Journal: Journal of Biomedical Science

Article Title: Guilty by association: direct interaction with the tetraspanin CD63 suggests a role for organic cation transporter 3 in histamine release from granulocytes

doi: 10.1186/s12929-025-01158-2

Figure Lengend Snippet: Panel A shows PCR analysis of mRNA expression of CD63 (lane 1), hOCT1 (lane 2), hOCT2 (lane 3), hOCT3 (lane 4), and GAPDH (lane 5) in three different preparations of fresh isolated human basophils. Panel B shows the effect of 24 h incubation with 20 ng/ml IL-3 in the presence or not of 1 mM MPP + , a known substrate of hOCT3, on the histamine release (ng/mg protein) into cell supernatant from fresh isolated human basophils. Panel C shows a PCR analysis of mRNA expression of hOCT1 (lane 1), hOCT2 (lane 2), hOCT3 (lane 3), MATE1 (lane 4), MATE2K (lane 5), hOCTN1 (lane 6), hOCTN2 (lane 7), CD63 (lane 8), CD9 (lane 9), and GAPDH (lane 10) in the human basophilic cell line KU812. Panel D shows histamine concentration in supernatants from KU812 cells with (grey columns) or without (open columns) stimulation with 5 µg/ml IgE and 0.2 µg/ml anti-IgE. The effects of the addition of 1 mM MPP. + or 1 mM corticosterone to experiments with stimulation (black columns) are also shown. Histamine concentration (ng/ml) is expressed as mean ± SEM. Every single point in the figures represents the result of an independent experiment. Asterisks indicate a statistically significant difference (****p < 0.0001, ***p = 0.0001, **p = 0.0011, *p = 0.0207. ANOVA test with Dunnett’s multiple comparison test)

Article Snippet: Further experiments were performed in the KU812 cell line (ATCC CRL-2099).

Techniques: Expressing, Isolation, Incubation, Concentration Assay, Comparison

Journal: Journal of Biomedical Science

Article Title: Guilty by association: direct interaction with the tetraspanin CD63 suggests a role for organic cation transporter 3 in histamine release from granulocytes

doi: 10.1186/s12929-025-01158-2

Figure Lengend Snippet: Immunofluorescence analysis of hOCT3 (green) and CD63 (red) distribution in KU812 cells without (w/o stimulation) or with stimulation (with stimulation) using 5 µg/ml IgE and 0.2 µg/ml anti-IgE (magnification 630x). Panels A and B and panels D and E show the labelling of hOCT3 and CD63 without and with stimulation, respectively. Panels C and F show the overlay of hOCT3, CD63, and DAPI labelling without and with stimulation, respectively. After stimulation, there is a clear translocation of hOCT3 and CD63 to the plasma membrane, where they co-localize. A 10 µm scale bar is also shown. Panel G shows Pearson’s coefficient for co-localization of hOCT3 and CD63 in the presence (stimulated) or absence (unstimulated) of histamine release stimulation with IgE/anti-IgE. Stimulation of histamine release significantly increased the hOCT3/CD63 co-localization coefficient measured using a Jacop plug-in in 88 and 70 unstimulated (open circles) and stimulated (closed circles) cells, respectively, from at least 3 independent experiments, each marked with different colors (****p < 0.0001, unpaired t-test)

Article Snippet: Further experiments were performed in the KU812 cell line (ATCC CRL-2099).

Techniques: Immunofluorescence, Translocation Assay, Clinical Proteomics, Membrane

Journal: Journal of Biomedical Science

Article Title: Guilty by association: direct interaction with the tetraspanin CD63 suggests a role for organic cation transporter 3 in histamine release from granulocytes

doi: 10.1186/s12929-025-01158-2

Figure Lengend Snippet: Cellular distribution of CD63 (green) and hOCT3 (red) before, at the end, and 24 h after end of incubation of KU812 cells to stimulate histamine release. The immunofluorescence analysis was conducted on stack images; histamine release was stimulated by 24 h incubation with 5 µg/ml IgE followed by 20 min incubation with 0.2 µg/ml anti-IgE (magnification 630x). Upper row represents the CD63 and hOCT3 labeling before the IgE/anti-IgE incubation, middle row immediately after IgE/anti-IgE incubation and the lower row 24 h after the end of incubation. DAPI labeling of cell nuclei (blue) and merge images are also presented. A 10 µm scale bar is included for reference in the merge picture

Article Snippet: Further experiments were performed in the KU812 cell line (ATCC CRL-2099).

