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

Journal: bioRxiv

Article Title: Functional Convergence of Genetically Diverse B-Cell Receptors in Simian-HIV Infected Rhesus Macaques

doi: 10.64898/2026.01.09.698730

Figure Lengend Snippet: (A) Kinetics of IgG production in culture supernatants from RM IgM + and IgG + single B cells cultured with the MS40L feeder cell line. Each dot represents an individual IgG + culture well. Dashed lines indicate detection limits, and error bars (in red) represent the median values. (B-C) Comparison of the original MS40L and engineered MEC-147 feeder cell lines in supporting single B-cell cultures from different B-cell populations. Panel (B) shows the culture of B cells isolated from the lymph nodes of an RM after DTaP immunization, while panel (C) shows the culture of B cells isolated from PBMCs of a healthy human donor. The top panels display the percentage of IgG + culture wells among all single-cell sorted wells. Error bars indicate the mean + SD across three 96-well culture plates. The bottom panels show OD450 values from IgG ELISA assays, with each dot representing an individual IgG + culture well. Dashed lines indicate detection limits, and error bars (in red) represent the median values. (D) Single nucleated cells were isolated from LN biopsies of infected RMs at various time points following SHIV infection. B cells of different phenotypes, including mature follicular (MF), IgM + Bmem, IgG + GC, and IgG + Bmem cells, were identified from isolated LN cells by flow cytometry and sorted into single B-cell cultures with MEC-147 feeder cells. Supernatants were harvested after 18 days of culture, and IgG-producing cultures were identified using standard ELISA assays. IgG-containing culture supernatants were tested for neutralization activity against autologous SHIV pseudo-viruses using standard TZM-bl assays. Each dot represents an individual single B-cell culture supernatant containing clonal IgG. Error bars in red indicate median values, while blue dashed lines indicate cutoff thresholds for defining neutralizing versus non-neutralizing cultures. Inserted red numbers represent the percentage of neutralizing cultures among IgG + cultures for the corresponding B-cell populations (see Table S2 for details).

Article Snippet: Garnett Kelsoe, Shengli Song, and Duke University, non-exclusively licensed the engineered feeder cell line MEC-147 to Moderna Inc .

Techniques: Cell Culture, Comparison, Isolation, Enzyme-linked Immunosorbent Assay, Infection, Flow Cytometry, Neutralization, Activity Assay