American Society of Hematology

Figure 4

$Figure 4. lincRNA-3q overexpression in BPDCN and AML drives G1/S and leukemia-driver gene expression programs. (A) RT-qPCR-derived lincRNA-3q expression profiles in normal bone marrow; normal pDC; BPDCN (CAL-1, GEN2.2), AML (U937, MUTZ-3), and chronic myeloid leukemia cell lines (K562); and in BPDCN and AML patient samples. AML patients are classified according to cytogenetic risk group. Cases presenting chromosome 3q abnormalities are identified in blue. (B) RT-qPCR and RT-PCR analysis of the subcellular localization of lincRNA-3q in CAL-1 BPDCN cells and U937 AML cells. RNA extracted from whole-cell and subcellular fractions (n = 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. (C) RT-qPCR-derived lincRNA-3q expression in CAL-1 cells transduced with control (shCtrl) or lincRNA-3q-targeting short hairpin constructs (sh1 and 2) (left). Cell cycle analysis in CAL-1 cells transduced with control (shCtrl) or lincRNA-3q sh1 and 2 constructs (upper right). *P < .05 by Wilcoxon test; n = 6. Representative histogram representation of percentage of cells in cell cycle phases (n = 4) (lower right). (D) GSEA plots obtained by comparing gene expression profiles of CAL-1 cells transduced with control (shCtrl) or lincRNA-3q-targeting short hairpin (shlincRNA-3q) (right) and associated heatmap (n = 4 for each group) (left). Genes mentioned in the text are marked with an arrow. (E) E2F cyclin E–luciferase (luc) reporter assay in H1299 cells transiently transfected with control or siRNA targeting lincRNA-3q with or without addition of exogenous E2F1 (100 ng) (upper). *P < .05, **P < .01 by Wilcoxon test; n = 6. Western blot using anti-E2F1 antibody in H1299 cells transduced with siCtrl or silincRNA-3q, as indicated (n = 2) (lower). (F) Table presenting clone frequency and associated statistics derived from in vitro limiting dilution clonogenicity for CAL-1 either nontransduced (NT) or transduced with control (shCtrl) or lincRNA-3q-targeting (shlincRNA-3q) shRNA. Adv, adverse; cyt, cytosolic fraction; Fav, favorable; Interm, intermediate; nuc, nuclear fraction; tot, whole cell extract.$

lincRNA-3q overexpression in BPDCN and AML drives G1/S and leukemia-driver gene expression programs. (A) RT-qPCR-derived lincRNA-3q expression profiles in normal bone marrow; normal pDC; BPDCN (CAL-1, GEN2.2), AML (U937, MUTZ-3), and chronic myeloid leukemia cell lines (K562); and in BPDCN and AML patient samples. AML patients are classified according to cytogenetic risk group. Cases presenting chromosome 3q abnormalities are identified in blue. (B) RT-qPCR and RT-PCR analysis of the subcellular localization of lincRNA-3q in CAL-1 BPDCN cells and U937 AML cells. RNA extracted from whole-cell and subcellular fractions (n = 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. (C) RT-qPCR-derived lincRNA-3q expression in CAL-1 cells transduced with control (shCtrl) or lincRNA-3q-targeting short hairpin constructs (sh1 and 2) (left). Cell cycle analysis in CAL-1 cells transduced with control (shCtrl) or lincRNA-3q sh1 and 2 constructs (upper right). *P < .05 by Wilcoxon test; n = 6. Representative histogram representation of percentage of cells in cell cycle phases (n = 4) (lower right). (D) GSEA plots obtained by comparing gene expression profiles of CAL-1 cells transduced with control (shCtrl) or lincRNA-3q-targeting short hairpin (shlincRNA-3q) (right) and associated heatmap (n = 4 for each group) (left). Genes mentioned in the text are marked with an arrow. (E) E2F cyclin E–luciferase (luc) reporter assay in H1299 cells transiently transfected with control or siRNA targeting lincRNA-3q with or without addition of exogenous E2F1 (100 ng) (upper). *P < .05, **P < .01 by Wilcoxon test; n = 6. Western blot using anti-E2F1 antibody in H1299 cells transduced with siCtrl or silincRNA-3q, as indicated (n = 2) (lower). (F) Table presenting clone frequency and associated statistics derived from in vitro limiting dilution clonogenicity for CAL-1 either nontransduced (NT) or transduced with control (shCtrl) or lincRNA-3q-targeting (shlincRNA-3q) shRNA. Adv, adverse; cyt, cytosolic fraction; Fav, favorable; Interm, intermediate; nuc, nuclear fraction; tot, whole cell extract.

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