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. Author manuscript; available in PMC: 2021 Dec 8.
Published in final edited form as: Sci Bull (Beijing). 2020 Mar 30;65(15):1281–1296. doi: 10.1016/j.scib.2020.03.040

Figure 2. YAP1 promotes proliferation, supports survival, but blocked gonadotropin-induced steroidogenesis of hGCs.

Figure 2.

(a) Left panel: Western blot showing high efficiency of YAP1 knockdown with YAP1-targeting siRNA1 and siRNA2. Non-targeting siRNA was used as control (siCTL); middle panel: representative pictures show morphological changes of cultured hGCs incubated in the growing medium for 5 days after knocking down of YAP1 protein, Scale bar: 200 μm; right panel: bar graph showing cell number change of hGCs incubated in growing medium for 5 days after knockdown of YAP1 protein. Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (P < 0.01). (b) Representative images showing changes in cell death rate in hGCs with or without (CTL) knockdown of YAP1 proteins. Cell death was determined with an Annexin V-FITC/PI staining kit and sorted by flow cytometry. Right bar graph showing the Annexin V positive hGCs with or without YAP1 knockdown. Each bar represents the mean ± SEM (n = 3). Bars with different letters are significantly different from each other (P < 0.01). (c) Left panel: western blot showing YAP1 and phosphorylated YAP1 (Ser127) expressions in hGC-MXIV, hGC-YAP, and hGC-YAPS127A cells. β-Actin was used as loading control; middle panel: Representative images showing the spheroids from hGC-MXIV, hGC-YAP, and hGC-YAPS127A cells incubated in a 3D-hanging drop culture system for 5 days, Scale bar: 500 μm; Right panel: Bar graph showing volumes of the spheroids from these cells. Each bar represents the mean ± SEM (n = 6). Bars with different letters are significantly different from each other (P < 0.01). (d) Hyperactivation of YAP1 compromised basal (CTL) and FSK-induced production of estrogen and progesterone in the primary culture of hGCs. Each bar represents the mean ± SEM (n ≥ 5). *: P < 0.05; ***: P < 0.001. FSK: forskolin (10 μmol/L). (e) Hyperactivation of YAP1 decreased basal (CTL) and FSK-induced transcription of CYP19A1 and CYP11A1 genes in the primary culture of human granulosa cells. Each bar represents the mean ± SEM (n ≥ 5). *: P < 0.05; ***: P < 0.001. (f) Hyperactivation of YAP1 reduced basal and FSH- and FSK-induced production of estrogen and progesterone in KGN cells. Each bar represents the mean ± SEM (n ≥ 4). ***: P < 0.001. (g) Western blot results showing that hyperactivation of YAP1 compromised FSH- and FSK-induced expression of key enzymes for steroidogenesis in KGN cells. Aroma: aromatase; P450SCC: P450 side-chain cleavage enzyme. β-Actin was used as a protein loading control.