Endogenous restoration of WNT4 in BJAB cells. Overexpression of WNT4 in the leukemia-derived BJAB cell line was performed by using a Doxycycline (Doxy)-inducible lentiviral expression system. After a 48-h incubation with Doxy, WNT4 expression was confirmed using qRT-PCR (A), and western blot assays (B). A) Graphs showing the amplification curves obtained for WNT4 and RPS18 in BJAB-Tet-WNT4 cells cultured in the presence or absence of Doxy. The WNT4 normalized relative ratio was calculated utilizing the non-treated BJAB-Tet cells as a control (set as 1), and by using ACTB, RPL32 and RPS18 as reference genes in two independent experiments. B) Western blot assays (50 μg total protein) showing the presence of WNT4 protein after a 48-h incubation with Doxy; beta actin (ACTB) was used a protein loading control. C &D) Relative expression levels of WNT4 were measured by qRT-PCR in PBMCs from healthy volunteers, CD3+ and CD19+ sorted cells obtained from healthy volunteers (C), as well as in parental BJAB cells and in BJAB WNT4-expressing cells (D). The average values of PBMCs (in C) and CD19+ sorted cells (in D) were used as controls. The graph depicts the means obtained with all reference genes ± standard deviations (SD).