mcl 1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: TET2 loss promotes premalignant survival and clonal selection in MYC-driven B cell lymphoma

doi: 10.64898/2026.03.20.712678

Figure Lengend Snippet: (A) GO-term analysis of RNA-seq data from FACS-sorted IgM + immature-like B cells from the comparison of premalignant EµMyc Tet2 −/− (n=5) versus EµMyc (n=6) mice. DEGs (adjusted p-value<0.05 and absolute log 2 (fold change)>1) were subjected to MSigDB Hallmark 2020 in Enrichr. The bar graph depicts the top enriched pathways ranked by p-value, with the number of DEGs contributing to each term indicated in italics at the end of each bar. (B) Volcano plot comparing transcriptional profiles, with a specific focus on apoptotic/BCL2-family gene panel from the RNA-seq analysis described in (A). Significance was defined as adjusted p-value<0.05 and absolute log 2 (fold change)>0.5. Downregulated genes in EµMyc Tet2 −/− subsets are shown in blue; upregulated genes are shown in red; apoptotic/BCL2-family genes, which are not differentially expressed in dark grey, and all other genes in bright grey. (C) Flow cytometric analysis determining the fraction of BCL2 + cells and MFI within the BCL2 + gate in IgM + immature-like B cells (B220 + CD19 + IgM + IgD − ) ( EµMyc : n=6, EµMyc Tet2 −/− : n=4). (D) MFI of BCL-XL and MFI of MCL1 in IgM + immature-like B cells, quantified by flow cytometry ( EµMyc : n=8, EµMyc Tet2 −/− : n=4). (E) Flow cytometric analysis determining the fraction of BIM hi cells and representative histograms for the BIM staining in the IgM + immature-like B cell compartment ( EµMyc : n=8, EµMyc Tet2 −/− : n=4). (F) Flow cytometric assessment determining the fraction of BCL2 + BIM hi cells and representative dot plots of the BCL2 + BIM hi population in IgM + immature-like B cells ( EµMyc : n=3, EµMyc Tet2 −/− : n=4). (G) Cell survival kinetics assessed in vitro for IgM + immature-like B cells (DAPI + B220 + CD19 + IgM + IgD − ) from EµMyc (n=3) and EµMyc Tet2 −/− (n=3) mice, at 0, 2, 6, and 10 hours of culture. Assessment of mitochondrial apoptotic sensitivity via cytochrome c release of IgM + immature-like B cells (ZombieDye − B220 + CD19 + IgM + IgD − cytochromec − ) treated with (H) 30 µM ABT-199/Venetoclax and (I) 1 µM S63845. For both treatments, cytochrome c release in DMSO controls and treated samples is shown as a line plot (left) and as a bar graph (right), depicting the fold change relative to DMSO ( EµMyc : n=3, EµMyc Tet2 −/− : n=4). Bar plots show median with interquartile range. Statistical significance was assessed using unpaired t-test (A-F, H, I), or two-way ANOVA (G) with Holm-Šidák correction for multiple comparisons. Normality was evaluated using the Shapiro-Wilk test. MFI = mean fluorescence intensity, ns = not significant, *p<0.05, **p<0.005.

Article Snippet: To assess BCL2 family members, cells were incubated with 30 μl αCD16/32 Fc-Block (1:20 in PermWash Buffer) for 15 min, followed by incubation with 30 μl of following antibodies: αBIM (1:100, Abcam, ab32158), αBCL2-PE (1:100, Biolegend, 633507), αMCL1 (1:100, Cell Signaling, 5453T) or αBCL-XL (1:100, Cell Signaling, 2764S) for at least 30 min. After a washing step with 100 μl PermWash Buffer, a goat anti-rabbit IgG (H+L) Alexa Fluor TM 647-conjugated antibody (1:1000, Invitrogen, A21245) was applied for 15 min. A final washing step with 100 μl PermWash Buffer was performed.

Techniques: RNA Sequencing, Comparison, Flow Cytometry, Staining, In Vitro, Fluorescence