m2 10b4 crl 1972 cells  (ATCC)

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: Casein kinase 1δ/ε (CK1δ/ε) inhibition attenuated primary chronic lymphocytic leukemia (CLL) cell responsiveness to stromal cells. (A) Scheme of a dual species co‐culture experiment. Human CLL patient ( N = 9) cells were or were not co‐cultured for 6 h with bone marrow (BM) murine cell line M2‐10B4. Cell lysates were subjected to RNA sequencing (RNAseq) and subsequently, human reads were analyzed; all data in this figure are based on this sample set, with the exception of the independent cohort of seven patients in panels (F) , (G) , and (K) . (B) Volcano plot of gene expression detected in CLL cells cultured with and without M2‐10B4 cells. Black dots—downregulated genes (adj. P‐value < 0.05, log 2 FC < −1, adj, adjusted) and red dots—upregulated genes (adj. P‐value < 0.05, log 2 FC > −1), and 10 most upregulated and 10 most downregulated genes are labeled, tested by the limma voom algorithm with Benjamini–Hochberg correction. (C) Linear discriminant analysis trained on a previously described dataset of primary CLL samples from BM, lymph node (LN), and peripheral blood (PB) ⁴⁶ ( N BM = 20, N LN = 14, and N PB = 26) and tested on RNAseq data of primary CLL cells cultured with and without M2‐10B4 cells. (D) Oncology‐related pathways (PROGENy) scored according to differential expression (DE) analysis of CLL cells cultured with and without M2‐10B4 cells. (E) NFKBIA expression in CLL cells cultured with and without M2‐10B4 cells according to RNAseq, tested by the limma voom algorithm with Benjamini–Hochberg correction. (F, G) Percentage of live (F) and IκBα + CLL (G) cells cultured with and without M2‐10B4 cells according to flow cytometry (FCM), tested by Friedman's test coupled with Dunn's post hoc test. (H) Volcano plot of gene expression detected in CLL cells co‐cultured with M2‐10B4 cells with and without PF‐670462 treatment. Blue dots—downregulated genes (adj. P‐value < 0.05, log 2 FC < −1) and red dots—upregulated genes (adj. P‐value < 0.05, log 2 FC > −1), with 10 most upregulated and 10 most downregulated genes labeled, tested by the limma voom algorithm with Benjamini–Hochberg correction. (I) Oncology‐related pathways (PROGENy) scored according to DE analysis of CLL cells co‐cultured with M2‐10B4 cells with and without PF‐670462 treatment. (J) NFKBIA expression in CLL cells co‐cultured with M2‐10B4 cells with and without PF‐670462 (PF‐67) treatment according to bulk RNAseq, tested by the limma voom algorithm with Benjamini–Hochberg correction. (K) Percentage of IκBα + CLL cells co‐cultured with M2‐10B4 cells with and without PF‐670462 treatment according to flow cytometry, tested by Friedman's test coupled with Dunn's post hoc test. (L–N) Heatmaps of expression changes in genes associated with nuclear factor κ B (NFκB) (L) , tumor necrosis factor α (TNFα) (M) , and phosphoinositide 3‐kinase (PI3K) (N) by the PROGENy model. CLL cells cultured with vs. without M2‐10B4 and CLL cells co‐cultured with M2‐10B4 treated with vs. without PF‐670462 were compared. (O) Seurat cell cycle scoring output shown for the G2/M phase, tested by Friedman's test coupled with Dunn's post hoc test (5 M cells were analyzed per sample).

Article Snippet: CLL cells were subjected to co‐culture with CD40L‐expressing HS5 stromal cells or with a BM stromal cell line M2‐10B4 (ATCC, #CRL‐1972).

Techniques: Inhibition, Co-Culture Assay, Cell Culture, RNA Sequencing, RNA sequencing, Gene Expression, Labeling, Quantitative Proteomics, Expressing, Flow Cytometry