In two patient-derived skin SCC cultures, 50 nM I-BET726 decreased viability at 72 h and proliferation endpoints at 48 h. Primary keratinocytes and fibroblasts showed no significant change in these endpoints under the tested conditions. In a separate A431 experiment, viability also decreased in BRD4-knockout cells, indicating that the cellular response cannot be attributed to BRD4 alone. [2]
BET tandem-bromodomain ligand displacement
Biochemical assay · BRD2, BRD3 and BRD4 constructs containing BD1 and BD2 [1]
I-BET726 displaced a fluorescent ligand from BET tandem-bromodomain constructs. Reported IC50 values are assay endpoints, not treatment doses or dissociation constants.
Literature-Reported Treatment Conditions
- Treatment concentration
- Compound titration; each BET protein 10 nM and Alexa 647 fluorescent ligand 50 nM.
- Exposure duration
- 1 h equilibration before TR-FRET detection
- Method
- TR-FRET ligand displacement and four-parameter IC50 fitting
- Co-treatment
- Single-agent I-BET726; no combination drug in the selected comparison.
- Reported limits
- Vehicle and replicate count for Figure 1C are not specified in the retrieved characterization method. The main text describes histone-peptide competition, whereas Methods S1 specifies an Alexa 647 derivative of a fluorescent ligand; adopted wording follows the experimental method.
Methods and source locations
Compound reported in the paper: GSK1324726A (I-BET726); Compound label in the paper: I-BET726
Evidence describes the paper’s tested I-BET726 material. It does not establish equivalence of an Adooq lot, purity or formulation.
Results s2.1; Figure 1C; Methods S1, Determination of BET Protein Binding Affinities to I-BET726
BRD2 tandem-bromodomain ligand displacement: TR-FRET; 10 nM protein, 50 nM fluorescent ligand — Figure 1C, BRD2; Methods S1 BET binding
BRD3 tandem-bromodomain ligand displacement: TR-FRET; 10 nM protein, 50 nM fluorescent ligand — Figure 1C, BRD3; Methods S1 BET binding
BRD4 tandem-bromodomain ligand displacement: TR-FRET; 10 nM protein, 50 nM fluorescent ligand — Figure 1C, BRD4; Methods S1 BET binding
Primary SCC-cell viability and proliferation with normal-cell comparators
Cell-based study · Primary human skin SCC cultures C1/C2 from two patients; primary skin keratinocytes and fibroblasts [2]
50 nM I-BET726 reduced MTT viability and BrdU/EdU proliferation endpoints in C1/C2. Keratinocytes and fibroblasts showed no significant change in the same endpoints under these conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- 50 nM
- Exposure duration
- 72 h for MTT; 48 h for BrdU and EdU
- Method
- MTT, BrdU incorporation ELISA and EdU/DAPI microscopy
- Vehicle
- Untreated control; compound solvent is not specified for Figure 2.
- Co-treatment
- Single-agent I-BET726; no combination drug in the selected comparison.
- Reported limits
- C1/C2 are two patient-derived cultures, not two animal experiments. Caption n=5 means replicate wells/dishes; experiments were repeated three times, not five independent donors. No general normal-tissue safety conclusion is supported.
Methods and source locations
Compound reported in the paper: GSK1324726A (I-BET726); Compound label in the paper: I-BET726
Evidence describes the paper’s tested I-BET726 material. It does not establish equivalence of an Adooq lot, purity or formulation.
Results Sec23 primary-cell paragraph; Figure 2A–C; Chemicals and reagents, Cell culture, MTT, BrdU, EdU and Statistical analysis methods; Figure 1 shared legend
MTT signal in C1/C2 versus untreated control: MTT OD at 550 nm; mean ± SD; n=5 replicate wells/dishes; experiments repeated three times — Figure 2A; Figure 1 shared legend; MTT and Statistical analysis methods
BrdU and EdU endpoints in C1/C2 versus untreated control: BrdU ELISA and EdU/DAPI ratio; mean ± SD; n=5 replicate wells/dishes — Figure 2B–C; Figure 1 shared legend; BrdU and EdU methods
Keratinocyte and fibroblast MTT/BrdU/EdU endpoints: Matched primary-cell endpoint comparisons; does not establish equivalence or a safety margin — Figure 2A–C; Results Sec23 primary-cell paragraph
Residual I-BET726 response in BRD4-knockout A431 cells
Cell-based study · A431 cells with two stable BRD4-knockout lines (sL1/sL2) and Cas9 control cells [2]
BRD4 depletion was confirmed by western blotting. I-BET726 further decreased MTT viability in both knockout lines, supporting a contribution beyond BRD4 alone without identifying a direct additional target.
Literature-Reported Treatment Conditions
- Treatment concentration
- 50 nM
- Exposure duration
- 72 h
- Method
- MTT assay in two BRD4-knockout lines with untreated and Cas9 controls; western blot verification
- Vehicle
- No-treatment controls; solvent not specified for Figure 4B–C.
- Co-treatment
- Single-agent I-BET726; no combination drug in the selected comparison.
- Reported limits
- The phenotype does not establish direct SphK1 binding or inhibition. It also does not exclude actions on other BET proteins. No viability percentage was read from bar heights.
Methods and source locations
Compound reported in the paper: GSK1324726A (I-BET726); Compound label in the paper: I-BET726
Evidence describes the paper’s tested I-BET726 material. It does not establish equivalence of an Adooq lot, purity or formulation.
Results Sec25 first paragraph; Figure 4B–C and legend; BRD4 knockout, Western blotting, MTT and Statistical analysis methods
BRD4 protein expression in knockout lines: BRD4 immunoblot with tubulin loading control — Figure 4B; BRD4 knockout and Western blotting methods
MTT viability in BRD4-knockout A431 lines after treatment: MTT OD normalized to Cas9 control; mean ± SD; shared n=5 replicate wells/dishes — Figure 4C and legend; Results Sec25 first paragraph; Figure 1 shared legend
