Figure 1.
Elevated expression of YBX1 is required for survival of human myeloid leukemia cells. (A) Expression profiling of 1068 RNA-binding protein-encoding genes in patients with AML using the TCGA database. (B) YBX1 mRNA had the highest level of expression in AML compared with other cancers among 1429 human cancer cell lines in the Cancer Cell Line Encyclopedia database (https://portals.broadinstitute.org/ccle). Horizontal bars represent the tumor types where 1429 cells lines belong. (C) qRT-PCR analysis was used to determine the expression of YBX1 in normal BM cells from healthy donors (n = 5) and patient-derived primary AML cells of various subtypes, including inv(16) (n = 8), normal karyotype (n = 23), t(11q23) (n = 13), and t(8;21) (n = 28). Horizontal bars represent the cells used for qPCR are derived from bone marrow cells from healthy samples and patient samples with different karyotypes. (D) Immunoblot showing YBX1 expression in bulk BM mononuclear cells from healthy donors (n = 2) and primary patients with AML (n = 5). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (E) Western blot analysis of YBX1 expression in various patient-derived myeloid leukemia cell lines; CD34+ cells derived from cord blood were used as a normal control and GAPDH served as the loading control. (F) Immunoblot showing YBX1 KD efficiency in leukemia cells, MOLM-13, and THP-1 after transduction with shRNA lentiviruses targeting YBX1. (G) Colony formation assay showing the clonogenic defect of primary Lin–CD34+ cells from 3 individual patients with AML after YBX1 KD. Horizontal bars (AML#171, AML#172, and AML#173) are names of 3 patient samples. (H) In vivo leukemic engraftment analysis at 8 weeks after xenotransplantation. Data from 3 independent experiments for 3 patients with AML were combined (right, 3 recipients per patient sample per group). Horizontal bars (AML#XH04, AML#ZN01, and AML#124) are names of 3 patient samples. (I) Growth curves of MOLM-13 and THP-1 leukemia cells after transduction with lentiviruses for shYBX1#1, shYBX1#2, or shControl. (J) Colony formation assay of MOLM-13 and THP-1 leukemia cells after transduction with indicated lentiviruses. Horizontal bars (YBX1-KD#1 and YBX1-KD#2) represent shYBX1#1 and shYBX1#2, respectively. (K) Growth curves showing the rescued growth of leukemia cells by ectopic expression of YBX1. Leukemia cells were transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression. (L) Colony formation assay showing the rescued clonogenic capability of leukemia cells by ectopic expression of YBX1. Horizontal bars represent leukemia cells transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression or shControl. (M) Cell cycle distribution of leukemia cells after transduction with the indicated lentiviruses. Cells were labeled with Hoechst 33342 and assessed by flow cytometry. Horizontal bars represent different phases of the cell cycle after leukemia cells were transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ plus YBX1 complementary DNA for rescuing YBX1 expression or shControl. (N) Percentages of apoptotic leukemia cells at day 4 after transduction. Horizontal bars represent leukemia cells transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression or shControl. (O) Kaplan-Meier plot showing the survival time of 3 cohorts of recipient mice transplanted with MOLM-13 cells (n = 4 to 5 recipient mice per group). Horizontal bars represent days after leukemia cells were transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression or shControl and injected to recipient mice. A log-rank test was performed, and panels F to N show 1 representative sample of 3 independent experiments. Error bars denote mean ± standard deviation (SD). Two-tailed Student t test: *P < .05; **P < .01; ***P < .001. FPKM, fragments per kilobase million; GFP, green fluorescent protein.

Elevated expression of YBX1 is required for survival of human myeloid leukemia cells. (A) Expression profiling of 1068 RNA-binding protein-encoding genes in patients with AML using the TCGA database. (B) YBX1 mRNA had the highest level of expression in AML compared with other cancers among 1429 human cancer cell lines in the Cancer Cell Line Encyclopedia database (https://portals.broadinstitute.org/ccle). Horizontal bars represent the tumor types where 1429 cells lines belong. (C) qRT-PCR analysis was used to determine the expression of YBX1 in normal BM cells from healthy donors (n = 5) and patient-derived primary AML cells of various subtypes, including inv(16) (n = 8), normal karyotype (n = 23), t(11q23) (n = 13), and t(8;21) (n = 28). Horizontal bars represent the cells used for qPCR are derived from bone marrow cells from healthy samples and patient samples with different karyotypes. (D) Immunoblot showing YBX1 expression in bulk BM mononuclear cells from healthy donors (n = 2) and primary patients with AML (n = 5). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (E) Western blot analysis of YBX1 expression in various patient-derived myeloid leukemia cell lines; CD34+ cells derived from cord blood were used as a normal control and GAPDH served as the loading control. (F) Immunoblot showing YBX1 KD efficiency in leukemia cells, MOLM-13, and THP-1 after transduction with shRNA lentiviruses targeting YBX1. (G) Colony formation assay showing the clonogenic defect of primary Lin–CD34+ cells from 3 individual patients with AML after YBX1 KD. Horizontal bars (AML#171, AML#172, and AML#173) are names of 3 patient samples. (H) In vivo leukemic engraftment analysis at 8 weeks after xenotransplantation. Data from 3 independent experiments for 3 patients with AML were combined (right, 3 recipients per patient sample per group). Horizontal bars (AML#XH04, AML#ZN01, and AML#124) are names of 3 patient samples. (I) Growth curves of MOLM-13 and THP-1 leukemia cells after transduction with lentiviruses for shYBX1#1, shYBX1#2, or shControl. (J) Colony formation assay of MOLM-13 and THP-1 leukemia cells after transduction with indicated lentiviruses. Horizontal bars (YBX1-KD#1 and YBX1-KD#2) represent shYBX1#1 and shYBX1#2, respectively. (K) Growth curves showing the rescued growth of leukemia cells by ectopic expression of YBX1. Leukemia cells were transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression. (L) Colony formation assay showing the rescued clonogenic capability of leukemia cells by ectopic expression of YBX1. Horizontal bars represent leukemia cells transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression or shControl. (M) Cell cycle distribution of leukemia cells after transduction with the indicated lentiviruses. Cells were labeled with Hoechst 33342 and assessed by flow cytometry. Horizontal bars represent different phases of the cell cycle after leukemia cells were transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ plus YBX1 complementary DNA for rescuing YBX1 expression or shControl. (N) Percentages of apoptotic leukemia cells at day 4 after transduction. Horizontal bars represent leukemia cells transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression or shControl. (O) Kaplan-Meier plot showing the survival time of 3 cohorts of recipient mice transplanted with MOLM-13 cells (n = 4 to 5 recipient mice per group). Horizontal bars represent days after leukemia cells were transduced with shRNA lentivirus targeting the YBX1 3′ UTR (shYBX1-3′ UTR) or shYBX1 3′ UTR plus YBX1 complementary DNA for rescuing YBX1 expression or shControl and injected to recipient mice. A log-rank test was performed, and panels F to N show 1 representative sample of 3 independent experiments. Error bars denote mean ± standard deviation (SD). Two-tailed Student t test: *P < .05; **P < .01; ***P < .001. FPKM, fragments per kilobase million; GFP, green fluorescent protein.

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