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

Journal: The Journal of Biological Chemistry

Article Title: HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis *

doi: 10.1074/jbc.M116.714147

Figure Lengend Snippet: HBP1-mediated activation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to HBP1-induced premature senescence and tumor inhibition. A , the protein levels of HBP1, p53, EZH2, and p21 during replicative senescence. Shown is an immunoblot ( IB ) of WI-38 cells in young and old stages. The expression of endogenous HBP1, p53, β-catenin, EZH2, Wnt2, p21, and p16 is shown. GAPDH was used as a loading control ( left panel ). WI-38 cells were lysed with IP lysis buffer and then subjected to immunoprecipitation with anti-HBP1 antibody followed by Western blotting analysis with anti-Mdm2 and anti-TCF4 antibodies ( right panel ). B , exogenous EZH2 inhibits p21 expression. The protein levels of EZH2, p53, and p21 were measured by immunoblotting in WI-38 cells (PD20) retrovirally transduced with EZH2 or control vector. C , EZH2 attenuates HBP1 elevation of p21 when p53 is knocked down by shRNA. The levels of EZH2, p53, p21, and GAPDH (as a control) were determined by Western blotting of WI-38 cells retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. D and E , EZH2 attenuates HBP1-induced cell growth arrest when p53 is knocked down by shRNA. MTT ( D ) and BrdU ( E ) incorporation assays were conducted with WI-38 cells retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. The means ± S.E. for three independent experiments are shown. OD , optical density. *, p < 0.05; **, p < 0.01. F , EZH2 attenuates HBP1-induced premature senescence when p53 is knocked down by shRNA. WI-38 cells (PD20) were retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. Cells were then stained for SA β-gal on day 14 after transduction ( left panel ). The percentage of cells positive for SA β-gal in WI-38 cells was determined in three independent experiments and expressed as mean ± S.E., with 300 cells/experiment. *, p < 0.05 ( right panel ). G , EZH2 attenuates HBP1 elevation of p21 in the absence of p53. Levels of EZH2, p53, p21, and GAPDH (as a control) were determined by Western blotting of A549 cells retrovirally transduced with control vector or HBP1 or H1299 cells retrovirally transduced with control vector, HBP1, or HBP1 + EZH2. H , EZH2 attenuates HBP1-induced growth inhibition. A549 and H1299 cells were stably transfected with either control vector, HBP1, or HBP1 + EZH2. After puromycin or G418 selection, the growth rates of cells were measured by MTT assay. The mean ± S.E. for three independent experiments are shown. I , EZH2 attenuates HBP1-induced premature senescence. A549 and H1299 cells stably transfected with either control vector, HBP1, or HBP1 + EZH2 were subjected to an SA β-gal staining assay ( left panel ). The percentage of cells positive for SA β-gal was determined in three independent experiments and expressed as mean ± S.E., with 300 cells/experiment. **, p < 0.01 ( right panel ). J , soft agar colony formation assay of A549 and H1299 cells stably transfected with control vector and HBP1 or HBP1 + EZH2. Cells were cultured in soft agar for 2 weeks ( left panel ). The colony numbers in three different microscope fields were counted and are shown as mean ± S.E. *, p < 0.05; **, p < 0.01 ( right panel ). K , A549 and H1299 cells stably transfected with control vector and HBP1 or HBP1 + EZH2 were subcutaneously injected into nude mice. Four weeks after injection, the tumors were weighed, and size was measured. Data are shown as mean ± S.E. ( n = 3). *, p < 0.05.

Article Snippet: The primary antibodies used for immunoblotting analysis were against HBP1 (11746-1-AP, Proteintech), p53 (DO-1, sc-126, Santa Cruz Biotechnology; 9282, Cell Signaling Technology), Mdm2 (sc-965, Santa Cruz Biotechnology), p21 (K0081-3, MBL), GAPDH (KM9002, Sungene), EZH2 (5246, Cell Signaling Technology), Wnt2 (11160-1-AP, Proteintech), DNMT1 (sc-10222, Santa Cruz Biotechnology), p16 (10883-1-AP, Proteintech), FLAG (F1804, Sigma-Aldrich), HA (MMS-101P, Covance), GST (IT003M, M&C Gene Technology), His (66005-1, Proteintech), histone 3 (17168-1-AP, Proteintech), H3K27me3 (A2363, ABclonal), GFP(CW8006, CW Biotech), β-catenin (9587P, Cell Signaling Technology), TCF4 (13838-1-AP, Proteintech), and HDAC1 (AH379, Beyotime).

