kir 3 1  (Alomone Labs)

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: Kaplan-Meier survival analysis displaying (A) overall survival probability, and (B) median time to death in days for all malignant mice ( EµMyc : n=27, EµMyc Tet2 +/− : n=44, EµMyc Tet2 −/− : n=27). (C) Spleen to body weight ratio in non-malignant (280-320 days) mice ( wildtype : n=7 , Tet2 +/− : n=13 , Tet2 −/− : n=6) and malignant mice, excluding animals without overt disease at 300 days ( EµMyc : n=13, EµMyc Tet2 +/− : n=11, EµMyc Tet2 −/− : n=23). (D) Splenic immune cell composition assessed by flow cytometry: monocytes/macrophages (CD11b + Gr1 − ), granulocytes (CD11b + Gr1 + ), erythroid progenitors (nucleated Ter119 + ), NK cells (TCRβ − NK1.1 + ), CD4 + T cells (TCRβ + CD4 + ), CD8 + T cells (TCRβ + CD8 + ), and malignant B cells (B220 + CD19 + ) ( EµMyc : n=10, EµMyc Tet2 −/− : n=12). (E) Lymphoma immunophenotype in the spleen. Mixed tumors were defined as tumors where neither IgM − nor IgM + cells constituted >80% of the total tumor population. (F) Volcano plots displaying RNA-seq-derived transcriptional profiles of FACS-sorted IgM − tumor (left; B220 + CD19 + IgM − IgD − ) and IgM + tumor (right; B220 + CD19 + IgM + IgD − ) cells. Comparison between EµMyc Tet2 −/− and EµMyc (IgM − tumors: n=4 vs. n=5, IgM + tumors: n=4 vs. n=3) mice was performed separately for each cell type. Significance was defined as adjusted p-value<0.05 and absolute log 2 (fold change)>1. Downregulated genes in EµMyc Tet2 −/− tumors are shown in blue; upregulated genes are shown in red. (G) DNA content and (H) polyploid cell fraction of splenic lymphoma cells, assessed by TO-PRO-3 staining via flow cytometry ( EµMyc : n=7, EµMyc Tet2 −/− : n=8). Flow cytometry assessment of (I) DNA double strand breaks by γH2AX staining ( EµMyc : n=6, EµMyc Tet2 −/− : n=7) and (J) apoptosis by cleaved Caspase-3 staining ( EµMyc : n=5, EµMyc Tet2 −/− : n=5) within splenic lymphoma cells. Bar graphs show median with interquartile range. Statistical significance was determined using (A) Mantel-Cox test, (B, C) one-way ANOVA, (D, G) Mann-Whitney test, or (H, I, J) unpaired t-test, depending on normality (Shapiro-Wilk test) with Holm-Šidák correction for multiple comparisons. ns = not significant, *p<0.05, **p<0.005.

Article Snippet: Following 48 h of culture, cells were gently resuspended, transferred to a 96-U-well plate, and stained (see section Cell surface staining ) with 30 μl of the following fluorochrome-conjugated anti-mouse antibodies diluted in FACS-B: αIgD-PerCP/Cy5.5 (1:200, Biolegend, 405710), αIgM-APC (1:1000, Jackson ImmunoResearch, 115-607-020), αCD19-BV711 (1:400, Biolegend, 115555), and αB220-BV785 (1:400, Biolegend, 103246).

Techniques: Flow Cytometry, RNA Sequencing, Derivative Assay, Comparison, Staining, MANN-WHITNEY

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: Analysis of splenocytes from premalignant (day 50) EµMyc and EµMyc Tet2 −/− mice. (A) Spleen to body weight ratio for each mouse and (B) absolute splenocyte counts (*10 7 ) ( EµMyc : n=35, EµMyc Tet2 −/− : n=26). (C) Splenic immune cell composition assessed by flow cytometry: monocytes/macrophages (CD11b + Gr1 − ), granulocytes (CD11b + Gr1 + ), erythroid progenitors (nucleated Ter119 + ), NK cells (TCRβ − NK1.1 + ), CD4 + T cells (TCRβ + CD4 + ), CD8 + T cells (TCRβ + CD8 + ), and B cells (B220 + CD19 + ) ( EµMyc : n=18, EµMyc Tet2 −/− : n=13). (D) Splenic B cell subsets were assessed via flow cytometry: IgM − progenitors (B220 + CD19 + IgM − IgD − ), IgM + IgD − immature-(like) (B220 + CD19 + IgM + IgD − ), and IgM + IgD + mature (B220 + CD19 + IgM + IgD + ) B cells. The upper panel summarizes all data ( wildtype : n=10, Tet2 −/− : n=11, EµMyc : n=18, EµMyc Tet2 −/− : n=13), the lower panel shows representative dot blots for the IgM/IgD gate. (E) Volcano plots display RNA-seq-derived transcriptional profiles of premalignant FACS-sorted splenic IgM − progenitors (left; B220 + CD19 + IgM − IgD − ) and IgM + immature(-like) (right; B220 + CD19 + IgM + IgD − ) B cells. Comparisons between EµMyc Tet2 −/− (n=5) and EµMyc (n=6) mice were performed separately for each cell type. Axis ranges were kept identical across volcano plots to allow direct comparison. Significance was defined as adjusted p-value<0.05 and absolute log 2 (fold change)>1. Downregulated genes in EµMyc Tet2 −/− subsets are shown in blue; upregulated genes are shown in red. Bar plots show median with interquartile range. Statistical significance was determined using (A) unpaired t-test or (B, C) Mann-Whitney test and (D) two-way ANOVA, with Holm-Šidák correction for multiple comparisons. Normality was assessed using the Shapiro-Wilk test. n.d. = not detected, ns = not significant, *p<0.05, **p<0.005, ***p<0.0005.

