Figure 3.
Figure 3. Pim2 induction by RAG DSBs promotes pre–B cell survival. (A) Western blot analysis of p-Akt and Pim1 in Artemis−/−:IgH:Bcl2 pre–B cells after withdrawal of IL-7 for indicated times. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to time 0 h and standardized to loading controls. (B and C) Western blot analysis of Pim2 expression at indicated times after IL-7 withdrawal. Arrows indicate the three Pim2 isoforms and the asterisk indicates a nonspecific band. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to RAG1−/−:IgH:Bcl2 (B) or Artemis−/−:IgH:Bcl2 (C) at time 0 h and standardized to loading controls. (D) Cell death assessed by 7AAD uptake 24 h after withdrawal of IL-7 from Artemis−/−:IgH and Artemis−/−:Pim2−/−:IgH pre–B cells. Shown is the mean and standard deviation for three replicates. (E) Artemis−/−:IgH pre–B cells were transduced with retroviral vector (red peaks) expressing hCD2 and shEGFP (control) or hCD2 and shPim2 then subsequently withdrawn from IL-7 as mixed co-cultures with nontransduced cells (white peaks). Histograms demonstrate percentage of transduced cells (hCD2+, red peak) before (+IL-7) and 72 h after (−IL-7) withdrawal of IL-7. The experiment was conducted in duplicate with P = 0.008 for shPim2 compared with shEGFP at 72 h. (F) RAG1−/−:IgH pre–B cells were transduced with empty retroviral vector (empty), retrovirus-expressing Pim2 (Pim2), or retrovirus-expressing kinase-dead version of Pim2 (Pim2-KD). IL-7 was withdrawn from mixed cultures of transduced and nontransduced cells. Retrovirally transduced cells were followed by Thy1.1 expression. Data represents the fold increase in the percentage of retrovirally transduced cells at 72 h after IL-7 withdrawal relative to the percentage in the presence of IL-7. Shown is the mean and standard deviation for duplicate cultures. (G) BAX mRNA expression assessed by RT-PCR in cells cultured in IL-7 (+) and 48 h after (−) IL-7 withdrawal. Shown is the mean and standard deviation for three replicates. (H) BAX mRNA expression assessed by RT-PCR at indicated times after IL-7 withdrawal. Shown is the mean and standard deviation for three replicates. (I) PUMA and NOXA mRNA expression assessed by RT-PCR in cells cultured in IL-7 (+) and 72 h after (−) IL-7 withdrawal. Shown is the mean and standard deviation for three replicates. (J) Western blot analysis of p-p53 after withdrawal from IL-7 for the indicated times. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to time 0 h for each genotype and standardized to loading controls. (K) Western blot analysis of phosphorylated Bad (p-Bad) 96 h after IL-7 withdrawal. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to RAG1−/−:IgH:Bcl2 and standardized to loading controls. All data are representative of at least three experiments.

Pim2 induction by RAG DSBs promotes pre–B cell survival. (A) Western blot analysis of p-Akt and Pim1 in Artemis−/−:IgH:Bcl2 pre–B cells after withdrawal of IL-7 for indicated times. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to time 0 h and standardized to loading controls. (B and C) Western blot analysis of Pim2 expression at indicated times after IL-7 withdrawal. Arrows indicate the three Pim2 isoforms and the asterisk indicates a nonspecific band. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to RAG1−/−:IgH:Bcl2 (B) or Artemis−/−:IgH:Bcl2 (C) at time 0 h and standardized to loading controls. (D) Cell death assessed by 7AAD uptake 24 h after withdrawal of IL-7 from Artemis−/−:IgH and Artemis−/−:Pim2−/−:IgH pre–B cells. Shown is the mean and standard deviation for three replicates. (E) Artemis−/−:IgH pre–B cells were transduced with retroviral vector (red peaks) expressing hCD2 and shEGFP (control) or hCD2 and shPim2 then subsequently withdrawn from IL-7 as mixed co-cultures with nontransduced cells (white peaks). Histograms demonstrate percentage of transduced cells (hCD2+, red peak) before (+IL-7) and 72 h after (−IL-7) withdrawal of IL-7. The experiment was conducted in duplicate with P = 0.008 for shPim2 compared with shEGFP at 72 h. (F) RAG1−/−:IgH pre–B cells were transduced with empty retroviral vector (empty), retrovirus-expressing Pim2 (Pim2), or retrovirus-expressing kinase-dead version of Pim2 (Pim2-KD). IL-7 was withdrawn from mixed cultures of transduced and nontransduced cells. Retrovirally transduced cells were followed by Thy1.1 expression. Data represents the fold increase in the percentage of retrovirally transduced cells at 72 h after IL-7 withdrawal relative to the percentage in the presence of IL-7. Shown is the mean and standard deviation for duplicate cultures. (G) BAX mRNA expression assessed by RT-PCR in cells cultured in IL-7 (+) and 48 h after (−) IL-7 withdrawal. Shown is the mean and standard deviation for three replicates. (H) BAX mRNA expression assessed by RT-PCR at indicated times after IL-7 withdrawal. Shown is the mean and standard deviation for three replicates. (I) PUMA and NOXA mRNA expression assessed by RT-PCR in cells cultured in IL-7 (+) and 72 h after (−) IL-7 withdrawal. Shown is the mean and standard deviation for three replicates. (J) Western blot analysis of p-p53 after withdrawal from IL-7 for the indicated times. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to time 0 h for each genotype and standardized to loading controls. (K) Western blot analysis of phosphorylated Bad (p-Bad) 96 h after IL-7 withdrawal. Gapdh is shown as a protein loading control. Numbers represent band intensity relative to RAG1−/−:IgH:Bcl2 and standardized to loading controls. All data are representative of at least three experiments.

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