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. 2018 Jun 1;32(11-12):849–864. doi: 10.1101/gad.307504.117

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© 2018 Kim et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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Figure 4. — RNAi-mediated Jak2 depletion induces an anti-proliferative response independent of cell death in Eµ-Crlf2/Jak2^R683G B-ALLs in vivo. (A) Schematic illustration of the experimental design. Splenocytes from moribund bound secondary recipients (mouse 7, mouse 8, and mouse 9 from recipients in Fig. 3F, referred to here as Jak2 knockdown-persistent clone 7 [C#7], clone 8 [C#8], and clone 9 [C#8]) of Eµ-Crlf2/Jak2^R683G-pREBIR-shJak2.3323 (Dox on) leukemia cells were harvested and engrafted into tertiary recipient mice. n = 6 for each clone. For each clone, mice were left untreated (Dox off; n = 3) or treated with Dox (Dox on; n = 3) from day 0 after transplantation. (B) Kaplan-Meier survival curve of Dox-treated (Jak2 off; n = 3 per clone) and untreated (Jak2 on; n = 3 per clone) recipient mice of Jak2 knockdown-persistent Eµ-Crlf2/Jak2^R683G-pREBIR-shJak2.3323 leukemia cells. Gray shading indicates the period of Dox administration. (C) Representative flow cytometric plot showing eBFP2/dsRed proportions of Jak2 knockdown-persistent Eµ-Crlf2/Jak2^R683G-pREBIR-shJak2.3323 cells (GFP⁺) in the spleens of Dox-treated (Dox-on) and untreated (Dox-off) recipients in B at terminal disease. (D) Immunoblotting against the indicated targets was performed using lysates isolated from splenocytes of individual moribund bound Dox-treated (Dox on; eBFP2⁺/dsRed⁺ cells; M5–M8) and untreated (Dox off; eBFP2⁺/dsRed⁻ cells; M1–M4) recipients in B. (E) Representative flow cytometric plot of BrdU incorporation and 7-AAD staining of eBFP2⁺/dsRed⁺ splenocytes from recipients of Eμ-Crlf2/Jak2^R683G-pREBIR-shRenilla.713 (shRen.713) and Eμ-Crlf2/Jak2^R683G-pREBIR-shJak2.3323 B-ALLs (shJak2.3323) at 48 h after in vivo shRNA induction. (F) Quantification of S-, G₀/G₁-, and G₂M-phase cell percentages of cell cycle analysis from Figure 6E. Error bars represent SEM (n = 2) performed in triplicate (three independent mice). (*) P < 0.05; (ns) nonsignificant (P > 0.05).