American Society of Hematology

Figure 2

$Figure 2. Decreased proliferation of primary human hematopoietic progenitor cells, increased apoptosis, and reduction of cells in the S phase following knockdown of HSPA9. (A) Transduced cells were seeded at equal concentrations after 4 days in erythroid culture conditions, and the total number of cells was counted for 3 consecutive days in the presence of puromycin selection medium. The fold change in cell counts was calculated relative to the number of cells on day 4 (n = 5 for each shRNA; *P < .001). (B) Representative flow cytometric plots for annexin V and 7-AAD in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each quadrant. (C) Quantification of annexin V+/7-AAD+ cells (a surrogate for apoptosis; n = 5 for each shRNA; *P ≤ .03; **P < .0001). (D) Representative flow cytometric plots for BrdU and 7-AAD in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each gate. Sub-G0 cells are in the low left gate. Cells in G0/G1 of the cell cycle are BrdU−/7-AAD−, S-phase cells are BrdU+, and G2/M cells are BrdU−/7-AAD+. (E) Quantification of the cell-cycle phases after exclusion of cells in the sub-G0 fraction (n = 5 for each shRNA; *P < .02; **P < .006). Empty mean no shRNA sequence was inserted into the pLKO.1 vector. LUC indicates luciferase; and NS, not significant. All data represent the means ± SD.$

Decreased proliferation of primary human hematopoietic progenitor cells, increased apoptosis, and reduction of cells in the S phase following knockdown of HSPA9. (A) Transduced cells were seeded at equal concentrations after 4 days in erythroid culture conditions, and the total number of cells was counted for 3 consecutive days in the presence of puromycin selection medium. The fold change in cell counts was calculated relative to the number of cells on day 4 (n = 5 for each shRNA; *P < .001). (B) Representative flow cytometric plots for annexin V and 7-AAD in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each quadrant. (C) Quantification of annexin V⁺/7-AAD⁺ cells (a surrogate for apoptosis; n = 5 for each shRNA; *P ≤ .03; **P < .0001). (D) Representative flow cytometric plots for BrdU and 7-AAD in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each gate. Sub-G0 cells are in the low left gate. Cells in G0/G1 of the cell cycle are BrdU⁻/7-AAD⁻, S-phase cells are BrdU⁺, and G2/M cells are BrdU⁻/7-AAD⁺. (E) Quantification of the cell-cycle phases after exclusion of cells in the sub-G0 fraction (n = 5 for each shRNA; *P < .02; **P < .006). Empty mean no shRNA sequence was inserted into the pLKO.1 vector. LUC indicates luciferase; and NS, not significant. All data represent the means ± SD.

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