LSD1 is barely expressed in normal HSPCs, but is overexpressed in leukemic cells. (A) Top panel, We isolated whole-cell lysates from normal human bone marrow cells (CD34+ HSPCs, CD34−/Lin− progenitors, and Lin+ cells), mature blood cells (granulocytes, monocytes, uncultured T lymphocytes, and 24-hour–stimulated T lymphocytes), T-LBL cell lines (TALL-1, MOLT-4, CEM, KOPT-K1, and Jurkat), myeloid leukemia cell lines (K562, HL-60, KG-1, THP-1, MV4-11, and PL-21), normal human fibroblasts, and the cervical carcinoma cell line HeLa for immunoblot analyses on the expression of LSD1 and GAPDH (internal control). Bottom panel, We isolated total cellular RNA from the indicated cells and subjected them to semiquantitative RT-PCR analysis for the expression of LSD1 and GAPDH. The PCR products of suboptimal amplification cycles, 20 to 35 cycles, were electrophoresed (5 μL per lane). (B) LSD1 mRNA expression in primary samples according to the Oncomine database (http://www.oncomine.org). Panel Bi: 0, normal bone marrow mononuclear cells (n = 6); 1, AML (n = 23); 2, B-cell acute lymphoblastic leukemia (n = 87); 3, T-LBL (n = 11). Panel Bii: 0, various normal somatic cells (n = 146); 1, T-LBL (n = 2); 2, AML (n = 2); 3, Burkitt lymphoma (n = 4); 4, colorectal cancer (n = 2); 5, chronic myelogenous leukemia (n = 2). *P < .05 determined by a 1-way ANOVA with the Bonferroni post hoc test. (C) The structure of 4 LSD1 isoforms generated by either the single or double inclusion of 2 alternative exons 2a (Ex2a) and 8a (Ex8a). *The FAD-binding domain. We designed PCR primers 1/2F and 3R (arrows) to detect LSD1 isoforms (−/−) and (+/−) discriminately based on differences in sizes (60 bp as shown in the box). (D) We performed semiquantitative RT-PCR for the expression of LSD1 isoforms in normal human hematopoietic progenitors, leukemic cell lines, and primary T-LBL cells from 3 patients (cases 2, 5, and 8). The cDNAs were amplified with primers covering the entire exons (1F and 19R; see supplemental Table 1 for nucleotide sequences), followed by nested PCR with primers 1/2F and 3R as reported by Zibetti et al.15 (E) The signal intensities of each band at 35 cycles in panel D were quantified by NIH Image J software, normalized to those of corresponding GAPDH, and shown as relative values setting the expression level of LSD1 (−/−) in CD34+/CD38− cells to 1.0. (F) We quantified the mRNA expression of total LSD1, mostly (−/−) and (+/−), with 1/2F and 3R primers and the isoform (+/−) with 1/2F and 2aR (within exon 2a) primers using the Power SYBR Green PCR Master Mix (Life Technologies). The expression level was normalized to that of GAPDH and quantified by the 2-ΔΔCt method. The means ± SD (bars) of 3 independent experiments are shown. (G) We isolated total cellular RNA from murine Lin–/Sca-1+/c-Kit+ and Lin−/Sca-1−/c-Kit+ cells, and subjected them to nested RT-PCR for the expression of murine LSD1 isoforms with primer pairs of 2F and 3R (top panel) or 2F and 8/9R (middle panel) (see supplemental Table 1 for nucleotide sequences). ANOVA, analysis of variance; CT, cycle threshold; SD, standard deviation.
