Figure 5.
Exogenous CALR-del52 is inducing cell growth and JAK/STAT signaling in BaF3 cells expressing TpoR and mutant CALR. (A) Short-term proliferation of parental BaF3 and stable BaF3 cells expressing the indicated constructs were analyzed after daily exposition of the indicated doses of rhCALR-del52 over 72 hours with CTG assay (Promega). Values shown represent the average of 5 experiments with at least 29 biological replicates ± standard error of the mean (SEM). (B) Short-term proliferation of BaF3 TpoR CALRmut cells was analyzed after daily exposition of 1 μg/mL of rhCALR-del52, 0.1 ng/mL of Tpo, or 10 ng/mL of Tpo over 72 hours with CTG assay (Promega). Average ± SD of 6 replicates. (A-B). Statistical analysis (Jmp pro14) was performed by the nonparametric multiple comparisons Steel test with a control group (vehicle). ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01. (C) BaF3 TpoR Calrmut cells were transiently transfected with the Spi-Luc luciferase STAT5 reporter and the internal control pRL-TK used for normalization. The cells were cultured with different concentrations of recombinant human CALR-del52 or CALR WT over 24 hours before performing a Dual-Luciferase assay (Promega) for STAT5 transcriptional activity. (D) BaF3 TpoR Calrmut cells transfected with Spi-Luc and pRL-TK were treated for 24 hours with vehicle or 1 μg/mL of rhCALR-del52 and indicated molar rations of rhTFRC and STAT5 transcriptional activity was measured by Dual-Luciferase assay. (C-D) Luciferase activity was normalized to vehicle condition (⌀). Values shown represent the average of 6 to 12 biological replicates ± SEM. Statistical analysis (Jmp pro14) was performed by the nonparametric multiple comparisons Steel test with a control group (⌀). ∗∗∗P < .001, ∗∗P < .01, ∗P < .05. Statistical analysis of 2 specific conditions (see dashed lines) were performed by the unpaired t test. (E) Western blots showing time-dependent phosphorylation of TpoR, STAT5, and extracellular signal-regulated kinase 1/2 (ERK1/2) in BaF3 TpoR Calrmut cells treated or not treated with 0.1 μg/mL rhCALR-del52 for various lengths of time (5, 10, 15, 30, 45, and 60 minutes). P-Y626-TpoR denotes phosphorylation of tyrosine residue 112 of the intracellular chain of TpoR. HA denotes detection of total HA-tagged TpoR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin (tag).

Exogenous CALR-del52 is inducing cell growth and JAK/STAT signaling in BaF3 cells expressing TpoR and mutant CALR. (A) Short-term proliferation of parental BaF3 and stable BaF3 cells expressing the indicated constructs were analyzed after daily exposition of the indicated doses of rhCALR-del52 over 72 hours with CTG assay (Promega). Values shown represent the average of 5 experiments with at least 29 biological replicates ± standard error of the mean (SEM). (B) Short-term proliferation of BaF3 TpoR CALRmut cells was analyzed after daily exposition of 1 μg/mL of rhCALR-del52, 0.1 ng/mL of Tpo, or 10 ng/mL of Tpo over 72 hours with CTG assay (Promega). Average ± SD of 6 replicates. (A-B). Statistical analysis (Jmp pro14) was performed by the nonparametric multiple comparisons Steel test with a control group (vehicle). ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01. (C) BaF3 TpoR Calrmut cells were transiently transfected with the Spi-Luc luciferase STAT5 reporter and the internal control pRL-TK used for normalization. The cells were cultured with different concentrations of recombinant human CALR-del52 or CALR WT over 24 hours before performing a Dual-Luciferase assay (Promega) for STAT5 transcriptional activity. (D) BaF3 TpoR Calrmut cells transfected with Spi-Luc and pRL-TK were treated for 24 hours with vehicle or 1 μg/mL of rhCALR-del52 and indicated molar rations of rhTFRC and STAT5 transcriptional activity was measured by Dual-Luciferase assay. (C-D) Luciferase activity was normalized to vehicle condition (⌀). Values shown represent the average of 6 to 12 biological replicates ± SEM. Statistical analysis (Jmp pro14) was performed by the nonparametric multiple comparisons Steel test with a control group (⌀). ∗∗∗P < .001, ∗∗P < .01, ∗P < .05. Statistical analysis of 2 specific conditions (see dashed lines) were performed by the unpaired t test. (E) Western blots showing time-dependent phosphorylation of TpoR, STAT5, and extracellular signal-regulated kinase 1/2 (ERK1/2) in BaF3 TpoR Calrmut cells treated or not treated with 0.1 μg/mL rhCALR-del52 for various lengths of time (5, 10, 15, 30, 45, and 60 minutes). P-Y626-TpoR denotes phosphorylation of tyrosine residue 112 of the intracellular chain of TpoR. HA denotes detection of total HA-tagged TpoR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin (tag).

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