Article Snippet: Following 48 h of culture, cells were gently resuspended, transferred to a 96-U-well plate, and stained (see section Cell surface staining ) with 30 μl of the following fluorochrome-conjugated anti-mouse antibodies diluted in FACS-B: αIgD-PerCP/Cy5.5 (1:200, Biolegend, 405710), αIgM-APC (1:1000, Jackson ImmunoResearch, 115-607-020), αCD19-BV711 (1:400, Biolegend, 115555), and αB220-BV785 (1:400, Biolegend, 103246).

Techniques: Flow Cytometry, RNA Sequencing, Derivative Assay, Comparison, MANN-WHITNEY

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 enrichment 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 GO-term biological process analysis in RStudio. The bar graph depicts the top enriched biological processes ranked by adjusted p-value, with the number of DEGs contributing to each term indicated in italics at the end of each bar. (B) DecoupleR analysis on the RNA-seq experiment performed in RStudio using the CollecTRI transcription factor (TF)-target network. Bar plot displays the top 14 TFs with at least 45 targets and p-value<0.05. (C) Volcano plot comparing transcriptional profiles, with a specific focus on a self-defined B-lineage regulatory/maturation gene panel from the RNA-seq analysis described in (A). Significance was defined as adjusted p-value<0.05 and absolute log2(fold change)>0.5. Downregulated genes in EµMyc Tet2 −/− subsets are shown in blue; upregulated genes are shown in red; B-lineage regulatory/maturation genes, which are not differentially expressed, are shown in dark grey, and all other genes in bright grey. (D-E) Flow cytometric validation of selected B-lineage regulatory and maturation genes in IgM − progenitors (filled symbols; B220 + CD19 + IgM − IgD − ) and IgM + immature-like B cells (empty symbols; B220 + CD19 + IgM + IgD − ). The upper panel displays the percentage of cells expressing the indicated markers, while the lower panel shows the geometric MFI within the respective marker-positive gate. (F) MFI of surface IgM and CD19 within IgM − progenitors (filled symbols; B220 + CD19 + IgM − IgD − ) and IgM + immature-like B cells (empty symbols; B220 + CD19 + IgM + IgD − ). Bar plots show median with interquartile range. Statistical significance was assessed using unpaired t-test for %CD21 + and CD21 MFI, %CD23 + , %CD80 + , %CD86 + , IgM MFI, CD19 MFI, or Mann-Whitney test for CD23 MFI, %CD9 + and CD9 MFI, CD80 MFI, CD86 MFI, 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, ****p<0.0001.

Article Snippet: Following 48 h of culture, cells were gently resuspended, transferred to a 96-U-well plate, and stained (see section Cell surface staining ) with 30 μl of the following fluorochrome-conjugated anti-mouse antibodies diluted in FACS-B: αIgD-PerCP/Cy5.5 (1:200, Biolegend, 405710), αIgM-APC (1:1000, Jackson ImmunoResearch, 115-607-020), αCD19-BV711 (1:400, Biolegend, 115555), and αB220-BV785 (1:400, Biolegend, 103246).

Techniques: RNA Sequencing, Comparison, Biomarker Discovery, Expressing, Marker, MANN-WHITNEY, Fluorescence

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: Following 48 h of culture, cells were gently resuspended, transferred to a 96-U-well plate, and stained (see section Cell surface staining ) with 30 μl of the following fluorochrome-conjugated anti-mouse antibodies diluted in FACS-B: αIgD-PerCP/Cy5.5 (1:200, Biolegend, 405710), αIgM-APC (1:1000, Jackson ImmunoResearch, 115-607-020), αCD19-BV711 (1:400, Biolegend, 115555), and αB220-BV785 (1:400, Biolegend, 103246).

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

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: Analysis of proliferative capacity in splenic IgM + immature-like B cells from EµMyc (n=9) and EµMyc Tet2 −/− (n=10) mice. Total splenocytes were labeled in vitro with a proliferation dye, and flow cytometric analysis was performed at 0 and 48 hours. (A) Representative dot plots showing an overlay of total cells (grey) and live IgM + immature-like B cells (red) at 0 and 48 hours. Two replicates of each genotype are depicted. (B) The fraction of dividing IgM + immature-like B cells was quantified based on proliferation dye dilution. Representative flow cytometry histograms of proliferation dye intensity in IgM + immature-like B cells from EµMyc (left) and EµMyc Tet2 −/− (right) mice. (C) Splenocytes from premalignant EµMyc (n=3) and EµMyc Tet2 −/− (n=3) were plated in methylcellulose with or without IL-7, and colonies were counted after 7 days. (D) Immunoglobulin heavy chain variable region ( Ighv ) gene usage in IgM + immature-like B cells was determined by RNA-seq. Each bar represents an individual mouse, with Ighv genes comprising >4% of total Ighv transcripts displayed in distinct colors with corresponding gene names. Bar plots show median with interquartile range. Statistical significance was determined using (B) unpaired t-test. Normality was assessed using the Shapiro-Wilk test. *p<0.05

Article Snippet: Following 48 h of culture, cells were gently resuspended, transferred to a 96-U-well plate, and stained (see section Cell surface staining ) with 30 μl of the following fluorochrome-conjugated anti-mouse antibodies diluted in FACS-B: αIgD-PerCP/Cy5.5 (1:200, Biolegend, 405710), αIgM-APC (1:1000, Jackson ImmunoResearch, 115-607-020), αCD19-BV711 (1:400, Biolegend, 115555), and αB220-BV785 (1:400, Biolegend, 103246).

Techniques: Labeling, In Vitro, Flow Cytometry, RNA Sequencing