Fig 1: High ETV6 or ETV6/PIM2 levels predict poor survival in B-cell lymphomas. Kaplan–Meier survival curves of the entire series of 223 DLBCL patients [25], analysed as a group (A) or excluding PBML cases (B). ETV6 (probe 235056_at) high cases (>median expression) or ETV6 low cases (Kaplan–Meier survival curves of the entire series of 223 DLBCL patients [25], analysed as a group (C) or excluding PBML cases (D). PIM2 (probe 204269_at) high cases (>median expression) or PIM2 low cases (Kaplan–Meier survival curves of the entire series of 223 DLBCL patients [25], analysed as a group (E) or excluding PBML cases (F). High ETV6/PIM2 ratio (>median level) or low ETV6/PIM2 ratio cases (Kaplan–Meier survival curves of a series [26] of DLBCL patients (left panel), mantle cell lymphoma (MCL) patients (middle panel) or Burkitt lymphoma (BL) patients (right panel). ETV6 high cases (>median expression) or ETV6 low cases (ETV6 high expressors (>2 mean expression Z score) across the B-cell lymphoma subtypes [26] (top panels), and these cases are associated with poor survival (bottom panel).
Fig 2: PIM2 regulates PFKFB3 protein stability. (A) HEK293T cells were cotransfected with HA-PFKFB3 and Flag-PIM2 (WT or KA) plasmid. Western blot analysis tested whole-cell lysate after 72-h transfection. (B) HEK293T cells were cotransfected with HA-PFKFB3 and Flag-PIM2 (0, 0.5, 1.0 µg) plasmid. Western blot analysis tested whole-cell lysate after 72-h transfection. (C and D) MCF7 or MB231 cells were cotransfected with Flag-PIM2 plasmid. Whole-cell lysate after 72-h transfection. (E and F) MCF7 or MB231 cells were knocked down PIM2 with shRNA. Total cell lysates were prepared. (G) MCF7 cells were transfected with HA-PFKFB3 and Flag-PIM2 plasmids, and then treated with CHX for indicated time. Total cell lysates were prepared. (H) MCF-7 cells with stable knockdown PIM2 proteins were treated with CHX for indicated time. Total cell lysates were prepared. (I and J) MCF7 or MB231 cells were knocked down PIM2 with shRNA. Confocal immunofluorescence microscopy was performed to observe the expression of PIM2 and PFKFB3. Red box mark was partially enlarged in the Figures S1B and S1C. (K) PIM2 gene knockout mice constructed and screened. (L) Detection of PIM2 and PFKFB3 expression in PIM2 knockout mice by WB. (M) MCF-7 cells were overexpressed the indicated both Flag-PIM2 and HA-PFKFB3 (WT, S478A, or S478D) proteins. Total cell lysates were prepared. All experiments were repeated at least 3 times
Fig 3: PIM2 expression is positively correlated with pS478‐PFKFB3 in BC. (A) Immunohistochemical expression of PIM2 or pSer478‐PFKFB3 in human BC tissues. (B) Pearson correlation analysis of PIM2 and pSer478‐PFKFB3 semi‐quantitative staining score. (C) A working model that schematic diagram of PIM2 regulating breast cancer glycolysis and paclitaxel resistance through PFKFB3 Ser478 site
Fig 4: ETV6 expression in malignant B-cells compared to normal B-cell subsets. (A) Heat map representation of ETV6 and PIM2 transcript levels in DLBCL samples and normal tonsil B-cell subsets [29]. (B) Comparison of ETV6 transcript levels between DLBCL subgroups (ABC, GCB and UNCL) and normal B-cell subsets obtained from tonsils. For statistical analysis, an unpaired t-test was used. (C) Comparison of PIM2 transcript levels between DLBCL subgroups (ABC, GCB and UNCL) and normal B-cell subsets obtained from tonsils. CC = centrocytes; CB = centroblasts; PB: plasmablasts. For statistical analysis, a nonparametric t-test was used. (D) ETV6 immunohistochemical staining of reactive tonsil (top): original magnification (×10) and 40× (insets). PIM2 immunohistochemical staining of reactive tonsil (top): original magnification (×10) and 40× (insets). (E) Immunohistochemical staining for ETV6 in representative cases of DLBCL patients showing low (+1), medium (+2) and high (+3) expression levels. Original magnification ×20; inset ×40. (F) OncoPrint visualization in cBioPortal of genetic alterations (top) affecting ETV6, MYC, BCL2 and BCL6 genes for selected B-lymphoma cell lines (source: Cancer Cell Line Encyclopedia (Broad, 2019) and Lymphoma cell lines (MSKCC,2020)). Heat map showing gene expression levels of selected genes is also depicted (bottom). (G) Heat map representation of relative ETV6 and PIM2 transcript levels (normalized to RPL19 housekeeping gene) in available B-cell lymphoma cell lines as determined by qRT-PCR. (H) Evaluation of ETV6 and PIM2 and protein expression levels in DLBCL cell lines of different molecular subtypes (ABC and GCB-type) and Burkitt lymphoma (BL) cell lines using immunoblotting. Tubulin is shown as loading control. Relative protein expressions (normalized to loading control) are shown on top of appropriate panels. kD = kilodaltons.
Fig 5: Kaplan–Meier curves of OS in DLBCL patients treated with chemotherapy and stratified according to various putative prognostic markers. (A) Kaplan–Meier curves of OS in two groups identified using the PIM2-ETV6 prognostic score (Group 0: score ≤ median; Group 1: score > median). (B) Kaplan–Meier curves of OS in the three groups identified using the tertiles of PIM2-ETV6 prognostic score (<25%, 25–75%, >75% of score). (C) Kaplan–Meier curves of OS in the same patients subdivided into GCB and non-GCB subgroups using the Hans immune-histochemical algorithm, which uses three markers (CD10, BCL6 and MUM1) to separate GCB DLBCL from non-GCB DLBCL. (D) Kaplan–Meier curves of OS in the same cohort of patients subdivided into COO subgroups (ABC, GCB, UNCL) using the Lymph2Cx assay. (E) Kaplan–Meier curves of OS in the same patients subdivided into 4 groups using the IPI clinical score. (F) IHC validation of ETV6 and PIM2 expression in an independent DLBCL patient cohort (n = 39). Kaplan–Meier curves of OS in this cohort, dividing the patients into two groups in relations to the ETV6/PIM2 ratio (high: ratio > 1; low < 1). (G) Bar graph showing the distribution of patients’ outcome (alive versus dead) in relation to ETV6 or PIM2 expression. High: staining intensity > 1; low: staining intensity ≤ 1. The Chi square test was used to determine significance. (H) Kaplan–Meier curves of OS in the four groups identified using the PIM2 (low versus high) and ETV6 (low versus high) IHC expression levels (cohort n = 39).
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