Cd24 apc

Peripheral blood from each mouse was subjected to complete blood count. Tibias, femurs, iliac crests, spines, ulnae, radii, and humeri were harvested for bone marrow cells. Spleen and thymus were collected for lymphocyte staining. Flow cytometry staining for all hematopoietic subpopulations including T cell developmental stages was performed as previously described (Yu et al., 2015a (link)). For B cell development, the following scheme was used: B220-Pacific Blue (BD Biosciences 558108), IgM-PE-Cy5 (eBiosciences 15-5790-82), CD43-fluorescein isothiocyanate (FITC) (eBiosciences 11-0431-82), CD24-APC (Biolegend 101814), and BP1-PE (eBiosciences 12-5891-83). Definitions of stages of B cell maturation were as follows: A′ pre-pro-B (IgM⁻B220⁺CD43⁺BP1⁻CD24⁻), B′ pro-B (IgM⁻B220⁺CD43⁺BP1⁻CD24⁺), C′ pro-B (IgM⁻B220⁺CD43⁺BP1⁺CD24^lo), C″ pro-B (IgM⁻B220⁺CD43⁺BP1⁺CD24^hi), pro-B (IgM⁻B220⁺CD43⁺), pre-B (IgM⁻B220⁺CD43⁻), B progenitors (IgM⁻B220⁺), immature B (IgM⁺B220^lo), and mature B (IgM⁺B220^hi). For cell-cycle analysis, bromodeoxyuridine-FITC and 7AAD, or Ki67-FITC and DAPI staining were coupled with staining of specific populations to reveal their cell-cycle status.

Human embryonic kidney (HEK 293T) cells, normal liver (LO2) cells, normal breast (MCF-10A) cells and breast cancer (MDA-MB-231, MDA-MB-468, BT549, T47D) cell lines were cultured in DMEM (Gibco, CA, USA) supplemented with 1% penicillin and streptomycin, and 10% fetal bovine serum (Gibco). Cells were incubated at 37°C/5% CO₂ in a humidified atmosphere. Compounds 1–14 (purity > 95%) were purchased from J&K Scientific Ltd. (Hong Kong, China). The positive control dinaciclib (15) was bought from Selleck (Houston, CA, USA). All the compounds were dissolved in dimethyl sulfoxide (DMSO). The CDK9 Assay Kit was obtained from BSP Bioscience (San Diego, CA, USA). Actinomycin D (ActD) and cycloheximide (CHX) were purchased from Beyotime (Shanghai, China). TurboFect™ Transfection Reagent was purchased from Thermal Fisher (Catalog number: R0532). The sources of the antibodies are indicated separately in the procedures below. CD44-FITC and CD24-APC were purchased from BioLegend (San Diego, CA, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Sigma–Aldrich (St. Louis, MO, USA).