Hesperadin’s checkpoint effects depend on the arrest condition and concentration. In HeLa cells, 100 nM Hesperadin induced mitotic exit within 1 h after taxol or monastrol arrest, while nocodazole-treated cells remained arrested for at least 3 h. A later HeLa study found longer arrest at high nocodazole with 100 nM hesperadin and markedly shorter arrest when hesperadin was increased to 0.5–1.0 μM. These experiments used spindle-poison co-treatment. [1,2]
In RPE1 cells treated for 10 h with 3.2 μM nocodazole, adding 100 nM Hesperadin did not significantly reduce kinetochore-associated Mad2. This negative result limits generalization of checkpoint disruption across cell models and treatment conditions. [3]
In a GFP-MELK localization experiment, 100 nM Hesperadin did not cause premature cortex association of GFP-MELK in prometaphase HeLa cells, unlike OTSSP167. The paper identifies the Hesperadin source as Adooq bioscience; this does not validate a particular catalog lot. [4]
Inhibition of immunoprecipitated human Aurora B
Biochemical assay · Human Aurora B immunoprecipitated from mitotic HeLa cell extracts; histone H3 phosphorylation assay. Cdk1/histone H1 assessed in parallel. [1]
Hesperadin reduced Aurora B-dependent histone H3 phosphorylation. The authors reported approximately 250 nM for half-maximal inhibition in this assay; this is an assay endpoint, not a cell treatment recommendation.
Literature-Reported Treatment Conditions
- Treatment concentration
- Hesperadin concentration series shown in Fig. 2D (1–5,000 nM for Aurora B).
- Method
- Added directly to in vitro kinase reactions.
- Vehicle
- 0.1% DMSO control.
- Co-treatment
- None; Cdk1 assayed separately.
- Reported limits
- Reaction duration, ATP concentration and replicate number are not established from the acquired main-text/caption scope. Detailed SI methods were not retrieved.
Methods and source locations
Compound reported in the paper: Hesperadin; Compound label in the paper: Hesperadin
Results apply to the reported experimental material and conditions; no equivalence to a particular Adooq lot is established.
Fig. 2D and caption; Results: Hesperadin inhibits Aurora B function; Fig. 1A structure
Half-maximal inhibition of histone H3 phosphorylation by immunoprecipitated Aurora B: In vitro kinase reaction with immunoprecipitated human Aurora B and histone H3; radiolabeled phosphorylation detected after electrophoresis. Controls: 0.1% DMSO, no substrate and nonspecific-antibody immunoprecipitate. — Results: Hesperadin inhibits Aurora B function, paragraph beginning Approximately 250 nM; Fig. 2D and caption
Spindle-poison-dependent mitotic exit in HeLa cells
Cell-based study · HeLa cells arrested with nocodazole, taxol or monastrol, then exposed to Hesperadin or DMSO. [1]
Addition of Hesperadin rapidly overcame taxol- or monastrol-associated arrest, whereas nocodazole-treated cells retained mitotic arrest for at least 3 h. These are conditional results in cells already exposed to spindle poisons.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 nM Hesperadin; arrest agents: 330 nM nocodazole, 10 μM taxol or 100 μM monastrol.
- Exposure duration
- Sampled after addition from 30 min through 7 h; monastrol images at 7 h marked not determined.
- Method
- Added after drug-induced mitotic arrest; chromosome spreads or immunofluorescence.
- Vehicle
- DMSO solvent controls; final percentage not specified for this experiment in the acquired main text.
- Co-treatment
- Each arrest agent retained as its own condition; no Hesperadin-alone outcome inferred.
- Reported limits
- Arrest induction duration and sample counts for Fig. 8A–D were not established in the acquired scope; SI detailed methods not retrieved.
Methods and source locations
Compound reported in the paper: Hesperadin; Compound label in the paper: Hesperadin
Results apply to the reported experimental material and conditions; no equivalence to a particular Adooq lot is established.
Fig. 8A–D and caption; corresponding Results subsection; Materials and methods: Cell culture and synchronization
Timing of mitotic exit following Hesperadin addition in spindle-poison-arrested HeLa cells: Chromosome spreading for nocodazole/taxol conditions; immunofluorescence for monastrol; DMSO controls under each arrest condition. Fig. 8D quantifies the same samples. — Fig. 8A–D and caption; Results: Hesperadin treatment rapidly overrides the spindle assembly checkpoint in taxol- and monastrol-treated cells but not in nocodazole-treated cells
Dose and nocodazole context alter checkpoint duration
Cell-based study · HeLa cells released from a single thymidine arrest; 5 h later nocodazole and inhibitors were added before live-cell imaging. [2]
At fixed 100 nM hesperadin, mitotic arrest lasted longer at high than at low nocodazole. Increasing hesperadin to 0.5 or 1.0 μM strongly shortened arrest at both 0.33 and 3.3 μM nocodazole. Reversine was a separate checkpoint-override comparator.
Literature-Reported Treatment Conditions
- Treatment concentration
- Hesperadin 100 nM in Fig. 2A; 0.1, 0.5 and 1 μM in Fig. 2B. Nocodazole: 0.33 or 3.3 μM in Fig. 2B; concentration range in Fig. 2A.
