stromal cells; # indicates statistical significance (P < 0.05). Reproductive BioMedicine Online 2018 36, 181-187DOI: (10.1016/j.rbmo.2017.11.001) Copyright © 2017 Reproductive Healthcare Ltd. Terms and Conditions