Journal: Journal of Biological Chemistry

Article Title: Xanthine Oxidoreductase Promotes the Inflammatory State of Mononuclear Phagocytes through Effects on Chemokine Expression, Peroxisome Proliferator-activated Receptor-γ Sumoylation, and HIF-1α

doi: 10.1074/jbc.m110.150847

Figure Lengend Snippet: FIGURE 9. Inhibition of XOR activity increases levels of HIF-1 protein in rat inflammatory MNP and in PMA-differentiated U937 cells. A, I-MNP were purified from rat lungs 24 h following insufflation of Th-1 cytokines and were plated in 12-well plates at 1.0 106 cells/well and grown under normoxic conditions. Nonadherent cells were removed by washing after 1 h, and cells were treated with the indicated doses of MIG132. Cells were harvested after 6 h; whole cell lysates were prepared and Western immunoblots run with antibody to HIF-1. In addition, washed, adherent cells were exposed to MIG132 (50 M) for the indicated times, and Western immunoblots were run on whole cell lysates and probed with antibody to HIF-1. Blots were subsequently stripped and re-probed with antibody to GAPDH to control for protein loading. B, I-MNP were purified and plated as in A. Plates were placed in either nor- moxic (21% O2, 5% CO2) or hypoxic culture (1% O2, 5% CO2, 94% N). After 1 h, cells were treated with the XOR inhibitors allopurinol (Allo, 150 M), oxypuri- nol (Oxy, 150 M), or Y-700 (50 nM). One h later cells were treated with MIG132 (50 M) and grown for 6 h. Whole cell lysates were then prepared and West- ern immunoblots run with independent duplicate samples as indicated. Blots were first probed with antibody to HIF-1 and subsequently with antibody to GAPDH. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of duplicate samples. C, I-MNP were purified, plated, and grown in normoxia in the presence of MIG132 as in A. Cells were exposed to the XOR inhibitor Y-700 (50 nM) for 1 h and subsequently treated with IL-1 (10 ng/ml), IFN- (20 ng/ml), or LPS (1.0 g/ml). Whole cell lysates were pre- pared and Western immunoblots run after 24 h of exposure to cytokines. Blots were run on triplicate samples, and representative blots are shown. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of triplicate blots. D, U937 cells were plated in 12-well plates at 1 106 cells/well and treated with PMA (30 nM) for 48 h. Cells were then washed; the medium was replaced, and cells were grown under normoxic or hypoxic conditions for 1 h in the presence of 50 M MIG132. Subsequently, cells were treated with Y-700 (50 nM) for 1 h and cytokines added as above. Whole cell lysates were prepared after 24 h of exposure to cytokine/MIG132, and Western immunoblots were run sequentially with antibody to HIF-1 and GAPDH. Representative blots are shown of three independent blots for each experiment. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of triplicate blots. E, I-MNP were purified and plated in normoxia as in B. Levels of VEGF were quantitated in the cell-free supernatant over a period of 48 h in culture. In addition, cells were treated independently with MIG132 in the absence or presence of allopurinol, oxypurinol, or Y-700 as in B, and levels of VEGF were measured in the cell-free supernatant 6 or 24 h after treatment. Data show the mean S.D. of three independent experiments. F, I-MNP were plated as above in the presence or absence of Y-700 (1.0 mM) and in the absence of MIG132. After 24 h, cell-free supernatants were collected and analyzed with the rat-specific proteome profiler. Experiments were performed in quadruplicate, and each cytokine was analyzed from two spots on each filter (boxed for VEGF), and thus all data reflect eight independent determinations for VEGF alone. Spots were quantitated as described by the supplier using an R&D transmission mode scanner and image analysis software from R&D. Data show the mean S.D. of light transmission signals (arbitrary units) from eight spots for VEGF only and both control and Y-700 groups and were normalized first to the mean positive control spots (A1, A2, A19, A20, D1, and D2) and subsequently to the signal obtained from the control samples, which was thereby set at 1.00. ***, p 0.02.

Article Snippet: VEGF was also measured using the rat specific proteome profiler array (catalog no. ARY008, R&D Systems, Inc.).