- Exposure duration
- Inhibitors added 5 h after release from single thymidine arrest; cells followed through mitosis.
- Method
- Nocodazole plus Hesperadin exposure followed by video microscopy.
- Vehicle
- DMSO comparison in Fig. 2B; final solvent percentage not established in the read scope.
- Co-treatment
- Nocodazole co-treatment; 1 μM reversine tested separately as positive comparator, not combined with Hesperadin in this adopted study.
- Reported limits
- Exact means/s.d. and condition-specific n in Supplementary Table SI were not retrieved; no exact values reconstructed. Hesperadin supplier/form characterization not established in the read scope.
Methods and source locations
Compound reported in the paper: Hesperadin; Compound label in the paper: hesperadin
Results apply to the reported experimental material and conditions; no equivalence to a particular Adooq lot is established.
Fig. 2A–B and caption; Results and discussion: Effects on mitotic arrest from inhibiting Aurora B in low or high nocodazole; Materials and methods: Cell culture and synchronization, Video microscopy
Time in mitosis under low/high nocodazole and increasing hesperadin: Time-lapse microscopy; mitotic rounding used to assess time in mitosis. Fig. 2A reports mean and s.d., at least 50 cells per condition; Fig. 2B follows the same filming approach. Numerical approximations here are from Results prose, not curve estimation. — Fig. 2A–B and caption; Results and discussion: Effects on mitotic arrest from inhibiting Aurora B in low or high nocodazole
Mad2 retention at kinetochores in nocodazole-treated RPE1 cells
Cell-based study · Human RPE1 cells treated with 3.2 μM nocodazole, with or without 100 nM Hesperadin, for 10 h. [3]
Under this condition, Hesperadin did not measurably reduce kinetochore-associated Mad2 relative to nocodazole alone. This is a model- and condition-specific negative result.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 nM Hesperadin with 3.2 μM nocodazole.
- Exposure duration
- 10 h.
- Method
- Cells fixed and immunostained for Mad2 and ACA; normalized kinetochore intensity compared with nocodazole-only cells.
- Vehicle
- All drugs diluted in DMSO according to Materials and methods; final concentration and exact vehicle matching not specified in read text.
- Co-treatment
- Nocodazole co-treatment, compared with nocodazole alone. MG132 conditions in Fig. 4E–G are not part of the adopted B–D experiment.
- Reported limits
- Hesperadin supplier/lot and exact error-bar definition for Fig. 4D not established in acquired text/caption; no power or equivalence conclusion made.
Methods and source locations
Compound reported in the paper: Hesperadin; Compound label in the paper: Hesperadin
Results apply to the reported experimental material and conditions; no equivalence to a particular Adooq lot is established.
Fig. 4B–D and caption; Results paragraph beginning If aurora B activity is required; Materials and methods: Cell culture, Immunostaining, Mad2 intensity quantification
Kinetochore-associated Mad2 fluorescence normalized to anti-centromere antibody (ACA): Immunofluorescence of Mad2 and ACA; same acquisition settings. 100 sister-kinetochore pairs examined in each of three separate experiments. Nocodazole-only mean normalized to 1. Exact bar heights and error values not extracted. — Fig. 4B–D and caption; Materials and methods: Mad2 intensity quantification
Hesperadin comparator in GFP-MELK localization
Cell-based study · GFP-MELK-transfected HeLa cells (HeLaM subline specified in shared methods), assessed in prometaphase. [4]
Unlike the premature cortical GFP-MELK localization observed after OTSSP167, Hesperadin-treated prometaphase cells did not show this redistribution. The comparison supports a narrow localization result, not a claim about MELK catalytic inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 nM Hesperadin.
- Exposure duration
- Exposure duration for Hesperadin in Fig. 7C is not explicit in the read caption; 2 h is stated for OTSSP167 in Fig. 7B only.
- Method
- GFP-MELK-transfected cells exposed to Hesperadin and examined by fluorescence microscopy.
- Vehicle
- Vehicle for Fig. 7C not explicitly specified; no DMSO percentage assigned.
- Co-treatment
- No drug combination inferred; OTSSP167, reversine and Plk1 inhibitor III were separate comparators.
- Reported limits
- Hesperadin exposure time, counted cell numbers and quantitative cortical/cytoplasmic ratios are not specified in the adopted Fig. 7C text/caption.
Methods and source locations
Compound reported in the paper: Hesperadin; Compound label in the paper: Hesperadin
Results apply to the reported experimental material and conditions; no equivalence to a particular Adooq lot is established.
Fig. 7A–C and caption; Results: GFP-MELK localization comparison; Materials and Methods: Cell culture, drug treatment and plasmids; Immunofluorescence and live cell imaging
Premature cortex association of GFP-MELK in prometaphase: Single-plane fluorescence images of GFP-MELK and DAPI; Fig. 7A shows untreated metaphase/anaphase localization, B shows OTSSP167, and C shows Hesperadin and separate kinase-inhibitor comparators. No numerical localization frequency reported in the adopted figure/caption. — Fig. 7A–C and caption; Results: GFP-MELK localization comparison; Materials and Methods: Cell culture, drug treatment and plasmids
