Fig. 1

Overexpression of C3G in K562 cells increases megakaryocytic markers. a May-Grünwald-Giemsa staining of the indicated K562 clones untreated or treated with 20 nM PMA for 72 h. Scale bars: 20 μm. b Representative images showing the morphology of HEL cells after 24, 48 and 72 h of PMA treatment. Images were obtained using a Zeiss Axiovert 135 inverted microscope. Scale bars: 50 μm, c Analysis of C3G expression in K562 and HEL cells stimulated with 20 nM PMA at the indicated times. Tubulin was used as loading control. C3G expression values are relative to non-treated cells and normalized against tubulin (C3G/Tub). d Expression of CD41, CD61 and GPA markers was analyzed by flow cytometry using specific fluorochrome-conjugated antibodies (CD41-APC, CD61-PE and GPA-FITC). Representative flow cytometry plots of untreated cells are shown. Histograms represent the mean ± SEM of the fluorescence intensity (relative units) of CD41, CD61 and GPA from at least 4 independent experiments of each clone, treated as indicated. 2-way ANOVA and Holm-Sidak analysis were done. **p < 0.01, ***p < 0.001. e Representative Western blots showing C3G expression in the pLTR2 and pLTR2/GFP clones. The numbers represent C3G expression values relative to empty vectors and normalized against tubulin (C3G/Tub), which was used as loading control. f Analysis of CD61 and GPA expression in untreated K562 clones, pLTR2-CT and pLTR2-C3G, by RT-PCR. The numbers represent expression values relative to pLTR2-CT expressing cells and normalized against GAPDH, which was used as housekeeping gene. g Western blot analysis of α-globin and C3G in the indicated clones of K562, treated or not with 2 μM STI-571, an inducer of erythroid differentiation in these cells, [46]. The expression of total ERK was used as loading control. α-globin/ERK ratios are shown. All values are relative to control, non-treated cells. STI: STI-571 (Imatinib mesylate); CT: control