Figure 5
Gene silencing of LMO2 in t(17;19)-ALL cell line by a lentiviral vector. (A) Schematic representation of a short hairpin RNA (shRNA)–expressing lentiviral vector. pLL3.7 lentiviral vector was engineered to co-express green fluorescent protein (GFP) as a reporter gene by cytomegalovirus-derived promoter–GFP expression cassette. pLL3.7 lentiviral vector and packaging vector were cotransfected into 293FT cells and the resulting supernatant was collected after 36 hours. Lentivirus was recovered after ultracentrifugation and infected to UOC-B1 cells. (B) LMO2 expression of GFP-positive population sorted from lentivirus-infected cells. On day 2 or 4 after infection, the GFP-positive population was sorted and processed for real-time RT-PCR analysis using the primer for exons 4 and 5 of LMO2 gene. The gray boxes indicate pLL control vector-infected cells and the purple boxes indicate shRNA-expressing cells. (C) Changes in GFP-positive populations in UOC-B1 and 697 cells on days 2 and 4 after infection. The percentage of the GFP positive population is indicated in each box. (D) Changes in the percentage of GFP-positive populations in UOC-B1 cells infected with shRNA-containing and control lentivirus on day 3 and 5 after infection. The P value in t test is indicated. (E) Flow cytometric analysis of BrdU/7-AAD double staining in the GFP-positive population of shRNA-expressing and control UOC-B1 cells 3 days after infection. Representative data of the percentage of apoptotic cells in the sub G0/G1 phase among the GFP-positive population and the percentage of living cells in the G0/G1, S, and G2/M phases are indicated. (F) Comparison of cell-cycle distribution between control virus-infected cells and shRNA virus-infected cells. The P value in t test is indicated. (G) Flow cytometric analysis of cleaved-caspase3 in the GFP-positive population of shRNA-expressing and control UOC-B1 cells 3 days after infection. Representative data of the percentage of cleaved-caspase3-positive cells among the GFP-positive population are indicated in the left panel. Percentages of cleaved-caspase3-positive cells are compared between control virus-infected cells and shRNA virus-infected cells. The P value in t test is indicated.

Gene silencing of LMO2 in t(17;19)-ALL cell line by a lentiviral vector. (A) Schematic representation of a short hairpin RNA (shRNA)–expressing lentiviral vector. pLL3.7 lentiviral vector was engineered to co-express green fluorescent protein (GFP) as a reporter gene by cytomegalovirus-derived promoter–GFP expression cassette. pLL3.7 lentiviral vector and packaging vector were cotransfected into 293FT cells and the resulting supernatant was collected after 36 hours. Lentivirus was recovered after ultracentrifugation and infected to UOC-B1 cells. (B) LMO2 expression of GFP-positive population sorted from lentivirus-infected cells. On day 2 or 4 after infection, the GFP-positive population was sorted and processed for real-time RT-PCR analysis using the primer for exons 4 and 5 of LMO2 gene. The gray boxes indicate pLL control vector-infected cells and the purple boxes indicate shRNA-expressing cells. (C) Changes in GFP-positive populations in UOC-B1 and 697 cells on days 2 and 4 after infection. The percentage of the GFP positive population is indicated in each box. (D) Changes in the percentage of GFP-positive populations in UOC-B1 cells infected with shRNA-containing and control lentivirus on day 3 and 5 after infection. The P value in t test is indicated. (E) Flow cytometric analysis of BrdU/7-AAD double staining in the GFP-positive population of shRNA-expressing and control UOC-B1 cells 3 days after infection. Representative data of the percentage of apoptotic cells in the sub G0/G1 phase among the GFP-positive population and the percentage of living cells in the G0/G1, S, and G2/M phases are indicated. (F) Comparison of cell-cycle distribution between control virus-infected cells and shRNA virus-infected cells. The P value in t test is indicated. (G) Flow cytometric analysis of cleaved-caspase3 in the GFP-positive population of shRNA-expressing and control UOC-B1 cells 3 days after infection. Representative data of the percentage of cleaved-caspase3-positive cells among the GFP-positive population are indicated in the left panel. Percentages of cleaved-caspase3-positive cells are compared between control virus-infected cells and shRNA virus-infected cells. The P value in t test is indicated.

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