Techniques: Activation Assay, Inhibition, Western Blot, Expressing, Control, Lysis, Immunoprecipitation, Transduction, Plasmid Preparation, shRNA, Staining, Stable Transfection, Transfection, Selection, MTT Assay, Soft Agar Assay, Cell Culture, Microscopy, Injection

Journal: Annals of clinical and translational neurology

Article Title: Clinical heterogeneity within the ALS-FTD spectrum in a family with a homozygous optineurin mutation.

doi: 10.1002/acn3.52075

Figure Lengend Snippet: Figure 4. (A–C): Rt-PCR revealed OPTN-mRNA expression in PBMCs from the brother of the index patient (listed as Pat2, A). On a protein level, optineurin could not be detected in PMBC lysates (B) or lysates from postmortem frontal cortex of the index patient (listed as Pat1) in western blot analysis (C). (D–I) Anti-phospho-TDP-43 immunohistochemistry on spinal cord sections of a sporadic case of ALS (D–F) and of the index patient ALS and the optineurin mutation (G–I). Phospho-TDP43-positive neuronal skein-like and compact cytoplasmic inclusions in motor neurons in the control case (D) are also immunopositive (arrows) for two different anti-optineurin antibodies (E: non-C-terminal; F: C-terminal). In contrast, in the patient with the optineurin mutation, the inclusions (G) do not immunoreact with any of the two anti-optineurin antibodies (H and I). Scale bars: 50 lm: D–F; 100 lm: G–I;

Article Snippet: The Trans-Blot TurboTM Transfer System was used for transfer of proteins onto nitrocellulose membranes at 25 V for 15 min. Membranes with loaded proteins were incubated with EveryBlot Blocking Buffer (Bio-Rad) at room temperature for 30–60 min, followed by incubation with primary antibodies at 4°C for at least 8 h. We used two OPTN polyclonal antibodies (Proteintech, Cat. No. 10837-1-AP, at a dilution of 1:10,000 & Cayman, Item No. 100000, at a dilution of 1:1000), of which one binds to the C-terminus of optineurin (Cayman), and a GAPDH antibody (Cell Signaling, Cat No. 2118, at a dilution of 1:1000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemistry, Mutagenesis, Control

Journal: Journal of materials science. Materials in medicine

Article Title: Microsphere-Based Scaffolds Encapsulating Tricalcium Phosphate And Hydroxyapatite For Bone Regeneration

doi: 10.1007/s10856-016-5734-1

Figure Lengend Snippet: Genes used for RT-qPCR analysis

Article Snippet: For quantification, the BLANK constructs at week 0 were designated as the calibrator group and GAPDH expression as the endogenous control. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Symbol TaqMan Assay ID Glyceraldehyde 3-phosphate dehydrogenase GAPDH Rn01775763_g1 Collagen type I COL1A1 Rn01463848_m1 Runt-related transcription factor 2 RUNX2 Rn01512298_m1 Bone gamma- carboxyglutamate protein BGLAP Rn00566386_g1 Integrin-binding sialoprotein IBSP Rn00561414_m1 Open in a separate window Genes used for RT-qPCR analysis 2.10 Histology and Immunohistochemistry (IHC) At 6 weeks, microsphere-based constructs (n = 3) were soaked in 30% w/v sucrose (Thermofisher Scientific, Waltham, MA) solution in PBS for 24 h. Afterward, the constructs were equilibrated in optimal cutting temperature embedding medium (OCT, Tissue-Tek, Torrance, CA) overnight at 37°C and then frozen at −20°C.

Techniques: TaqMan Assay