Fig. 1

In vitro functional analysis of HOXC8-overexpressing immortalized endometrial stromal cells (HOXC8-imESCs). (A) HOXC8 mRNA expression in Control-imESCs and HOXC8-imESCs analyzed by real-time RT-PCR. Experiments were performed three times in triplicate, with GAPDH used as an internal control. The relative HOXC8 expression in HOXC8-imESCs was normalized to Control-imESCs as 1 and is presented as mean ± SD. *p < 0.05. (B) Western blot analysis of HOXC8 protein in Control-imESCs and HOXC8-imESCs. TUBB was used as an internal control. The images of HOXC8 and TUBB are cropped from different blots. The original blot images are presented in Supplementary Figure S1. (C) Cell proliferation assay in Control-imESCs and HOXC8-imESCs. Cell numbers were counted at 24, 48, 72, and 96 hours after seeding and are shown as mean ± SD. Experiments were performed three times in triplicate. (D) Cell migration assay in Control-imESCs and HOXC8-imESCs. Representative images for each cell line are shown. Scale bar = 100 μm. Migrated cells were counted in four randomly selected fields at 200× magnification and are shown as mean ± SD. Experiments were repeated three times. *p < 0.05. (E) Cell invasion assay in Control-imESCs and HOXC8-imESCs. Representative images for each cell line are shown. Scale bar = 100 μm. Invaded cells were counted in four randomly selected fields at 200× magnification. Experiments were repeated three times. Invasion rate (%) was calculated by dividing the number of invaded cells by the number of migrated cells, multiplied by 100, and shown as mean ± SD. *p < 0.05. (F) Wound healing assay in Control-imESCs and HOXC8-imESCs. Representative images at 0 and 24 hours after scratching are shown. Scale bar = 50 μm. Wound healing rate (%) was calculated as the area covered by migrating or proliferating cells at 24 hours divided by the initial wound area, multiplied by 100, and presented as mean ± SD. Experiments were performed three times in triplicate. *p < 0.05 . (G) Collagen gel contraction assay in Control-imESCs and HOXC8-imESCs. Representative images at 24 hours after collagen gel culture are shown. The contraction area was calculated by subtracting the gel area from the whole well area. The contraction rate (%) was calculated by dividing the contraction area by the whole well area, multiplied by 100, and presented as mean ± SD. Experiments were performed three times in triplicate. *p < 0.05