ki67 primary ab antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: GPER1 signaling restricts macrophage proliferation and accumulation in human hepatocellular carcinoma

doi: 10.3389/fimmu.2024.1481972

Figure Lengend Snippet: GPER1 expression negatively correlates with macrophage proliferation in HCC tumors. (A–F) Human monocyte-derived macrophages were treated with 20% culture supernatant from SK-Hep-1 cells (TSN) or control media (Med) for 48 hours. The proliferation level of macrophages was detected using flow cytometry analysis, and Ki67 - and Ki67 + macrophages were gated (A) . The expression levels of sex hormone receptors were examined using qPCR (B , n = 5). The expression levels of GPER1 in Ki67 - and Ki67 + macrophages gated in (A) were determined using flow cytometry (C , n = 6). The expression of Ki67, GPER1 and ERα in TSN-treated macrophages was visualized using confocal microscopy. Then the fluorescence intensity of GPER1 or ERα staining was compared between Ki67 - and Ki67 + macrophages (D-F , n = 4). The scale bar is 50 μm. (G) Representative dot plot and statistical analysis of the proliferation level of GPER1 - or GPER1 + macrophages isolated from fresh tumor tissues of HCC patients (n = 7). (H) HCC tumor samples were stained with anti-human CD68, GPER1 and Ki67 antibodies, and were then analyzed using confocal microscopy. The scale bar is 50 μm. Patients were divided into two groups according to the median frequency of GPER1 + macrophages among the total CD68 + macrophages, and the percentage of Ki67 + macrophages among the total CD68 + macrophages was compared between the two groups (n = 20). (I) 17β-estradiol concentrations (ng/g tissue) in HCC tumor tissues were examined using Enzyme-Linked Immunosorbent Assay (ELISA) (male, n = 4; female, n = 4). The results shown are represented as mean ± SEM. P values were obtained using paired or nonpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: For double staining of Ki67 and GPER1 or ERα, the cells were simultaneously incubated with primary Abs against human Ki67 and GPER1 (PA5-109319, Thermo Fisher Scientific), or ERα (ab16660, Abcam).

Techniques: Expressing, Derivative Assay, Control, Flow Cytometry, Confocal Microscopy, Fluorescence, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: GPER1 signaling restricts macrophage proliferation and accumulation in human hepatocellular carcinoma

doi: 10.3389/fimmu.2024.1481972

Figure Lengend Snippet: GPER1 activation restricts macrophage proliferation. (A–D) Human monocyte-derived macrophages were either untreated or treated with 20% TSN from Huh7, HepG2 or SK-Hep-1 cells for 48 hours, in the presence or absence of indicated concentrations of G-1 (1 μM for A and B ). The percentages of Ki67 + macrophages were assessed using flow cytometry (n = 7). (E–I) Human monocyte-derived macrophages were either untreated (Med) or treated with 20% Huh7-TSN for 48 hours, in the presence or absence of G-1 (1 μM). Ki67 + (E) and EdU + (F) macrophages were visualized using confocal microscopy. The scale bar is 50 μm in (E) and 300 μm in (F) . The statistical analysis of the percentages of Ki67 + ( G , n = 4) and EdU + ( H , n = 4) macrophages is shown. Densities of macrophages were determined using a CCK-8 assay, and the optical density (OD) values of each donor were normalized relative to the corresponding value of the group without TSN treatment and then compared among different groups ( I , n = 4). (J) Human monocyte-derived macrophages were either untreated or treated with Huh7-TSN for 48 hours, in the presence or absence of G-1 (1 μM) or G15 (0.1 μM). Ki67 + macrophages were assessed using flow cytometry (n = 6). The results shown are represented as mean ± SEM. P values were obtained using one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: For double staining of Ki67 and GPER1 or ERα, the cells were simultaneously incubated with primary Abs against human Ki67 and GPER1 (PA5-109319, Thermo Fisher Scientific), or ERα (ab16660, Abcam).

