Journal: HemaSphere

Article Title: Casein kinase 1δ/ε inhibition suppresses CLL proliferation through cell‐intrinsic and microenvironmental mechanisms

doi: 10.1002/hem3.70343

Figure Lengend Snippet: Validation of the cell cycle and proliferation effects of casein kinase 1δ/ε (CK1δ/ε) inhibition in vivo and in vitro. (A) Percentages of EdU‐Alexa Fluor 647+ leukemic B cells within the spleen (SPL) of treated and control TCL1 adoptive transfer (AT) recipient mice ( N (AT CTRL) = 3; N (AT + PF‐670462) = 4), tested by the t ‐test. (B) Relative cell counts (% of CTRL) originating from in vitro treated MEC‐1 wild‐type (WT) cells after a 72 h treatment with PF‐670462 or MU1742 (performed on the following biological replicates: N (CTRL) = 6; N (3µM PF‐670462) = 3; N (10µM PF‐670462) = 6; N (3µM MU1742) = 3; and N (10µM MU1742) = 3), tested by the Kruskal–Wallis test with post hoc pairwise Wilcoxon rank sum tests with Benjamini–Hochberg correction. (C) Relative cell counts (% of CTRL) originating from in vitro treated HG‐3 WT cells after 72 h treatment with PF‐670462 or MU1742 (performed on the following biological replicates: N (CTRL) = 4; N (3µM PF‐670462) = 4; N (10µM PF‐670462) = 4; N (3µM MU1742) = 4; and N (10µM MU1742) = 4), tested by the Kruskal–Wallis test with post hoc pairwise Wilcoxon rank sum tests with Benjamini–Hochberg correction. (D) Cell cycle assay setup with initial CK1 inhibitor treatment and mitotic arrest with nocodazole and the representative example of cell cycle alterations between analyzed conditions in MEC‐1 and HG‐3 cell lines. (E) Cell cycle phase distribution in MEC‐1 WT cells upon 9 h pre‐treatment with 3 µM PF‐670462 and 10 µM PF‐670462 and subsequent mitotic arrest with nocodazole (performed on the following biological replicates: N (CTRL) = 11; N (NOCODAZOLE) = 11; N (3µM PF‐670462) = 7; and N (10µM PF‐670462) = 11); for all cases together, the generalized linear mixed‐effects model, followed by estimated marginal means calculation (P‐value < 0.05), was used separately for comparison of CTRL versus NOCODAZOLE and NOCODAZOLE versus PF‐670462 and corrected due to usage of 2 models. (F, G) Cell cycle phase distribution in MEC‐1 WT cells upon 9 h pre‐treatment with 3 and 10 µM concentrations of MU1742 and AH078 (respectively) and subsequent mitotic arrest with nocodazole (performed on the following biological replicates: N (CTRL) = 11; N (NOCODAZOLE) = 11; N (3µM) = 3; and N (10µM) = 3); for all cases together, the generalized linear mixed‐effects model followed by estimated marginal means calculation (P‐value < 0.05), was used separately for comparison of CTRL versus NOCODAZOLE and NOCODAZOLE versus MU1742/AH078 and corrected due to usage of 2 models. (H–J) Cell cycle phase distribution in HG‐3 WT cells upon 9 h pre‐treatment with 3 and 10 µM concentrations of PF‐670462, MU1742, and AH078 (respectively) and subsequent mitotic arrest with nocodazole (performed on the following biological replicates: N (CTRL) = 4; N (NOCODAZOLE) = 4; N (3µM) = 4; and N (10µM) = 4); for all cases together, the generalized linear mixed‐effects model, followed by estimated marginal means calculation (P‐value < 0.05), was used separately for comparison of CTRL versus NOCODAZOLE and NOCODAZOLE versus PF‐670462/MU1742/AH078 and corrected due to usage of 2 models. DMSO, dimethyl sulfoxide; PI, propidium iodide.

Article Snippet: CLL cell lines MEC‐1 WT (DSMZ, #ACC497) and HG‐3 WT (DSMZ, #ACC765) were treated with PF‐670462 (DC Chemicals, #DC2086), an in‐house CK1δ/ε inhibitor MU1742 or CK1δ/ε degrader AH078, and subjected to cell proliferation tracking and cell cycle tracking via PI staining, EdU Click‐iT assays, and/or western blotting, as described in more detail in the Supporting Information S1: .

Techniques: Biomarker Discovery, Inhibition, In Vivo, In Vitro, Control, Adoptive Transfer Assay, Cell Cycle Assay, Comparison