In cultured Aplysia neuronal growth cones, 100 μM CK666 for 20 min reduced leading-edge Arp3 enrichment. The inactive analogue CK689 lacked this effect, and enrichment recovered after 30 min in control medium. [3]
In RSL3-exposed HT1080 cells, CK-666 at 100 μM preserved viability over 24 h. CK-689 did not protect, while other Arp2/3 inhibitors also failed to reproduce the protection. These comparisons caution against attributing every CK-666 phenotype solely to Arp2/3 inhibition. [4]
Inhibition of the activating Arp2–Arp3 conformation
Biochemical assay · Purified budding-yeast Arp2/3 cysteine-pair reporters Arp-021 and Arp-024 [1]
CK-666 reduced the short-pitch crosslink. Single-cysteine controls lacked the product; reporter complexes retained NPF-responsive activity but were less inhibitor-sensitive than wild type.
Literature-Reported Treatment Conditions
- Treatment concentration
- 200 μM
- Exposure duration
- Crosslink reaction quenched after 10 s
- Method
- BMOE crosslinking and anti-Arp3 immunoblot
- Co-treatment
- GST-VCA and latrunculin-B-bound actin are assay components, not a therapeutic combination.
- Reported limits
- Inhibitor preincubation and vehicle details not established from retrieved main methods; SI unavailable.
Methods and source locations
Compound reported in the paper: CK-666; Compound label in the paper: CK-666
Applies to the reported research compound and model; no salt conversion, clinical efficacy claim or Adooq lot validation.
Figure 5B–H; Results: Developing a biochemical assay; CK-869 and CK-666 block formation of the short pitch dimer; Experimental Procedures: Dual cysteine crosslinking assays
Arp2–Arp3 short-pitch crosslink: Ratio of crosslinked to uncrosslinked Arp3; no numerical fraction inferred from bars. — Figure 5G–H and caption
Preformed actin branches did not undergo accelerated dissociation
Biochemical assay · Purified bovine Arp2/3-generated actin branches [1]
CK-666 did not increase branch dissociation over the observed time window.
Literature-Reported Treatment Conditions
- Treatment concentration
- 200 μM
- Exposure duration
- Branches formed for 6 min before wash; displayed observation to 12 min after wash
- Method
- TIRF branch-survival imaging after flushing with inhibitor or DMSO buffer
- Vehicle
- DMSO control
- Reported limits
- Additional TIRF methods in unread SI were not established.
Methods and source locations
Compound reported in the paper: CK-666; Compound label in the paper: CK-666
Applies to the reported research compound and model; no salt conversion, clinical efficacy claim or Adooq lot validation.
Figure 3G–H and caption; Results: Inhibitors do not influence interactions with sides or ends of filaments or disassemble preformed branches
Survival of preformed branch junctions: TIRF microscopy; n denotes branches, not independent biological replicates. — Figure 3G–H and caption
Reversible loss of leading-edge Arp3 enrichment
Cell-based study · Cultured Aplysia bag-cell neuron growth cones [3]
CK666 reduced leading-edge Arp3 enrichment; CK689 lacked the effect, and enrichment recovered after washout.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 μM; inactive CK689 control also 100 μM
- Exposure duration
- 20 min treatment; 30 min recovery in control medium
- Method
- Arp3 immunofluorescence; leading-edge enrichment ratio
- Vehicle
- DMSO
- Reported limits
- Vehicle percentage not extracted.
Methods and source locations
Compound reported in the paper: CK-666; Compound label in the paper: CK666
Applies to the reported research compound and model; no salt conversion, clinical efficacy claim or Adooq lot validation.
Figure 1B–H; first Results subsection; Materials and methods: Cell culture and chemicals, Immunocytochemistry, Quantification of Arp2/3 complex enrichment, Statistical analysis
Leading-edge Arp3 enrichment: Ratio of average Arp3 intensity in peripheral slices 1 and 4; two-tailed unpaired t test, P < 0.01 for marked differences; counts in Figure 1E are growth cones, not biological replicates. — Figure 1B–E and caption; enrichment quantification methods
Differential effects of Arp2/3 inhibitors on ferroptotic cell viability
Cell-based study · HT1080 cells exposed to RSL3 [4]
CK-666 and CK-636 preserved viability, while inactive CK-689 did not. CK-869 slightly sensitized cells; benproperine and pimozide did not protect. These pharmacological contrasts limit target-specific attribution.
Literature-Reported Treatment Conditions
- Treatment concentration
- CK-666, CK-636, CK-689 and CK-869: 100 μM; benproperine and pimozide: 10 μM
- Exposure duration
- 24 h
- Method
- Hoechst 33342/propidium iodide imaging of total and dead cells
- Co-treatment
- RSL3 concentration series induces ferroptosis; CK-666 is tested as cotreatment.
- Reported limits
- Vehicle concentration not established; genetic depletion validation and SI-dependent mechanism claims not adopted.
Methods and source locations
Compound reported in the paper: CK-666; Compound label in the paper: CK-666
Applies to the reported research compound and model; no salt conversion, clinical efficacy claim or Adooq lot validation.
Figure 2A–C; Results sec1.2; Experimental procedures: Chemicals and Cell viability measurement
Cell viability during RSL3 exposure: Imaging-based viability; no IC50 or curve-derived viability percentage estimated. — Figure 2B–C and caption
ArpC1 isoform-dependent inhibition of actin branching
Biochemical assay · Defined recombinant human Arp3-containing Arp2/3 complexes with ArpC1A or ArpC1B and ArpC5 or ArpC5L [2]
At 100 μM, CK-666 inhibited ArpC1A-containing complexes but did not significantly inhibit ArpC1B-containing complexes in the branching assay.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 μM for direct comparison; concentration series for IC50
- Exposure duration
- 1 h inhibitor preincubation; representative branching images at 2 min
- Method
- GST-N-WASP-VCA-driven pyrene polymerization and TIRF branching assays
- Vehicle
- Matched DMSO control
- Co-treatment
- GST-N-WASP-VCA is the activating assay component.
- Reported limits
- No universal potency inferred across isoforms or activators. Methods describe branching-rate time units inconsistently (per minute versus per second); no absolute branching rate adopted.
Methods and source locations
Compound reported in the paper: CK-666; Compound label in the paper: CK-666
Applies to the reported research compound and model; no salt conversion, clinical efficacy claim or Adooq lot validation.
Main Figure 2A–C (HTML/XML id Fig3); Results Sec4; Methods Sec13–15
VCA-driven branching of ArpC1A/C5L-containing complex: TIRF branch generation with fitted concentration-response; points: mean ± SD from at least two independent measurements. — Main Figure 2C (HTML/XML id Fig3) and Results Sec4
Branching rate by defined iso-complex: Paired two-tailed t test; preserves the negative result without converting plotted rates. — Main Figure 2B (HTML/XML id Fig3) and caption
