Figure 2.
Ybx1 is required to maintain murine myeloid leukemia cell survival in vitro and in vivo. (A) Experimental scheme for assays in panels C to J. Lin– BM cells were transduced with MLL-AF9-yellow fluorescent protein (MLL-AF9-YFP) retrovirus and transplanted into sublethally irradiated recipient mice. YFP+ leukemia cells from AML mice were sorted and used for experiments as indicated. (B) Immunoblot showing Ybx1 KD efficiency in MLL-AF9-YFP leukemia cells after transduction with shRNA lentiviruses targeting Ybx1. (C) Growth curves of murine leukemia cells after transduction with lentiviruses for shYbx1#1, shYbx1#2, or shControl. (D-E) Colony formation assay (D; Control, shControl; P1 and P2, plating 1 and serial replating 2, respectively; Ybx1-KD#1 and Ybx1-KD#2, shYbx1#1 and shYbx1#2, respectively) and representative colony images (E; original magnification, ×100) of murine leukemia cells after transduction with the indicated lentiviruses; cells were cultured in MethoCult M3434 for 7 days. (F) Quantitative summary of flow cytometry analysis of cell cycles after leukemia cells were transduced with the indicated lentiviruses. Cells were labeled with Hoechst 33342 and were assessed by flow cytometry. (G) Percentage of apoptotic murine leukemia cells at day 4 after transduction. (H) Representative flow cytometry plot showing YFP+ leukemia cells in PB at 4 weeks after transplantation. (I) Percentages of YFP+ leukemia cells in PB at the indicated time after transplantation (n = 5 per group). (J) Kaplan-Meier survival curves of recipient mice transplanted with YFP+ leukemia cells after KD of Ybx1 (n = 5). A log-rank test was performed. (K) Experimental scheme for MLL-AF9–induced AML model. (L) Kaplan-Meier survival curves for recipients of MLL-AF9–transduced Lin– BM cells from WT or Ybx1cKO donor mice (n = 10; from 2 independent experiments). A log-rank test was performed. (M-N) Colony formation assay of leukemia cells (M) and cell numbers (N) from WT and Ybx1cKO AML mice. (B-G) Shown is 1 representative of ≥3 independent experiments. Error bars denote mean ± SD. Two-tailed Student t test: *P < .05; **P < .01; ***P < .001. BMT, BM transplantation.

Ybx1 is required to maintain murine myeloid leukemia cell survival in vitro and in vivo. (A) Experimental scheme for assays in panels C to J. Lin– BM cells were transduced with MLL-AF9-yellow fluorescent protein (MLL-AF9-YFP) retrovirus and transplanted into sublethally irradiated recipient mice. YFP+ leukemia cells from AML mice were sorted and used for experiments as indicated. (B) Immunoblot showing Ybx1 KD efficiency in MLL-AF9-YFP leukemia cells after transduction with shRNA lentiviruses targeting Ybx1. (C) Growth curves of murine leukemia cells after transduction with lentiviruses for shYbx1#1, shYbx1#2, or shControl. (D-E) Colony formation assay (D; Control, shControl; P1 and P2, plating 1 and serial replating 2, respectively; Ybx1-KD#1 and Ybx1-KD#2, shYbx1#1 and shYbx1#2, respectively) and representative colony images (E; original magnification, ×100) of murine leukemia cells after transduction with the indicated lentiviruses; cells were cultured in MethoCult M3434 for 7 days. (F) Quantitative summary of flow cytometry analysis of cell cycles after leukemia cells were transduced with the indicated lentiviruses. Cells were labeled with Hoechst 33342 and were assessed by flow cytometry. (G) Percentage of apoptotic murine leukemia cells at day 4 after transduction. (H) Representative flow cytometry plot showing YFP+ leukemia cells in PB at 4 weeks after transplantation. (I) Percentages of YFP+ leukemia cells in PB at the indicated time after transplantation (n = 5 per group). (J) Kaplan-Meier survival curves of recipient mice transplanted with YFP+ leukemia cells after KD of Ybx1 (n = 5). A log-rank test was performed. (K) Experimental scheme for MLL-AF9–induced AML model. (L) Kaplan-Meier survival curves for recipients of MLL-AF9–transduced Lin– BM cells from WT or Ybx1cKO donor mice (n = 10; from 2 independent experiments). A log-rank test was performed. (M-N) Colony formation assay of leukemia cells (M) and cell numbers (N) from WT and Ybx1cKO AML mice. (B-G) Shown is 1 representative of ≥3 independent experiments. Error bars denote mean ± SD. Two-tailed Student t test: *P < .05; **P < .01; ***P < .001. BMT, BM transplantation.

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