Protein abundance of AKT and ERK pathway components governs cell-type-specific regulation of proliferation (2017)
Figure 1

Figure 1. Characterization of Epo-induced proliferation and signaling.

A DNA content of mCFU-E and BaF3-EpoR cells in response to different Epo concentrations. [3H]-Thymidine incorporation was measured after 14 h (mCFU-E) or 38 h (BaF3-EpoR). Data represented as mean ± standard deviation, N=3. Lines represent sigmoidal regression. EC50 values are given.

B Cell doubling with time in response to 50 U/ml Epo. Cell numbers were determined by manual counting using trypan blue exclusion assay. Data represented as mean ± standard deviation, N=3. Lines represent exponential regression. Doubling time is indicated.

C Size determinations of mCFU-E and BaF3-EpoR cells. Exemplary fluorescence microscopy pictures upon Hoechst staining for nucleus visualization with 60x objective. The bar respresents 10 µm distance (upper panel). Cell diameter was measured by Imaging flow cytometry. Cytoplasm was stained with Calcein, nuclei were stained with DRAQ5. Probability density function of size distribution with indicated mean diameter of mCFU-E and BaF3EpoR cells. All cells were growth-factor deprived and unstimulated.

D Epo-induced phosphorylation of EpoR, AKT and ERK of mCFU-E and BaF3-EpoR cells. Above each panel the number of growth-factor deprived cells examined per time point is indicated as well as the concentration of Epo applied for stimulation. To the left the position of the molecular weight marker is indicated in kDa and arrowheads indicate the position of the protein of interest. For the detection of the EpoR, 5x106 mCFU-E cells were stimulated with 2.5 U/ml Epo and 5x106 BaF3-EpoR cells were stimulated with 5 U/ml Epo. Cells were lysed, subjected to immunoprecipitation with anti-EpoR and were analyzed by immunoblotting using either anti-pTyr (pEpoR) or anti-EpoR (EpoR) antibodies. For the detection of AKT, mCFU-E cells were stimulated with 2.5 U/ml Epo and BaF3-EpoR cells were stimulated with 5 U/ml Epo. Per time point cellular lysates equivalent to 5x105 mCFU-E cells and 1x106 BaF3-EpoR cells were analyzed by immunoblotting using anti-pAKT and anti-AKT antibodies. For the detection of ERK, cells were stimulated with 50 U/ml Epo. Per time point cellular lysates equivalent to 8x105 cells were analyzed by immunoblotting. Immunoblot detection was performed with chemiluminescence utilizing a CCD camera device (ImageQuant).

E Absolute concentrations of pEpoR, pAKT and ppERK in mCFU-E and BaF3-EpoR cells with time in response to 50 U/ml Epo. Experimental data of a representative experiment is depicted with filled circles, dashed lines represent splines. Error bars indicate standard deviation estimated by an error model.

F Expression of cell-cycle indicator genes of mCFU-E and BaF3-EpoR cells in response to 5 U/ml Epo. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, * p<0.05.

G Fold change of fractions of cells in S/G2/M phase of the cell cycle with respect to 0 U/ml Epo. Growth-factor deprived cells were stimulated with indicated Epo doses for given time. Fractions in sub-G1, G1, S, G2/M were determined by propidium iodide (PI) staining for DNA content. N=3. Welch Modified Two-Sample t-Test, n.s. = not significant, * p < 0.05 , ** p < 0.01 , *** p < 0.005. The percentage of cells in S/G2/M phase in unstimulated cells is additionally indicated.

Table 1. Cytoplasmic concentration of pathway components of mCFU-E, BaF3-EpoR and 32D-EpoR cells.

Cytoplasmic volumes were estimated using imaging flow cytometry excluding the nucleus. 1,000 EpoR molecules on the surface of mCFU-E cells were reported (D'Andrea and Zon, 1990). 15,500 EpoR molecules on the surface of BaF3-EpoR cells were determined by a saturation-binding assay with 125I-labeled Epo (Becker et al, 2010). Number of EpoR molecules on the surface of 32D-EpoR cells was calculated in comparison to BaF3-EpoR cells using flow cytometry. All other concentrations were determined as fold differences to BaF3-EpoR expression level. ‘-‘ indicates the absence of protein. For details, see Appendix G.