Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells
Fig 1
Functional and signaling-competent EPOR in the NSCLC cell line H838 and CFU-E cells.
(A) Left panel: H838 and H838 cells stably overexpressing HA-tagged human EPOR (H838-HA-hEPOR) were either left untreated (-) or stimulated with 10 U/ml EPO beta (+). Cells were lysed after 10 min and hEPOR proteins were subjected to immunoprecipitation (IP, MAB 307, R&D) and phosphorylated (p) EPOR (4G10, Merck Millipore) and total EPOR (C-20, Santa Cruz) were detected by quantitative immunoblotting (IB). The complete immunoblot is shown in S1 Fig. Right panel: murine (m) CFU-E cells were either left untreated (-) or stimulated with 5 U/ml Epo alfa (+). Cells were lysed after 10 min and mEPOR proteins were subjected to IP (M-20, Santa Cruz). pEPOR (4G10, Merck Millipore) and total EPOR (M-20, Santa Cruz) were detected by IB with chemiluminescence by CCD camera. The experiment was performed in biological triplicates. (B) H838 and H838-HA-hEPOR cells were treated with the indicated doses of EPO beta. Cells were lysed after 10 min and JAK2 proteins were subjected to IP. pJAK2 and total JAK2 were detected by IB with chemiluminescence by CCD camera. (C) H838 and H838-HA-hEPOR cells were treated with the indicated doses of EPO beta. Cells were lysed after 20 min and STAT5 proteins were subjected to IP. The degree of phosphorylation of STAT5 was measured with mass spectrometry. For H838 cells, the average degree of STAT5 phosphorylation based on biological duplicates is shown. (D) H838 and H838-HA-hEPOR cells were treated for three days with 5 mg/l cisplatin or left untreated. Additionally, cells were treated with 10 U/ml EPO beta and the cell viability was measured with CellTiter-Blue assay. The error bars represent standard deviation of biological replicates (n ≥ 5). The experiment was performed on two independent days (second replicate in S4 Fig). (E) H838-HA-hEPOR cells expressing the Casper3-GR FRET-based sensor (H838-HA-hEPOR-Casper3-GR) were treated with 5 mg/l cisplatin, EPO beta, a combination of both or left untreated. Staurosporine (10 μM) was used as positive control for induction of apoptosis. Casper3-GR FRET signal was measured by life-cell imaging for 60 hours. Caspase-3 activity was determined based on the green-to-red ratio and normalized to the untreated control (n = 2, second replicate in S5 Fig).