Figure 6
Figure 6. R17C mutant THPO protein is deficient in maintaining UT7-TPO cells. (A) Conditioned media were collected from human embryonic kidney (293T) cells transfected with murine WT-Thpo, mutagenized R17C-Thpo, and control (no Thpo) constructs. The transfected cells were analyzed by RT-PCR. In the top, both WT and R17C mutant mThpo transfections show equivalent expression and none with the control construct (no Thpo). There is no significant endogenous expression of hTHPO in mutant transfected human embryonic kidney cells (middle). The faint hTHPO background band visible in the WT-Thpo transfected sample is due to increased overall transcript levels for WT-Thpo compared with R17C-Thpo and no-Thpo, as seen with control hOAZ1 transcript levels (bottom). (B) 1 × 104 UT7-TPO cells, starved for commercial THPO for 16 hours, were treated with indicated volumes of WT, R17C mutant, or control conditioned media in a 96-well plate. Following 3 days of growth, proliferation was measured using MTT assays. WT THPO-containing conditioned media is able to maintain significantly increased UT7-TPO cell proliferation than the R17C mutant conditioned media at volumes of 1 μL (P < .03), 2.5 μL (P < .0002), and 5 μL (P < .02). Conditioned media with no exogenous THPO were not able to maintain cell proliferation. All treatments were done in triplicate. Results indicate mean ± standard error of the mean from 4 independent experiments. (C) 1 × 104 UT7-TPO cells were treated with indicated amounts of WT and R17C mutant conditioned media as above. MTT assay results following 3 days of growth are shown compared with baseline proliferation in the absence of THPO (lane 1). WT THPO shows significantly greater proliferation of UT7-TPO cells than mutant R17C THPO (lane 2 vs 4; *P < .01). Mutant R17C THPO is not able to reduce proliferation by WT THPO (lane 2 vs 6) even when twice as much mutant THPO is added (lane 2 vs 7). n/s, not significant. All treatments were done in triplicate. Results indicate mean ± standard error of the mean from 6 independent experiments.

R17C mutant THPO protein is deficient in maintaining UT7-TPO cells. (A) Conditioned media were collected from human embryonic kidney (293T) cells transfected with murine WT-Thpo, mutagenized R17C-Thpo, and control (no Thpo) constructs. The transfected cells were analyzed by RT-PCR. In the top, both WT and R17C mutant mThpo transfections show equivalent expression and none with the control construct (no Thpo). There is no significant endogenous expression of hTHPO in mutant transfected human embryonic kidney cells (middle). The faint hTHPO background band visible in the WT-Thpo transfected sample is due to increased overall transcript levels for WT-Thpo compared with R17C-Thpo and no-Thpo, as seen with control hOAZ1 transcript levels (bottom). (B) 1 × 104 UT7-TPO cells, starved for commercial THPO for 16 hours, were treated with indicated volumes of WT, R17C mutant, or control conditioned media in a 96-well plate. Following 3 days of growth, proliferation was measured using MTT assays. WT THPO-containing conditioned media is able to maintain significantly increased UT7-TPO cell proliferation than the R17C mutant conditioned media at volumes of 1 μL (P < .03), 2.5 μL (P < .0002), and 5 μL (P < .02). Conditioned media with no exogenous THPO were not able to maintain cell proliferation. All treatments were done in triplicate. Results indicate mean ± standard error of the mean from 4 independent experiments. (C) 1 × 104 UT7-TPO cells were treated with indicated amounts of WT and R17C mutant conditioned media as above. MTT assay results following 3 days of growth are shown compared with baseline proliferation in the absence of THPO (lane 1). WT THPO shows significantly greater proliferation of UT7-TPO cells than mutant R17C THPO (lane 2 vs 4; *P < .01). Mutant R17C THPO is not able to reduce proliferation by WT THPO (lane 2 vs 6) even when twice as much mutant THPO is added (lane 2 vs 7). n/s, not significant. All treatments were done in triplicate. Results indicate mean ± standard error of the mean from 6 independent experiments.

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