Fig. 3: KDM5B-mediated cell cycle arrest and inhibition of cytokinetic abscission.
From: Persister state-directed transitioning and vulnerability in melanoma
a Propidium iodide cell cycle analysis of Cpd1-treated WM3734 (above) or Dox-treated WM3734Tet3G-KDM5B cells (below) after 72 h. Shown are representative data (n = 4). b Quantitation of the G1 (upper panel) and S/G2/M (lower panel) cell cycle duration by real-time cell cycle imaging of FUCCI-WM164 cells pretreated with Cpd1 (10 µM) vs. DMSO or Neg4 controls (10 µM) for 72 h. Scatter dot plots represent mean ± SD (n = 3); Kruskal–Wallis test. c Time-lapse imaging analysis of WM3734 cell numbers during treatment with Cpd1 (10 µM) vs. DMSO or Neg4 controls (10 µM) up to 72 h (15 different areas, n = 2). d Time-lapse microscopic movies were analyzed for the time to complete cytokinesis (15 different cells, n = 2). e Quantitation and representative immunofluorescence staining of midbodies in Cpd1-treated (72 h) WM164 cells (left) and Dox-treated WM3734Tet3G-KDM5B cells (right) vs. respective controls. Depicted are single fluorescence channels: blue (DAPI, nucleus); yellow (α-tubulin, microtubules); green (Aurora B kinase, midbodies); red (phalloidin, F-actin). Dot plots show the ratio of midbodies/cell per field of view, 12–16 fields, n = 3. Mean ± SD, left, one-way ANOVA: right, two-sided t-test. f Quantitation of midbody decrease in the KDM5B-PROTAC model. Proteasomal degradation of KDM5B was induced by SLF′-thalidomide (SLF′-t) concurrently applied with Cpd1 (“con”) or applied after Cpd1 pre-treatment for 3 days (“pre”). Neg4 was used as control. Dot plots show the ratio of midbodies/cell nuclei as median values with the interquartile range (4 coverslips, n = 2; one-way ANOVA). g Venn diagram and gene ontology analysis of significantly regulated genes upon Cpd1 (10 µM) treatment of WM3734 cells as detected by mass spectrometry and RNA sequencing. h Quantitation of downregulation of selected cytokinesis regulators as assessed by mass spectrometry and RNAseq (left, n = 1) or qPCR (right, mean ± SD, n = 2) in Cpd1-treated WM3734 and Dox-treated WM3734Tet3G-KDM5B cells. Source data are provided as a Source Data file.
