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. 2019 Jan 21;9(3):620–632. doi: 10.7150/thno.30655

Figure 5.

Figure 5

ANTXR2 promotes YAP1 activation via inhibition of Hippo pathway. (A) ANTXR2 was knocked down by siRNA (40 nM) for 48 h in ectopic stromal cells. Representative Western blots (left panel) showed the levels of ANTXR2, phosho-YAP1 (P-YAP1), and total-TAP1 (T-YAP1) in ectopic stromal cells. Quantified results (right panel) showed the ratio of phosho-YAP1 (P-YAP1) and total-TAP1 (T-YAP1) in ectopic stromal cells (n = 3). (B) Representative Western blots (left panel) and quantified result (right panel) showed the levels of phosho-YAP1(P-YAP1) and total-TAP1 (T-YAP1) in ectopic stromal cells treated with different doses of PGG for 24 h (n = 3). (C) Immunocytochemistry staining shows the retention of YAP1 in cytosol of cells treated with PGG. Cells were treated with 10% DMSO or PGG (5 μM in 10%DMSO) for 24 h. Scale bar: 20 μm. (D, E) Levels of YAP1 downstream target genes (CTGF and BIRC2) were analyzed in knockdown control (siNC) and ANTXR2-knocked down (siANTXR2) ectopic stromal cells (D) and PGG treated ectopic stromal cells (E) by using quantitative RT-PCR (n = 4). (F) YAP1 downstream target genes associated with cell adhesive function were analyzed in eutopic stromal cells treated with control or ANTXR2 siRNA with or without YAP1 expression construct (n = 4). (G) Ectopic endometriotic stromal cells were treated with control or siRNA against YAP1 (40 nM) for 48 h. Then, ChIP-PCR was performed to quantify the binding of YAP1 on CD44 and Col5A2 loci (n = 4). (H) Heat map shows the correlation between ANTXR2, YAP1, and its potential downstream target genes associated with cell adhesion in clinical endometriotic dataset (GSE51981). Correlation values were calculated by Pearson's correlation test. Asterisk (*) indicates P < 0.05. Results were presented as means ±SEM.