Techniques: Activation Assay, Derivative Assay, Flow Cytometry, Confocal Microscopy, CCK-8 Assay

Journal: Frontiers in Immunology

Article Title: GPER1 signaling restricts macrophage proliferation and accumulation in human hepatocellular carcinoma

doi: 10.3389/fimmu.2024.1481972

Figure Lengend Snippet: GPER1 signaling restrains macrophage proliferation by inhibiting the MEK/ERK/cyclin pathway. (A–D) Human monocyte-derived macrophages were either untreated or treated with 20% Huh7-TSN for 48 hours, in the presence or absence of G-1(1 μM). The levels of p-ERK, ERK, p-Akt, Akt, cyclin D1, cyclin E1, CDK2 and CDK4 were determined using immunoblotting (A, C) . Quantitative analysis of protein expression levels, normalized to β-actin, was performed and plotted ( B, D , n = 4 or 6). The results shown in (B, D) are represented as mean ± SEM. P values were obtained using one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01; ns, not significant.

Article Snippet: For double staining of Ki67 and GPER1 or ERα, the cells were simultaneously incubated with primary Abs against human Ki67 and GPER1 (PA5-109319, Thermo Fisher Scientific), or ERα (ab16660, Abcam).

Techniques: Derivative Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: GPER1 signaling restricts macrophage proliferation and accumulation in human hepatocellular carcinoma

doi: 10.3389/fimmu.2024.1481972

Figure Lengend Snippet: GPER1 expression positively correlates with the survival of HCC patients. (A, B) HCC patients were divided into two groups according to the median percentage of GPER1 + macrophages among the total macrophages in the tumor tissues. The cumulative overall survival (A) and recurrence-free survival (B) of patients were analyzed using the Kaplan-Meier method and compared by the log-rank test (n = 48). (C, D) Forest plot illustrating the associations between overall survival (C) or recurrence-free survival (D) and the clinical characteristics of HCC patients. A multivariate cox proportional hazards regression model was applied, incorporating variables that exhibited a significant univariate association with the outcome ( p < 0.05). HR, hazard ratio; CI, confidence interval. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For double staining of Ki67 and GPER1 or ERα, the cells were simultaneously incubated with primary Abs against human Ki67 and GPER1 (PA5-109319, Thermo Fisher Scientific), or ERα (ab16660, Abcam).

Techniques: Expressing

Journal: iScience

Article Title: Iloprost requires the Frizzled-9 receptor to prevent lung cancer

doi: 10.1016/j.isci.2022.104442

Figure Lengend Snippet: Iloprost requires Fzd 9 to prevent murine adenoma development (A) Schematic of urethane and inhaled iloprost lung cancer chemoprevention model. (B) Change in weights of mice in the urethane and inhaled iloprost model. (C) Average adenoma number per mouse in the urethane exposed groups. (D) Diameter of adenomas in urethane exposed groups. (E) Average number of Ki67 expressing cells/mm 2 adenoma area per mouse exposed to urethane. Urethane mice are compared to their genotype saline control. (F) microCT image of Fzd 9 −/− urethane iloprost mouse lung. (G) H&E of a representative adenoma generated by the 15-week urethane model. WUS, wild type urethane saline; WUI, wild type urethane iloprost; FUS, Fzd 9 −/− urethane saline; FUI, Fzd 9 −/− urethane iloprost. Data are represented as mean ± SEM. One-way ANOVA with Tukey’s post hoc test was used to measure significance. ∗p < 0.05. Scale bar is 100uM.

Article Snippet: Sections were incubated in Ki67 primary Ab (1:2000 dilution, Abcam 15580) for 1 hour at room temperature, followed by anti-rabbit universal antibody for 30 minutes and ABC reagent for 30 minutes at room temperature (Vector Laboratories PK-7200).

