Fig 2: Effects of S100A8 on the distribution of Ki-67-positive proliferating cells in the uteri and the proliferation of endometrial cells. (a) Ki-67-positive proliferating cells were distributed in the endometrium or uterine interior wall in all groups (control, injury, P407, and S100A8 + P407 groups). Scale bar is 100 or 400 μm. (b) Number of Ki-67-positive cells in the endometrium of each group (n = 20, from ten rats, the number was counted from two immunohistochemical images of each sample, mean ± SEM). (c) Number of Ki-67-positive cells in the uterine interior wall of each group (n = 20, from ten rats, the number was counted from two immunohistochemical images of each sample, mean ± SEM). (d) Effects of S100A8 and its receptor inhibitor (FPS-ZM1) on the proliferation of cultured endometrial cells in vitro (n = 5, mean ± SEM). Scale bar is 100 μm. * Results with P values < 0.05 were considered significant. ** Results with P values < 0.01 were considered very significant.

Fig 3: Effects of S100A8 on tight junctions of endometrial epithelium or endometrial cells. (a) Immunohistochemical staining showing Claudin-1 distribution in the endometrium of different groups (control, injury, P407, and P407 + S100A8 groups); black arrows show Claudin-1 accumulated below the cell membrane of the lateral contact zone between adjacent cells and red arrows show cells expressing Claudin-1 in the stroma. Scale bar is 100 μm. (b) Claudin-1 expression in the endometrium of different groups (n = 10, mean ± SEM). (c) Western blot showing ZO-1 expression in endometrial cells of three different groups (control, S100A8, and S100A8 + FPS-ZM1 groups, n = 3, mean ± SEM). Actin was used as the loading control. The full-length bands can be found in supplementary Fig. 1. The bands of ZO-1 and actin cropped from different parts of the same gel. (d) Immunofluorescence showing the co-expression of Claudin-1 (red) and ZO-1 (green) in the endometrial cells of the three groups (n = 5, mean ± SEM). The nucleus is stained blue. The scale bar is 200 μm. * Results with P values < 0.05 were considered significant. ** Results with P values < 0.01 were considered very significant.

Fig 4: Effects of uterine cavity administration of S100A8 on the distribution of S100A8-PICs and endometrial morphology. (a) Distribution of S100A8-positive cells in the endometrium of four different groups with estrogen treatment (control, injury, P407, and S100A8 + P407 groups). Black arrows show S100A8-PICs. S100A8-PICs were distributed around the glands and blood vessels in the control group. S100A8-PICs in the site of injury or adhesion in the injury group and in the P407 group. S100A8-PICs reverse migrated to the glands or blood vessels in the S100A8 + P407 group. Scale bar is 100 μm or 1 mm. (b) Endometrial thickness in the four groups (n = 15, from 5 rats; 3 position was measured randomly in an image of each rat, mean ± SEM). (c) The number of endometrial glands in each group (n = 10, from 10 rats, glands were counted from a uterine horn cross-section image at 40 × of each rat, mean ± SEM). (d) The interior wall integrity of uterine cavity in each group (n = 10, from 10 rats, data was measured from a uterine horn cross-section image at 40 × of each rat, mean ± SEM). (e) The number of endometrial crypts in each group (n = 10, from 10 rats, crypts were counted from a uterine horn cross-section image at 40 × of each rat, mean ± SEM). * Results with P values < 0.05 were considered significant. ** Results with P values < 0.01 were considered very significant.

Fig 5: Effects of S100A8 on endometrial “double-featured” cells. (a) Vimentin (red) and ZO-1 (green) immunofluorescent double staining of cultured endometrial cells. Scale bar is 200 μm. (b) Western blot showing CK-18 and vimentin expression in endometrial cells from each group (control, S100A8, and FPS-ZM1 groups; n = 3, mean ± SEM). The full-length bands can be found in supplementary Fig. 1. Actin was used as the loading control. As the target proteins have a similar molecular weight to that of the loading control, the bands of CK18/vimentin and actin were cropped from different gels. (c) Immunofluorescence showing vimentin expression in endometrial cells of each group (n = 5, mean ± SEM). Scale bar is 200 μm. * Results with P values < 0.05 were considered significant. ** Results with P values < 0.01 were considered very significant.