Figure 2
Figure 2. HSP27 depletion delays erythroid differentiation of human primary erythroblasts and K562 cells. (A) Human primary erythroblasts were induced to differentiate with Epo, IL-3,and TGFβ (2.5 ng/mL) after 24 hours of transfection with HSP27 siRNA (siHSP27) or control siRNA (Ctrl,) (20nM). Percentages of cell transfection and survival were approximately 60% to 70% and 80%, respectively. At the days indicated (day 0 to day 3) the percentage of differentiated cells was evaluated by benzidine assay. Insert, cell lysate from day 2 was resolved on a gel and blotted with the indicated antibodies. (Middle) Differentiating cells at day 2 were visualized by microscopy after cell fixation and staining with May-Grünwald-Giemsa (magnification ×40; bar, 10μm). → indicates polychromatic. One representative image is shown. (Bottom) Percentages of basophilic and polychromatic cells. Quantification, at day 2, from a total number of 300 cells in randomly chosen microscopic fields. (B) CD34+ cells, growing in presence of cytokines (IL-3, Epo), were transduced with shRNA specific for HSP27 or shRNA control (Ctrl). At day 5, CD36+ green fluorescent protein–positive (GFP+) cells were sorted and differentiated in the presence of 30% SVF or TGFβ (2.5 ng/μL) to allow the production of reticulocytes. (Insert) Western blot analysis of HSP27 expression after shRNA transduction. Actin serves as a loading control. Reticulocytes were quantified at the indicated times as the ratio of their number to a total number of 300 cells chosen randomly in different microscopic fields. (Right) Reticulocytes visualized by microscopy after cell fixation and staining with May-Grünwald-Giemsa (magnification ×20; bar, 10 μm). → indicates reticulocytes. Note that HSP27 siRNA and shRNA target different sequences of HSP27 mRNA. (C) Human K562 cells were induced to differentiate with hemin (40μM) after 24 hours of transfection with HSP27 siRNA (siHSP27; ) or scrambled (Ctrl; ). The percentage of differentiated cells was determined by benzidine assay. (Insert) Cell lysate from days 0 and 2 was resolved on a gel and blotted with the indicated antibodies.

HSP27 depletion delays erythroid differentiation of human primary erythroblasts and K562 cells. (A) Human primary erythroblasts were induced to differentiate with Epo, IL-3,and TGFβ (2.5 ng/mL) after 24 hours of transfection with HSP27 siRNA (siHSP27) or control siRNA (Ctrl,) (20nM). Percentages of cell transfection and survival were approximately 60% to 70% and 80%, respectively. At the days indicated (day 0 to day 3) the percentage of differentiated cells was evaluated by benzidine assay. Insert, cell lysate from day 2 was resolved on a gel and blotted with the indicated antibodies. (Middle) Differentiating cells at day 2 were visualized by microscopy after cell fixation and staining with May-Grünwald-Giemsa (magnification ×40; bar, 10μm). → indicates polychromatic. One representative image is shown. (Bottom) Percentages of basophilic and polychromatic cells. Quantification, at day 2, from a total number of 300 cells in randomly chosen microscopic fields. (B) CD34+ cells, growing in presence of cytokines (IL-3, Epo), were transduced with shRNA specific for HSP27 or shRNA control (Ctrl). At day 5, CD36+ green fluorescent protein–positive (GFP+) cells were sorted and differentiated in the presence of 30% SVF or TGFβ (2.5 ng/μL) to allow the production of reticulocytes. (Insert) Western blot analysis of HSP27 expression after shRNA transduction. Actin serves as a loading control. Reticulocytes were quantified at the indicated times as the ratio of their number to a total number of 300 cells chosen randomly in different microscopic fields. (Right) Reticulocytes visualized by microscopy after cell fixation and staining with May-Grünwald-Giemsa (magnification ×20; bar, 10 μm). → indicates reticulocytes. Note that HSP27 siRNA and shRNA target different sequences of HSP27 mRNA. (C) Human K562 cells were induced to differentiate with hemin (40μM) after 24 hours of transfection with HSP27 siRNA (siHSP27; ) or scrambled (Ctrl; ). The percentage of differentiated cells was determined by benzidine assay. (Insert) Cell lysate from days 0 and 2 was resolved on a gel and blotted with the indicated antibodies.

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