Fig. 2

Inhibition of PRLR decreases AML clonogenicity, sparing the healthy counterpart. A AML cell lines (HL-60, MonoMac-1, SKM-1, KG-1, U-937, Kasumi-1) (n = 6) were treated with the vehicle (grey), PRL (blue) or G129R (red) at 500 ng/mL for 72 h and cell viability was analysed by flow cytometry (one-way ANOVA, Dunnett’s multiple comparison test, triplicates). B PRLR-transduced MonoMac-1 (MM), HL-60 and SKM-1 cells were validated by qPCR (mRNA expression, bars represent 2^−ΔCt ± SEM) and Western Blot with GAPDH as loading control (protein, a representative membrane is shown, and arrows showed the interesting band). Grey, control; Orange, PRLRwt-transduced cells. ***p < 0.001; ****p < 0.0001 (two-way ANOVA, Šidák’s multiple comparison test, triplicates). C Parental and PRLR-overexpressing MonoMac-1 (MM), HL-60 and SKM-1 cells were treated during 4 days with the vehicle (grey), PRL (blue) or G129R (red) at 500 ng/mL and proliferation (DiI mean fluorescence intensity) was assessed by flow cytometry. **p < 0.01; ***p < 0.001 (two-way ANOVA, Šidák’s multiple comparison test, duplicates). D AML patient cells (n = 19) were treated with the vehicle (C, grey), PRL (blue) or G129R (red) at 250 and 500 ng/mL and cell viability was assessed after 72 h in the CD45⁺ cell population by flow cytometry. **p < 0.01 (non-parametric one-way ANOVA, Holm-Šidák’s multiple comparison test). E Healthy donor PB-MNCs (n = 7) were treated with the vehicle (grey), PRL (blue) or G129R (red) at 250 and 500 ng/mL and cell viability was assessed after 72 h in CD45⁺ total blood population, CD13⁺ myeloid cells, CD3⁺ T cells and CD19⁺ B cells by flow cytometry. Statistics were done as in D. F AML patient cells (n = 10) were treated with the vehicle (C, grey), PRL (blue) or G129R (red) at 250 and 500 ng/mL for 18 h, colonies were counted after 14 days based on cellularity and morphology. Statistics were done as in D. **p < 0.01. G Lineage-depleted umbilical cord blood cells (n = 4) were treated with the vehicle (grey), PRL (blue) or G129R (red) at 250 or 500 ng/mL for 18 h, colonies were counted after 14 days based on cellularity and morphology. Statistics were done as in D. H PRLR expression of parental U-937 (grey) and three different PRLR-directed CRISPR/Cas9-transduced clones (orange) were phenotyped for PRLR surface expression by flow cytometry (one-way ANOVA, Dunnett’s multiple comparison test, triplicates). I Parental cells (grey) and transduced clones (orange) were cultured in a semisolid medium enriched with cytokines; colonies were counted after 7 days based on cellularity and morphology. **p < 0.01 (one-way ANOVA, Dunnett’s multiple comparison test, n = 6). In all graphs, bars represent the mean ± SEM of each individual experiment