LIF promotes proliferation via respiration.Figure 4(A) Flow cytometry analysis after double staining with Annexin-V and Propidium Iodide in ES cells. The combination of Annexin V- FITC and Propidium Iodide allows the distinction between viable cells (unstained) bottom left quadrants, early apoptotic cells (Annexin V-FITC positive) bottom right quadrants, late apoptotic and/or necrotic cells (Annexin V-FITC and Propidium Iodide positive) top right quadrants. ES cells were treated with increasing concentrations of rotenone from 50nM to 200nM for 48 hours. A slight increase in cell death could be observed only at a concentration of 200nM of rotenone. In each quadrant the mean and s.e.m. of 3 independent experiments is indicated. (B) Proliferation assay of Stat3+/+ and Stat3-/- cells cultured in 2i. Cells were seeded and treated for 48 hours with LIF and rotenone (orange bars), a Complex I inhibitor, as indicated. Scores were normalized to Stat3+/+ cells treated with LIF and DMSO. Proliferation was enhanced by LIF treatment in Stat3+/+ cells and reduced by rotenone treatment. Stat3-/- cells resulted more sensitive to rotenone treatment. Mean and s.e.m. of at least 2 independent experiments is shown. Unpaired t-test: * P<0.05 *** P<0.001. n.s. :non significant. See also Appendix Figure S4A. (C) Gene expression analysis of Stat3+/+ cells transfected with control shRNA (SCR, dark green), and two independent shRNAs for a Complex I subunity (NDUFS3) (SH1 and SH2). Note that shRNAs for NDUFS3 downregulates gene expression of about 70%. Mean and st.dev. of two independent experiments is shown. Unpaired t-test: * P<0.05 ** P<0.01. (D) Relative changes in oxygen consumption after 200nM FCCP treatment of Stat3+/+ cells transfected with control shRNA (dark green) and two different shRNAs for NDUFS3. Note that downregulation of the Complex I subunit results in decreased respiration. Mean and s.e.m. of >4 technical replicates are shown. Unpaired t-test: * P<0.05 *** P<0.001. See also Appendix Figure S4D for full Seahorse profiles. (E) Proliferation assay of control shRNA cells (dark green) and cells with downregulation of NDUFS3. Note that downregulation of the Complex I subunit results in decreased proliferation. Mean and s.e.m. of three independent experiments is shown. (F) Proliferation assay of Stat3+/+ and Stat3-/- cells cultured in presence of LIF showing the reduction of proliferation after 48hours treatment with increasing concentrations of Antimycin A (50nM, 100nM or 200nM). See also Appendix Figure S4F. Scores were normalized to WT cells treated with a vehicle (EtOH). Mean and s.e.m. of two independent experiments is shown. Unpaired t-test: ** P<0.01. See also Appendix Figure S4E. (G) Proliferation assay showing the effect of long-term treatment of rotenone on cell proliferation. Stat3+/+ cells cultured in 2i and 2i+LIF with or without rotenone were scored for 4 subsequent passages. The cumulative number of cells has been calculated and shown on log scale. Rotenone at 50nM or 100nM reduced the number of cells after 4 passages by 4.8 and 16.7 times, respectively. Mean and st.dev of two technical replicates of a representative experiment is shown. See also Appendix Figure S6E. (H) Gene expression analysis of Stat3+/+ cells cultured in 2i+LIF (blue bars) and with 50nM rotenone for 48 hours (orange bars) showing that rotenone does not affect transcription of main LIF targets in ES cells. Data were normalized to the highest value for each condition. Mean and st.dev. of 2 biological replicates is shown. (I) Bright field images showing that long-term treatment with rotenone causes reduction in proliferation, shown by the reduced size of individual colonies, but does not cause differentiation in mES cells. Scale bar, 50 µm.