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VDAC-driven mitophagy in human erythropoiesis
VDAC1 expression, both at the mRNA (53.6 ± 7.6%) and protein level (78.5 ± 5.9%) was observed as compared to shSCR-transduced cells (Online Supplementary Figure S1B and C). Of note, no significant difference was observed in the mRNA expression of two other VDAC, VDAC2 and
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VDAC3, confirming the specificity of the shRNA con- struct (Online Supplementary Figure S1D).
The effect of VDAC1 downregulation on erythroblast maturation was assessed by quantifying the percentages of erythroblasts at different stages of differentiation, as
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EFG
Figure 1. VDAC1 knockdown in human erythroid progenitors accelerates erythroid differentiation. (A) Schematic of the ex vivo erythroid differentiation protocol fol- lowing short hairpin RNA (shRNA)-mediated downregulation of VDAC1. CD34+ progenitors were isolated from cord blood and transduced at day 4 with a lentiviral vec- tor harboring either the VDAC1 shRNA or scramble shRNA (shSCR), together with the EGFP transgene. EGFP+ cells were sorted at day 7 and differentiated until day 17. (B) Representative May-Grünwald-Giemsa (MGG) images of erythroid progenitors and quantification of cells at different stages of differentiation are shown for day 7 and (C) day 10 of differentiation (n=3). Scale bar =20 mm. (D) Representative a4-integrin/Band3 flow cytometry profiles of GPA+ cells at day 10 are shown, allowing different stages of differentiation to be distinguished. (E) Band3 and (F) a4-integrin surface expressions were evaluated on glycophorin A positive (GPA+) cells following introduction of shSCR (black) and shVDAC1 (red) at days 8, 10, 14, and 16 of erythroid differentiation. The percentages at each time point were quan- tified and means ± standard error are shown (n=9). (G) Erythroblast proliferation in control progenitors and shVDAC1-transduced progenitors was monitored at days 7, 10, 14 and 17 (n=4). *P<0.05, **P<0.01, ***P<0.001. CBMC: cord blood mononucleated cells; shVDAC1: VDAC1 shRNA; D: day. ProE: proerythroblast; BasoE: basophilic erythroblasts; PolyE: polychromatic erythroblasts; OrthoE: orthochromatic erythroblasts; Retic: reticulocytes.
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