Techniques: Incubation, Immunofluorescence, Labeling

Journal: Oncogene

Article Title: β-Catenin interacts with canonical RBPs including MSI2 to associate with a Wnt signalling mRNA network in myeloid leukaemia cells

doi: 10.1038/s41388-025-03415-y

Figure Lengend Snippet: A Immunoblots showing MSI2, β-catenin and LEF1 level in K562 and KU812 cells harbouring MSI2 shRNA or non-targeting shRNA controls. GAPDH indicates protein loading. B Representative flow cytometric histograms showing intensity of the TCF-dependent expression of Venus Yellow Fluorescent Protein (YFP) from the β -catenin a ctivated r eporter (BAR) reporter, or negative control ‘ f ound u nresponsive’ BAR (fuBAR; containing mutated promoter binding sites) in K562 and KU812 cells ± MSI2 shRNA following treatment with 5 μM CHIR99021 overnight. The fuBAR (dashed), non-targeting control shRNA (grey filled), and two MSI2 shRNAs (blue or red) histograms are shown. Summary graphs showing the median fluorescence intensity (MFI) generated from the BAR/fuBAR in C K562 and D KU812 cells ±MSI2 shRNA with ±5 μM CHIR99021. E Immunoblots showing total β-catenin, LEF-1, and MSI2 subcellular localisation in K562 and KU812 cells lentivirally transduced with two different MSI2 shRNAs ±5 μM CHIR99021. Lamin A/C and α-tubulin indicate the purity/loading of the nuclear (N) and cytosol (C) fractions respectively. Densitometric quantitation of LEF-1 (relative to nuclear Lamin A/C, AU = arbitrary units) present in nuclear fractions of F K562 and G KU812 cells ± MSI2 shRNA with ±5 μM CHIR99021. Summary graph showing the fold change in LEF1 and TCF7L2 mRNA expression as assessed by RT-qPCR in ( H ) K562 and ( I ) KU812 cells expressing MSI2 shRNA relative to non-targeting control shRNA (represented by dotted line at y = 1). Fold change is relative to matched respective controls (black dashed line) and overall expression was normalised to the housekeeping gene β-actin ( ACTB ). Data represent mean ± 1 s.d ( n = 3). Statistical analysis is denoted by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 as deduced from a student’s t test or one-sample t-test for RT-qPCR data.

Article Snippet: The myeloid cell lines K562, HL60, HEL, U937, PLB-985, NOMO1, OCI-AML3, EOLI, ML-1, THP-1, KU812 (The European Collection of Authenticated Cell Cultures) and OCI-AML2, MV4-11, KG-1, KG1a SET2, NB4 and MONOMAC6 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were confirmed mycoplasma-free and authenticated via short-tandem repeat (STR) analysis prior to general culture as previously [ ]. β-Catenin was stabilised using the GSK-3β inhibitor CHIR99021 (Merck-Millipore) as previously described [ ], whilst transcription was inhibited via 2 μg/ml Actinomycin D (Merck-Millipore) at the stated timepoints.

Techniques: Western Blot, shRNA, Expressing, Negative Control, Binding Assay, Control, Fluorescence, Generated, Transduction, Quantitation Assay, Quantitative RT-PCR

Journal: Journal of Clinical Medicine

Article Title: Tyrosine Kinase Inhibitor Therapy Enhances Stem Cells Profile and May Contribute to Survival of Chronic Myeloid Leukemiastem Cells

doi: 10.3390/jcm14020392

Figure Lengend Snippet: Imatinib treatment increases protein levels of SOX2 in K562 and KU812. ( A , B ) The green signal corresponding to Sox2 or OCT 3/4 showed a strong nuclear increase after IM treatment in alive cells extracted with Ficoll gradient. Red propidium (PI) is used to detect nuclei (63X magnification). ( C ) ROS levels in cytosolic extract of cells treated with IM, ROS is expressed as nmol/mg of proteins. ( D ) Heatmap of the most significant expressed metabolites in the groups of alive and total cells after Imatinib treatment. Significantly reduced activities are represented in blue while those with a significantly increased activity are in red. K562 IM alive R1, 2, and 3 corresponded to viable cells condition triplicates while K562 IM tot R1, 2, and 3 represented the total cells after treatment condition triplicates. ( E ) Pathway enrichment analysis obtained with MetaboAnalyst and KEGG for pathway identification. The significance of a particular pathway was expressed as enrichment ratio. The p -value is represented with a red scale. * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: The K562 and KU812 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), grown in RPMI 1640 medium supplemented with L Glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 10% inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), and 0.1% penicillin/streptomycin (EuroClone, Milan, Italy), and maintained at 37 °C with 5% of CO 2 .

Techniques: Activity Assay