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V. Caraffini et al.
prominently expressed in healthy CD34+ HSPC (Figure 1B). RKIP expression levels in lymphocytes were similar to those observed in HSPC (P=0.244), while they were signif- icantly reduced in monocytes (P=0.001) and granulocytes (P<0.001).
Finally, we sought to confirm these findings in a murine
setting. We therefore performed a database retrieval via the Gene Expression Omnibus (GEO) database and re- analyzed RKIP mRNA in a previous publication of Konuma et al.31 (GEO data set GDS3997) who performed microarray analyses in different hematopoietic cell and progenitor compartments in C57BL/6 mice. In agreement
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Figure 2. RAF kinase inhibitor protein (RKIP) is functionally involved in myeloid lineage differentiation. (A) RKIP shRNA knockdown (KD) in CD34+ human hematopoi- etic stem and progenitor cells (HSPC) increased the myelomonocytic differentiation induced by a granulocyte-macrophage colony-stimulating factor (GM-CSF)/flt-3 ligand (FL)/stem cell factor (SCF)/tumor necrosis factor-α (TNFα) cytokine mix. (Left) Immunoblot showing the successful knockdown of RKIP. Control KD denotes control transduced cells. Representative flow cytometry plots showing increased expression of the myelomonocytic surface markers CD11b and CD14 in CD34+ HSPC with RKIP KD shown in the middle. (Right) Results of all six experiments performed; median is also shown. Statistical significance was calculated by Student´s t-test. Of note, RKIP KD as a single event without the addition of cytokines was insufficient to induce differentiation (data not shown). (B) RKIP KD in HL-60 AML cells increased 1,25D3-induced myelomonocytic differentiation, as assessed by flow cytometric expression of CD11c. (Left) Immunoblot showing the successful knockdown of RKIP; (right) mean CD11c expression of three independent experiments ±Standard Deviation (SD); expression values are given as x-fold expression of HL-60 control KD cells, statistical significance was evaluated by Student’s t-test. (C) RKIP overexpression (RKIP OE) in HL-60 AML cells reduced 1,25D3 induced myelomonocytic differentiation, as assessed by flow cytometric expression of CD11c. HL-60 cells were transduced with eGFP-C1-6xG-hRKIP (RKIP OE) or empty vec- tor (control OE). (Left) Immunoblot showing successful RKIP overexpression; (right) mean CD11c expression of three independent experiments ±SD. Expression val- ues are given as x-fold expression of HL-60 control OE cells. Statistical significance was evaluated by Student’s t-test. Note that HL-60 cells were treated with 10nM 1,25D3.
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haematologica | 2020; 105(2)