Fig. 7: Validation of F5 as a tubulin-targeting compound.

a, Structures of the six class F compounds that have >85% similarity to the tubulin cluster profile. b, Tubulin cluster profile relative to class F compounds that have >85% similarity to the tubulin cluster profile. The selected profiles are those that have >85% similarity to the tubulin cluster profile and >20% induction at the lowest concentrations per compound. The reference profile is the first profile (100% biosimilarity) for which all subsequent profiles are compared. The tubulin profile has 424 features and is divided into three segments: cell, cytoplasm and nuclei. Biosim, biosimilarity to the tubulin cluster profile; ind, induction (percentage of significantly changed features relative to DMSO controls); and conc, concentration. The induction value reported is relative to the full CPA profiles with 579 features. c, Influence on the microtubule network. U2OS cells were treated for 24 h with DMSO (control) or F5 before staining for tubulin (green) and DNA (blue). Scale bar, 50 µm. d, Quantification of mitotic cells via immunocytochemistry. U2OS cells were treated with DMSO (negative control), F5, nocodazole (noc, positive control) or colchicine (col, positive control) for 24 h before staining of cells for phospho-histone H3 and DNA. Cells in mitosis were quantified as the percentage of phospho-histone H3-positive cells. Data are mean values ± s.d. of three independent replicates (n = 3). Statistical analyses were performed relative to the DMSO control by employing unpaired two-tailed t-tests (****P < 0.0001). The P values relative to cells treated with DMSO are <0.0001 for F5 (10 µM)-treated, <0.0001 for F5 (30 µM)-treated, <0.0001 for noc-treated and <0.0001 for col-treated cells. e, In vitro tubulin polymerization assay. The polymerization was initiated upon addition of guanosine triphosphate (GTP) to porcine tubulin and quantified by means of turbidity measurement at 340 nm and 37 °C. DMSO was used as a negative control and noc was used as a positive control for tubulin destabilization. Data are representative of three independent experiments (n = 3).