Figure 5. TGF-β expression is regulated by a PI3K/ERK dependent pathway.

(A, B) Western blot showing phospho-JAK2 / total JAK2 (A) and phospho-STAT3 (Y705) /total STAT3 (B) with β-actin as loading control. Protein was isolated from 32D cells transfected with MPL and CALR^WT or CALR^del52 after overnight IL3 starvation, three individual harvest for each condition.

(C) Scatter dot plot showing fold change of nuclear phospho-STAT3 signal detected by immunohistochemistry in 32D-MPL-CALR^del52 cells compared to 32D-MPL-CALR^WT cells after overnight IL3 starvation. Pooled data from three independent experiments.

(D) Scatter plot showing luciferase activity in 32D-MPL-Calr^del52 cells, transfected with pGL3-TGFβ1 promotor vector, treated overnight with indicated concentrations of STAT3 inhibitor after IL-3 withdrawal. Pooled data from two independent experiments. P values were calculated using repeated-measures (RM) one-way ANOVA.

(E) Scatter plot showing fold change MFI of L-TGF-β1 on 32D-STAT3^WT or STAT3^V640F. Pooled data from two independent experiments. P values were calculated using Mann-Whitney test.

(F, G) Representative histogram (F) and scatter dot plot (G) showing L-TGF-β1 expression on 32D-MPL-CALR^del52 cells treated with indicated concentration of ruxolitinib (JAK1/2-inhibitor). Pooled data from three independent experiments.

(H) Scatter dot plot showing relative luciferase activity in 32D-MPL-Calr^del52, transfected with pGL3-TGFβ1 promotor vector and treated with indicated concentration of ruxolitinib overnight after IL-3 withdrawal. Pooled data from two independent experiments with two technical replicates for each condition. P values were calculated using ordinary one-way ANOVA.

(I, J) Relative expression of L-TGF-β1 (I) and cell viability (J) of 32D-MPL-CALR^del52 cells treated with either PI3K inhibitors (Buparlisib, Pictilisib), MEK inhibitors (Selumetinib, Trametinib), ERK inhibitor (Ulixertinib) or STAT3-inhibitor for 24h. Pooled data from three independent experiments.