Figure 4. Down-regulation of SALL4 by shRNA leads to decreased cell viability of primary human AML cells in culture and reduced leukemic development in vivo. (A) Down-regulation of SALL4 RNA in primary AML cells using shRNA. Control scr shRNA-infected cells and SALL4 shRNA-infected cells (shRNA1 and shRNA2) were analyzed 48 hours after transduction. The expression of SALL4 RNA in AML cells infected with SALL4 shRNA-expressing retroviruses was reduced to 35% of those infected with scr pRS control vectors, evaluated by quantitative RT-PCR after normalized to GAPDH (n = 3 biologic samples). Error bars indicate SD. (B) Increased apoptosis and cell death were observed via flow cytometry analysis of annexin V/PI staining in AML cells on SALL4 knock-down (shRNA1 and shRNA2). Data were derived from 3 independent experiments. Viable cells were defined as the double-negative (annexin V−/PI−) population. The viability was set as 100 for the control group. (C) Xenotransplantation showed increased survival of mice receiving SALL4-reduced leukemic cells. A total of 1.5 million primary human AML cells were transduced as described in the Methods and cultured for 48 hours before transplantation via tail vein injection. Although the median survival of recipient mice with control scr shRNA retrovirus-transduced primary human AML cells (n = 7) was 33 days, the median survival of recipient mice with SALL4 shRNA retrovirus-infected primary human AML cells (n = 6) was 109 days. The log-rank (Mantel-Cox) P = .03 and the Gehan-Breslow-Wilcoxon P = .01. (D-F) Leukemia development in xenotransplant recipient mice. AML is defined as a blast count more than 20% in the peripheral blood and/or BM with multiple organ involvements observed in recipient mice. Blasts were present in liver (D, 200×), spleen (E, 200×), and BM (F, 200×) as assessed by H&E staining. (G-H) AML cells in xenograft recipients were human CD45+. Flow cytometry was performed on BM from recipients after transduction with control scr (G) or SALL4 shRNA (H) retrovirus. The red line represents isotype control and the blue line represents anti–human CD45 antibody.
Figure 4

Down-regulation of SALL4 by shRNA leads to decreased cell viability of primary human AML cells in culture and reduced leukemic development in vivo. (A) Down-regulation of SALL4 RNA in primary AML cells using shRNA. Control scr shRNA-infected cells and SALL4 shRNA-infected cells (shRNA1 and shRNA2) were analyzed 48 hours after transduction. The expression of SALL4 RNA in AML cells infected with SALL4 shRNA-expressing retroviruses was reduced to 35% of those infected with scr pRS control vectors, evaluated by quantitative RT-PCR after normalized to GAPDH (n = 3 biologic samples). Error bars indicate SD. (B) Increased apoptosis and cell death were observed via flow cytometry analysis of annexin V/PI staining in AML cells on SALL4 knock-down (shRNA1 and shRNA2). Data were derived from 3 independent experiments. Viable cells were defined as the double-negative (annexin V/PI) population. The viability was set as 100 for the control group. (C) Xenotransplantation showed increased survival of mice receiving SALL4-reduced leukemic cells. A total of 1.5 million primary human AML cells were transduced as described in the Methods and cultured for 48 hours before transplantation via tail vein injection. Although the median survival of recipient mice with control scr shRNA retrovirus-transduced primary human AML cells (n = 7) was 33 days, the median survival of recipient mice with SALL4 shRNA retrovirus-infected primary human AML cells (n = 6) was 109 days. The log-rank (Mantel-Cox) P = .03 and the Gehan-Breslow-Wilcoxon P = .01. (D-F) Leukemia development in xenotransplant recipient mice. AML is defined as a blast count more than 20% in the peripheral blood and/or BM with multiple organ involvements observed in recipient mice. Blasts were present in liver (D, 200×), spleen (E, 200×), and BM (F, 200×) as assessed by H&E staining. (G-H) AML cells in xenograft recipients were human CD45+. Flow cytometry was performed on BM from recipients after transduction with control scr (G) or SALL4 shRNA (H) retrovirus. The red line represents isotype control and the blue line represents anti–human CD45 antibody.

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