primary antibodies against ki 67  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Roburic Acid as a Therapeutic Candidate: Antiproliferative Activity and Secondary Cell Death Response in Colorectal Cancer Cells

doi: 10.3390/ijms27052478

Figure Lengend Snippet: Effect of roburic acid (RA) on Ki-67 expression in colorectal cancer (CRC) cell lines. Ki-67 immunofluorescence staining in DLD-1, HCT-116, and HT-29 following 48 h of exposure to RA at concentrations corresponding to 0.5 × IC 50 and IC 50 . Quantitative data are presented as medians with interquartile ranges, minimums, and maximums. Statistical analysis was performed using the Kruskal–Wallis test, followed by Dunn’s post hoc test; an asterisk indicates significant differences relative to untreated controls (*) ( p < 0.05).

Article Snippet: Samples were then incubated for 1 h with a rabbit anti-Ki-67 primary antibody (Thermo Fisher, MA5-14520; 1:250 dilution in PBS supplemented with 1% bovine serum albumin and 0.5% Tween-20), followed by a 1 h incubation with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher, A-11008; 1:500 dilution).

Techniques: Expressing, Immunofluorescence, Staining

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: In vivo demonstration of glucose-activated PKA promoting glycolysis-dependent progression A. Immunoblot analysis of PRKAR2B in KPC cells with or without PRKAR2B knockout. (B–D) Representative images of colony formation by KPC cells, with or without PRKAR2B knockout, cultured in normal or low-glucose medium (B). Colony size (C) and number of colonies (D) were graphed ( n = 3 independent repeats). Data were expressed as mean ± SD. Scale bars, 50 μm ∗, p < 0.05, ∗∗, p < 0.01, NS, no significance. (E) Representative H&E-stained pancreatic sections from mice with tamoxifen-inducible, pancreas-specific KRAS expression, harvested 3 weeks after tamoxifen or vehicle injection. Scale bars, 1 mm (F–H) PKA activity (F), pyruvic acid level (G), and lactate level (H) in pancreatic tissues from mice, harvested 3 weeks after tamoxifen or vehicle injection, assessing the effect of tamoxifen-inducible, pancreas-specific KRAS expression ( n = 4 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01. (I) Colorimetric analysis of PKA activity in wild-type and diabetic mice ( n = 4 independent samples). Data were expressed as mean ± SD. ∗∗, p < 0.01. (J) Representative immunoblot of CREB, pCREB, and KLF5 in wild-type and diabetic mice ( n = 4 independent samples; see F for all replicates). (K) mRNA expression level of HK2 in wild-type and diabetic mice ( n = 4 independent samples). Data were expressed as mean ± SD. ∗∗, p < 0.01. (L) Schematic diagram of the experimental design in mice. Pan02 cells with or without PRKAR2B knockout was injected into wild type or diabetic mouse through the tail vein ( n = 4 independent samples). (M) Changes in mouse body weight following tail vein injection of PRKAR2B -knockout or wild-type Pan02 cells ( n = 4 independent samples). Data were expressed as mean ± SD. ∗∗, p < 0.01. (N) Colorimetric analysis of PKA activity in tumor allografts from wild-type and diabetic mice with or without PRKAR2B knockout ( n = 4 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗, p < 0.01. (O) Representative image of Ki67 IHC staining in Pan02-control and Pan02- PRKAR2B -sg allografts from wild-type or diabetic mice ( n = 4 independent samples). Scale bars, 150 μm (P) Representative H&E-stained lung sections at the experimental endpoint. Scale bars, 3 mm (Q–R) Quantification of metastatic lesion number (Q) and total metastatic burden (R) at the experimental endpoint across the indicated treatment groups ( n = 4 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05; NS, no significance.

Article Snippet: Primary antibody against Ki67 (1:500 dilution, cat. 12202, Cell Signaling Technology, MA, USA) was prepared in SignalStain Antibody Diluent (cat. 8112, Cell Signaling Technology) and applied to the sections overnight at 4°C.

Techniques: In Vivo, Western Blot, Knock-Out, Cell Culture, Staining, Expressing, Injection, Activity Assay, Immunohistochemistry, Control

Journal: Gut Pathogens

Article Title: Phenotypic characterization and complete genome of a tumorigenic pathobiont Escherichia coli LI60C3

doi: 10.1186/s13099-025-00732-1

Figure Lengend Snippet: Phenotypic characterization of the invasive feature and tumorigenic ability of E. coli strains. (A) Schematic diagram of microbial isolation from intestinal epithelial cells for in vitro and in vivo phenotypic assays. The invasive features, proliferation-inducing ability, and tumorigenic characteristics of the intraepithelial bacteria obtained from mouse colonocytes were evaluated in human cell lines and experimental murine models. (B) The intraepithelial E. coli strains (LI60C3, HM784C1, HM926C2, and HM936C4), adherent-invasive E. coli strains (LF82 and NC101), and nonpathogenic laboratory strains (MG1655, DH5α and JM109) were exposed to Caco-2 cells at MOI = 10 for 4 h. (a) Intracellular bacterial counts and (b) apical bacterial counts in the epithelial cultures are shown. (C) Cell cycle progression of Caco-2 cells after bacterial exposure for 24 h. The cell ratios in (a) S/G2/M to G1/G0 phase and (b) high to low Ki67 staining intensity are shown. Data are expressed as mean ± SEM ( N = 5–8/group). Each dot represents the data of one sample. * P < 0.05 vs. w/o, and # P < 0.05 vs. LI60C3. (D) A murine model with chemically induced colon cancer was inoculated with PBS vehicle, LI60C3, or LF82 at 10 9 CFU per mouse via the orogastric route. Representative images of intestinal tumors in each mouse group are displayed. Bar: 2 mm (macroscopic images) and 200 μm (histological images, H&E staining). (E) Increased tumor load was observed in mice inoculated with LI60C3, but not LF82. The tumor (a) number, (b) area, and (c) grade were determined for each mouse. The percentages of tumors with low- and high-grade dysplasia and carcinoma in each group are presented. Data are expressed as mean ± SEM ( N = 8–10/group). Each dot represents the data of one mouse. * P < 0.05 vs. PBS, and # P < 0.05 vs. LI60C3. The statistical significance of all panels is calculated by ANOVA followed by Tukey’s multiple comparison test, except those of (B-a), (C-a) and (E-a) are determined by the Kruskal–Wallis test. Experiments were repeated at least twice.

Article Snippet: The cells were then fixed with ice-cold 70% ethanol overnight, followed by washing with PBS containing 1% bovine serum albumin (BSA) (Cat. #A2153, Sigma-Aldrich) and centrifuged at 1500 × g for 5 min. After decanting the supernatant, the cells were stained with a primary antibody rabbit anti-human Ki67 (Cat. #9129) (1:400, Cell Signaling, Beverly, MA, USA) in PBS containing 0.5% TWEEN-20 (Cat. #TWN508, BioShop, Burlington, ON, Canada) and 1% BSA for 1 h at room temperature.

Techniques: Isolation, In Vitro, In Vivo, Bacteria, Staining, Comparison