Flavopiridol at 125 nM for 24 h altered cell-cycle distributions in three anaplastic thyroid cancer cell lines. KMH2 accumulated in G1/G0, whereas CAL62 and BHT-101 accumulated in G2/M relative to the vehicle comparator. The arrest phase therefore varied by cell model. [2]
Transient disruption of productive transcription at Hsp70
Cell-based study · Heat-shocked Drosophila melanogaster Kc cells; Hsp70 chromatin immunoprecipitation [1]
At 500 nM, flavopiridol rapidly depleted Pol II from downstream Hsp70 regions while retaining promoter-proximal Pol II. Ser2 phosphorylation near the promoter decreased more than Ser5 phosphorylation. Time-course controls showed recovery of gene-body Pol II despite continued drug exposure; the effect was not a permanent shutdown.
Literature-Reported Treatment Conditions
- Treatment concentration
- 500 nM
- Exposure duration
- Acute samples 1 and 3 min after drug addition at 7 min of heat shock; recovery experiment sampled 2, 8, 10 and 30 min of heat shock with drug given 1 min before or 7 min after heat shock
- Method
- ChIP for total Rpb3/Pol II and phosphorylated CTD; time-matched heat-shocked cells without FP; HSF control in recovery experiment
- Reported limits
- Drug vehicle, CAS, salt form and analytical stereochemical verification were not specified in the read material.
Methods and source locations
Compound reported in the paper: Flavopiridol; Compound label in the paper: FP
Evidence for the material named in the original paper; no Adooq lot validation, stereochemical equivalence testing or salt conversion is implied.
Figures 1A-E and 3A-C; Materials and Methods: ChIP; Results: immediate depletion, Ser2/Ser5 phosphorylation and Pol II recovery; author contributions
Promoter-proximal versus gene-body Pol II and CTD phosphorylation: Rpb3, Ser2-P and Ser5-P ChIP; two independent experiments, error bars show ranges — Figure 1B-E and caption; Results: P-TEFb inhibition rapidly depletes elongating Pol II and Levels of phosphorylated Ser2
Time-dependent recovery of gene-body Pol II at Hsp70 +946: ChIP; three independent experiments with standard error bars; untreated and pre-/post-heat-shock drug timing controls — Figure 3A-C and caption; Results: Pol II levels across Hsp70 recover with time
Model-dependent redistribution of the cell cycle
Cell-based study · KMH2, CAL62 and BHT-101 anaplastic thyroid cancer cell lines [2]
After 24 h at 125 nM, KMH2 accumulated in G1/G0, whereas CAL62 and BHT-101 accumulated in G2/M. These phase changes are model dependent and do not establish that all cancer cells arrest at the same phase or that cell-cycle arrest alone proves apoptosis.
Literature-Reported Treatment Conditions
- Treatment concentration
- 125 nM
- Exposure duration
- 24 h
- Method
- BrdU incorporation and propidium iodide flow cytometry; minimum 10,000 events; three biological replicates per cell line
- Vehicle
- DMSO-only comparator; drug prepared as a DMSO stock
- Reported limits
- Final DMSO percentage was not specified in the read methods.
Methods and source locations
Compound reported in the paper: Flavopiridol; Compound label in the paper: flavopiridol
Evidence for the material named in the original paper; no Adooq lot validation, stereochemical equivalence testing or salt conversion is implied.
Figure 2B and embedded tables/caption; Materials and methods: Drug treatment, Cell cycle analysis, Statistical analysis; Results: Flavopiridol downregulates cell cycle proteins and induces cell cycle arrest and apoptosis
KMH2 cells in G1/G0: BrdU/PI flow cytometry; n = 3 biological replicates; unpaired two-tailed Student t-test — Figure 2B KMH2 embedded table and caption; Cell cycle analysis
CAL62 cells in G2/M: BrdU/PI flow cytometry; n = 3 biological replicates; unpaired two-tailed Student t-test — Figure 2B CAL62 embedded table and caption; Cell cycle analysis
BHT-101 cells in G2/M: BrdU/PI flow cytometry; n = 3 biological replicates; unpaired two-tailed Student t-test — Figure 2B BHT-101 embedded table and caption; Cell cycle analysis
CDK9 depletion does not confer flavopiridol resistance
Cell-based study · KMH2 and CAL62 cells with CDK9 siRNA or scrambled siRNA control [2]
CDK9 depletion was confirmed by immunoblotting. It reduced the 72-h proliferation-associated signal in KMH2 but not significantly in CAL62. Drug-response curves did not shift toward resistance after depletion in either cell line. This limits attribution of flavopiridol responses exclusively to CDK9; partial knockdown is not a definitive target-rescue experiment.
Literature-Reported Treatment Conditions
- Treatment concentration
- Flavopiridol 0.06–4.0 μM dose range
- Exposure duration
- 72 h drug exposure for dose-response readout; knockdown-associated proliferation monitored through 72 h
- Method
- siCDK9 versus scrambled siCT, followed by PrestoBlue readout and CDK9 immunoblot with α-tubulin control
- Vehicle
- DMSO stock; dose-response signals normalized to untreated samples
- Co-treatment
- CDK9 siRNA perturbation, not a second drug
- Reported limits
- Figure 3 specifies technical replication; independent biological replication of the dose-response comparison is not established. Final DMSO percentage not specified.
Methods and source locations
Compound reported in the paper: Flavopiridol; Compound label in the paper: flavopiridol
Evidence for the material named in the original paper; no Adooq lot validation, stereochemical equivalence testing or salt conversion is implied.
Figure 3A-C and caption; Materials and methods: RNA interference and drug treatment, Drug treatment, Immunoblotting; Results: siRNA knockdown of CDK9 results in variable impairment of proliferation and does not result in resistance to flavopiridol
Proliferation-associated signal after CDK9 depletion: PrestoBlue normalized RFU; Figure 3B reports three technical replicates and SD; unpaired two-tailed Student t-test — Figure 3A-B and caption; Results: siRNA knockdown of CDK9
Flavopiridol sensitivity after CDK9 depletion: 72-h PrestoBlue drug-response curves; Figure 3C specifies three technical replicates per dose and SD — Figure 3C and caption; RNA interference and drug treatment; Results: siRNA knockdown of CDK9