Techniques: Inhibition, Activity Assay, Purification, Western Blot, Control, Transmission Assay, Software, Positive Control

Journal: Scientific Reports

Article Title: S100A8 regulated by estrogen improves injured endometrial epithelium reconstruction by promoting tight junction formation and stromal cell transformation

doi: 10.1038/s41598-025-08530-0

Figure Lengend Snippet: Effects of estrogen treatment on S100A8 expression in endometrium and epithelium morphology. ( a ) S100A8 immunohistochemical staining before and after estrogen treatment (1d/7d); black arrows indicate S100A8-positive immune cells (S100A8-PICs).The blue arrow indicates the release of S100A8 from S100A8-PICs. Scale bar is 100 or 400 μm. ( b ) Rat endometrial hematoxylin–eosin staining before and after estrogen treatment (1d/7d); red arrows show disrupted cell junctions. Scale bar is 100 μm. ( c ) S100A8-PICs number in the endometrium (1/mm 2 ) before and 1 d/7d after estrogen treatment (n = 18, from six rats, the number was counted from three immunohistochemical images of each sample , mean ± SEM). ( d ) S100A8 expression in the epithelium before and 1 d/7d after estrogen treatment (n = 18, from six rats, the expression level was measured from three immunohistochemical images of each sample, mean ± SEM). * Results with P values < 0.05 were considered significant. ** Results with P values < 0.01 were considered very significant.

Article Snippet: The cultured cells were randomly divided into three groups according to the experimental design: the control, S100A8 (0.5 μg/mL, HY- P71275 ; MCE, Shanghai, China), and S100A8 + FPS-ZM 1 (5 μM, HY-19370; MCE, Shanghai, China) groups.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: S100A8 regulated by estrogen improves injured endometrial epithelium reconstruction by promoting tight junction formation and stromal cell transformation

doi: 10.1038/s41598-025-08530-0

Figure Lengend Snippet: 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.

Article Snippet: The cultured cells were randomly divided into three groups according to the experimental design: the control, S100A8 (0.5 μg/mL, HY- P71275 ; MCE, Shanghai, China), and S100A8 + FPS-ZM 1 (5 μM, HY-19370; MCE, Shanghai, China) groups.

Techniques: Control

Journal: Scientific Reports

Article Title: S100A8 regulated by estrogen improves injured endometrial epithelium reconstruction by promoting tight junction formation and stromal cell transformation

doi: 10.1038/s41598-025-08530-0

Figure Lengend Snippet: 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.

Article Snippet: The cultured cells were randomly divided into three groups according to the experimental design: the control, S100A8 (0.5 μg/mL, HY- P71275 ; MCE, Shanghai, China), and S100A8 + FPS-ZM 1 (5 μM, HY-19370; MCE, Shanghai, China) groups.

Techniques: Control, Immunohistochemical staining, Cell Culture, In Vitro

Journal: Scientific Reports

Article Title: S100A8 regulated by estrogen improves injured endometrial epithelium reconstruction by promoting tight junction formation and stromal cell transformation

doi: 10.1038/s41598-025-08530-0

Figure Lengend Snippet: 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.

Article Snippet: The cultured cells were randomly divided into three groups according to the experimental design: the control, S100A8 (0.5 μg/mL, HY- P71275 ; MCE, Shanghai, China), and S100A8 + FPS-ZM 1 (5 μM, HY-19370; MCE, Shanghai, China) groups.

Techniques: Immunohistochemical staining, Staining, Control, Membrane, Expressing, Western Blot, Immunofluorescence

Journal: Scientific Reports

Article Title: S100A8 regulated by estrogen improves injured endometrial epithelium reconstruction by promoting tight junction formation and stromal cell transformation

doi: 10.1038/s41598-025-08530-0

Figure Lengend Snippet: 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.

Article Snippet: The cultured cells were randomly divided into three groups according to the experimental design: the control, S100A8 (0.5 μg/mL, HY- P71275 ; MCE, Shanghai, China), and S100A8 + FPS-ZM 1 (5 μM, HY-19370; MCE, Shanghai, China) groups.

Techniques: Double Staining, Cell Culture, Western Blot, Expressing, Control, Molecular Weight, Immunofluorescence