Cat.No. : CSC-RT0467
Host Cell: HEK293T Target Gene: BRD4
Size: 1x10^6 cells/vial, 1 mL Validation: Sequencing
| Cat. No. | CSC-RT0467 |
| Cell Line Information | This cell line is a stable cell line with a homozygous knockout of human BRD4 gene using CRISPR/Cas9. |
| Target Gene | BRD4 |
| Gene ID | 23476 |
| Genotype | BRD4 (-/-) |
| Host Cell | HEK293T |
| Size | 1x10^6 cells/vial, 1 mL |
| Sequencing Result | Homozygous: 2 bp deletion in exon1 |
| Species | Homo sapiens (Human) |
| Revival | Rapidly thaw cells in a 37°C water bath. Transfer contents into a tube containing pre-warmed media. Centrifuge cells and seed into a 25 cm2 flask containing pre-warmed media. |
| Media Type | Cells were cultured in DMEM supplemented with 10% fetal bovine serum. |
| Growth Properties | Cells are cultured as a monolayer at 37°C in a humidified atmosphere with 5% CO2. Split at 80-90% confluence, approximately 1:3-1:6. |
| Freeze Medium | Complete medium supplemented with 10% (v/v) DMSO |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
Small molecule inhibitors of bromodomain and extra-terminal domain (BET) family proteins are a promising option for cancer therapy. However, current BET inhibitors are limited by their potency or oral bioavailability. Here, researchers report a BET inhibitor, NHWD-870. NHWD-870 caused tumor shrinkage or significantly inhibited tumor growth in nine xenograft or syngeneic models. In addition to its ability to downregulate c-MYC and directly inhibit tumor cell proliferation, NHWD-870 also blocked the proliferation of tumor-associated macrophages (TAMs) through multiple mechanisms, in part by reducing tumor cell expression and secretion of macrophage colony-stimulating factor CSF1. NHWD-870 inhibited CSF1 expression by inhibiting BRD4 and its target HIF1α. Taken together, these results reveal a mechanism by which BRD4 inhibition suppresses tumor growth and support further development of NHWD-870 for the treatment of solid tumors.
BRD4 knockout (KO) deficiency significantly reduced HIF1α protein expression under hypoxia (Figure 1a, b), while NHWD-870 inhibition of BRD4 also reduced HIF1α protein expression (Figure 1c, d). BRD4 knockout, NHWD-870, or OTX-015 treatment significantly reduced HIF1 reporter gene activity in A2780 and A375 cells (Figure 1e). In addition, BRD4 deficiency or NHWD-870 treatment significantly reduced CSF1 mRNA levels (Figure 1f). These results raise the possibility that BRD4 regulates CSF1 expression by directly regulating HIF1A (HIF1α gene) transcription. Consistent with this possibility, NHWD-870 treatment reduced HIF1A mRNA levels (Figure 1g). Analysis of published ChIP-seq data showed that BRD4 binds to the HIF1α promoter in A375 cells, while JQ1 inhibition of BRD4 reduced BRD4 binding to the HIF1α promoter in MDA-MB 231 cells (Figure 1h). Importantly, ChIP-qPCR analysis showed that BRD4 binds to the HIF1A promoter, while NHWD-870 inhibition of BRD4 reduced BRD4 binding to the HIF1A promoter in A2780 cells (Figure 1i). Overexpression of wild-type HIF1α under hypoxic conditions significantly increased the mRNA levels of VEGFA (a well-known HIF target gene) and CSF1 in BRD4 knockout cells, even to levels much higher than those in control cells (Figure 1j). These data suggest that BRD4 directly regulates HIF1α-induced CSF1 expression in tumor cells, thereby promoting CSF1R signaling in TAMs (Figure 1k).
Figure 1. NHWD-870 inhibited CSF1 expression through suppressing BRD4 and HIF1α in tumor cells. (Yin, Mingzhu, et al. 2020)
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