Fig. 4

In AML cells, PRLR induces signaling through Jak2/Stat5. A CISH and pTATA reporters were transfected in HEK293T cells and treated with the vehicle (grey), PRL (blue) or G129R (red) at 500 ng/mL, in starving conditions. The luciferase activity (normalized against renilla signal) is shown in a representative replicate. **p < 0.01 (two-way ANOVA, Tukey’s multiple comparison test, triplicates). B PRLR-transduced MonoMac-1 (MM PRLR wt) cells were treated with the vehicle, PRL (blue) or G129R (red) at 250 and 500 ng/mL for 2 h and total protein were extracted. Total and phosphorylated Stat3 and Stat5 were analysed by Western Blot; GAPDH was used as loading control and a representative membrane is shown (n = 3). C PRLR-transduced and control MonoMac-1 (MM) were treated for 2 h with the vehicle, PRL or G129R at 500 ng/mL. Nuclear protein fraction was extracted and total and phosphorylated Stat5 were analysed by Western Blot; Lamin B1 was used as loading control and a representative membrane is shown (n = 3). D PRLR wt-expressing HEK293T cells were transfected with the CISH reporter and treated with increasing doses of PRL (blue) or G129R (red) for 24 h. Luciferase activity is shown. *p < 0.05 (paired t test, triplicates). E MonoMac-1 and HL-60 cells were treated with an inhibitor of Stat3, Stat5 and Jak2 for 72 h at different doses and viability was analysed by flow cytometry. EC50 value is shown in the table, triplicates. F PRLR-transduced and control MonoMac-1 (MM) cells were treated for 72 h with a Stat5 inhibitor (10 and 50 µM) alone (green) or in combination with 500 ng/mL PRL (squared green). Viability was assessed by flow cytometry. *p < 0.05 (one-way ANOVA. Dunnet’s multiple comparison test, triplicates). G Stat3 (209991_at) and Stat5 (203010_at) mRNA expression in healthy donors (grey) and AML patients (red) from GSE13159 database. Ns, not significant; ****p < 0.0001 (unpaired t test). In all graphs, bars represent the mean ± SEM