Techniques: Expressing, Saline, Control, Generated

Journal: iScience

Article Title: Iloprost requires the Frizzled-9 receptor to prevent lung cancer

doi: 10.1016/j.isci.2022.104442

Figure Lengend Snippet:

Article Snippet: Sections were incubated in Ki67 primary Ab (1:2000 dilution, Abcam 15580) for 1 hour at room temperature, followed by anti-rabbit universal antibody for 30 minutes and ABC reagent for 30 minutes at room temperature (Vector Laboratories PK-7200).

Techniques: Recombinant, Western Blot, Plasmid Preparation, Electrophoresis, Reporter Assay, BIA-KA, Reverse Transcription, Immunodetection, Knock-Out, Transgenic Assay, Software

Journal: The Journal of Pathology

Article Title: Distinct contributions of cathelin‐related antimicrobial peptide ( CRAMP ) derived from epithelial cells and macrophages to colon mucosal homeostasis

doi: 10.1002/path.5572

Figure Lengend Snippet: Reduced epithelial cell proliferation in the colon of epithelial Cnlp −/− mice. (A) Shortened colon crypts in epithelial Cnlp −/− mice. Left panel: representative images to show H&E staining of colon sections from naïve epithelial Cnlp −/− and epithelial Cnlp +/+ mice. Scale bar = 50 μm. Right panel: colonic crypt length ( n = 8 per group). *** p < 0.001. (B) No change in the length of colonic crypts of myeloid Cnlp −/− mice compared with myeloid Cnlp +/+ mice. Left panel: representative images are shown. Scale bar = 50 μm. Right panel: quantitative analysis of colon crypt length of naïve myeloid Cnlp −/− and myeloid Cnlp +/+ mice. n = 8 per group. (C) Reduced numbers of Ki67 + cells in the colonic crypts of epithelial Cnlp −/− mice. Left panels: representative images of immunofluorescence for Ki67 are shown. Red: Ki67 + cells; blue: DAPI. Scale bar = 100 μm. Right panel: numbers of Ki67 + cells per crypt ( n = 8 per group). *** p < 0.001. (D) No change in the numbers of Ki67 + cells in the colon crypts of myeloid Cnlp −/− mice compared with myeloid Cnlp +/+ mice. Left panels: red: Ki67 + cells; blue: DAPI. Scale bar = 60 μm. Right panel: quantitative analysis of Ki67 + cells per crypt ( n = 8 per group). ns = no significance between myeloid Cnlp −/− mice and myeloid Cnlp +/+ mice.

Article Snippet: For detection of Ki67 and Muc2 expression, primary Ki67 Ab (Cat #: ab16667, 1/200 dilution; Abcam, Cambridge, MA, USA) and Muc2 Ab (Cat #: ab272692, 1/1000 dilution; Abcam) were used.

Techniques: Staining, Immunofluorescence

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: The Second Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model

doi: 10.1158/1940-6207.CAPR-17-0050

Figure Lengend Snippet: JF32 mouse lung adenocarcinoma cells were serum-starved for 24 h prior to incubation with increasing doses of PGE2 (A) or treprostinil (B) in serum-free media. Cells were harvested by trypsinization and counted; *p < 0.05 vs control. (C) Iloprost pre-treatment prevented PGE2 and treprostinil-mediated increases in JF32 cell proliferation; ***p < 0.01 vs. PGE2 or treprostinil alone. Graphs represent 2 independent experiments with at least 3 samples/group in each experiment. (D) Ki67+ staining was used to generate a proliferative index (E). Ki67 nuclear staining (red arrowhead) was counted in each tumor. Area of each tumor was determined using CellSens Entry software, and the number of Ki67+ nuclei/mm2 tumor was determined in each group. No differences were detected between treatment groups.

Article Snippet: Sections were incubated with Ki67 primary Ab (1:25 dilution, Abcam 15580) for 30 min. at room temperature, and Ki67 + nuclei were detected using Biocare Medical’s Mach 2 detection kit (MRCT523).

Techniques: Incubation, Staining, Software