Figure 4
Figure 4. THPO/MPL signaling is essential for AE cells. (A) AE cells incubated with indicated cytokines were counted biweekly. One representative experiment was shown. The experiment was repeated 3 times using 2 AE clones with consistent results. (B) AE cells were transduced with NT shRNA, MPL shRNA194, or MPL shRNA195. The percentage of transduced AE cells were determined by flow cytometry at indicated time points, followed by normalization as described in Figure 1D. Data represent mean ± SD (n = 3; *P < .05) compared with day 3. (C) Scatter plots showing percentage change in Venus(+) cells at week 6 compared with initial transduction efficiency at day3. Total BM cells were obtained by flushing the injected bone with PBS using a 28-G needle, followed by red blood cell lysis and labeling of the remaining cells with antibody against human CD45 surface antigen for flow cytometry analysis. Each dot represents one mouse. Red bars indicate mean. (D) Cells were seeded in methylcellulose media containing KG36E (THPO−) or KG36ET (THPO+) at 5 × 104 per dish in triplicate. Colony numbers were scored after 14 days under a light microscope. One representative experiment is shown. Results were confirmed with 3 additional AE clones. Data represents mean ± SD (n = 3). (E) AE cells incubated with KF36 ± THPO for 3 days were stained with annexin V (left), or permeabilized and stained for active caspase 3 (right), followed by flow cytometry analysis. (F) AE cells transduced with indicated shRNAs were permeabilized and stained for active caspase 3, followed by flow cytometry analysis. (G) AE cells treated with respective anti-MPL antibodies with protocols described for Figure 3C were stained with annexin V and 7-AAD for flow cytometry analysis. (H) CD34+ UCB cells with or without AE transduction were cultured in media in KF36 ± THPO. Cell counting was performed biweekly using a hemocytometer. Data shown represents 1 UCB and 1 AE clone. The curves representing UCB THPO+ and THPO− groups overlap with each other. Consistent results were obtained from 3 additional UCB clones and 2 additional AE clones. At day 28, an aliquot of AE cells were labeled with anti-CD34 antibodies for flow cytometry analysis or were cytocentrifuged for Wright-Giemsa staining.

THPO/MPL signaling is essential for AE cells. (A) AE cells incubated with indicated cytokines were counted biweekly. One representative experiment was shown. The experiment was repeated 3 times using 2 AE clones with consistent results. (B) AE cells were transduced with NT shRNA, MPL shRNA194, or MPL shRNA195. The percentage of transduced AE cells were determined by flow cytometry at indicated time points, followed by normalization as described in Figure 1D. Data represent mean ± SD (n = 3; *P < .05) compared with day 3. (C) Scatter plots showing percentage change in Venus(+) cells at week 6 compared with initial transduction efficiency at day3. Total BM cells were obtained by flushing the injected bone with PBS using a 28-G needle, followed by red blood cell lysis and labeling of the remaining cells with antibody against human CD45 surface antigen for flow cytometry analysis. Each dot represents one mouse. Red bars indicate mean. (D) Cells were seeded in methylcellulose media containing KG36E (THPO−) or KG36ET (THPO+) at 5 × 104 per dish in triplicate. Colony numbers were scored after 14 days under a light microscope. One representative experiment is shown. Results were confirmed with 3 additional AE clones. Data represents mean ± SD (n = 3). (E) AE cells incubated with KF36 ± THPO for 3 days were stained with annexin V (left), or permeabilized and stained for active caspase 3 (right), followed by flow cytometry analysis. (F) AE cells transduced with indicated shRNAs were permeabilized and stained for active caspase 3, followed by flow cytometry analysis. (G) AE cells treated with respective anti-MPL antibodies with protocols described for Figure 3C were stained with annexin V and 7-AAD for flow cytometry analysis. (H) CD34+ UCB cells with or without AE transduction were cultured in media in KF36 ± THPO. Cell counting was performed biweekly using a hemocytometer. Data shown represents 1 UCB and 1 AE clone. The curves representing UCB THPO+ and THPO− groups overlap with each other. Consistent results were obtained from 3 additional UCB clones and 2 additional AE clones. At day 28, an aliquot of AE cells were labeled with anti-CD34 antibodies for flow cytometry analysis or were cytocentrifuged for Wright-Giemsa staining.

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