Figure 2.
Active conformations of hTpoR differ in presence of JAK2 WT and JAK2 V617F. (A-B) STAT5 transcriptional activity measured by luciferase assay in HEK293T transiently transfected with hJAK2 WT (A) or hJAK2 V617F (B) and the indicated constructs. Values represent the mean (±SD) of 4 independent experiments performed in triplicate. The active interfaces are shown in green, inactive interfaces in red, and partially active interfaces in orange. Controls (empty vector and hTpoR WT) are shown in blue. (C) Helical wheel diagram of cc-hTpoR constructs with amino acids at the interface for each of the 7 cc-hTpoR. The gradient of active orientations for JAK2 V617F (brown) and JAK2 WT (blue) is illustrated. The inactive interface is shown in red. (D) Relative messenger RNA (mRNA) of hJAK2 WT, hJAK2 V617F, and endogenous mJAK2 WT of Ba/F3 cells. Values represent the mean (±SD) mRNA levels quantified by qPCR from 3 independent experiments performed in triplicate (N = 3, n = 9). (E) Left: STAT5 phosphorylation in Ba/F3 cells expressing either hJAK2 WT or hJAK2 V617F together with indicated constructs. Values represent the mean fluorescence intensity (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT5 phosphorylation in Ba/F3 cells expressing hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. (F) Proliferation assay of Ba/F3 cells stably transduced with hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. Values represent the mean (±SD) of 3 independent experiments performed in triplicate (N = 3, n = 9). (G) Relative mRNA of hJAK2 WT, hJAK2 V617F, and endogenous hJAK2 WT of TF-1 cells. Values represent the mean (±SD) mRNA levels quantified by qPCR from 3 independent experiments performed in triplicate (N = 3, n = 9). (H) Left: STAT5 phosphorylation in TF-1 cells expressing either hJAK2 WT or hJAK2 V617F together with the indicated constructs. Values represent mean fluorescence intensities (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT5 phosphorylation in TF-1 cells expressing hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. (I) Luminescent viability assay (CellTiter-Glo) was used to measure the proliferation of TF-1 cells stably transduced with hJAK2 WT (blue) or hJAK2 V617F (red) with the indicated constructs. Values represent the mean (±SD) of 8 biological replicates (n = 8). (A-B, D-I) Statistics: two-way analysis of variance followed by the SIDAK multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Active conformations of hTpoR differ in presence of JAK2 WT and JAK2 V617F. (A-B) STAT5 transcriptional activity measured by luciferase assay in HEK293T transiently transfected with hJAK2 WT (A) or hJAK2 V617F (B) and the indicated constructs. Values represent the mean (±SD) of 4 independent experiments performed in triplicate. The active interfaces are shown in green, inactive interfaces in red, and partially active interfaces in orange. Controls (empty vector and hTpoR WT) are shown in blue. (C) Helical wheel diagram of cc-hTpoR constructs with amino acids at the interface for each of the 7 cc-hTpoR. The gradient of active orientations for JAK2 V617F (brown) and JAK2 WT (blue) is illustrated. The inactive interface is shown in red. (D) Relative messenger RNA (mRNA) of hJAK2 WT, hJAK2 V617F, and endogenous mJAK2 WT of Ba/F3 cells. Values represent the mean (±SD) mRNA levels quantified by qPCR from 3 independent experiments performed in triplicate (N = 3, n = 9). (E) Left: STAT5 phosphorylation in Ba/F3 cells expressing either hJAK2 WT or hJAK2 V617F together with indicated constructs. Values represent the mean fluorescence intensity (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT5 phosphorylation in Ba/F3 cells expressing hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. (F) Proliferation assay of Ba/F3 cells stably transduced with hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. Values represent the mean (±SD) of 3 independent experiments performed in triplicate (N = 3, n = 9). (G) Relative mRNA of hJAK2 WT, hJAK2 V617F, and endogenous hJAK2 WT of TF-1 cells. Values represent the mean (±SD) mRNA levels quantified by qPCR from 3 independent experiments performed in triplicate (N = 3, n = 9). (H) Left: STAT5 phosphorylation in TF-1 cells expressing either hJAK2 WT or hJAK2 V617F together with the indicated constructs. Values represent mean fluorescence intensities (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT5 phosphorylation in TF-1 cells expressing hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. (I) Luminescent viability assay (CellTiter-Glo) was used to measure the proliferation of TF-1 cells stably transduced with hJAK2 WT (blue) or hJAK2 V617F (red) with the indicated constructs. Values represent the mean (±SD) of 8 biological replicates (n = 8). (A-B, D-I) Statistics: two-way analysis of variance followed by the SIDAK multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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