Mitochondrial Stat3 enhances transcription of mitochondrial genes and proliferation of ES cells.Figure 5(A) Experimental approach used to characterize the functional role of Stat3 on cell proliferation and mitochondrial activity. (B) Western blot of Stat3+/+ cells cultured in presence or absence of LIF, Stat3-/- cells cultured in 2i+LIF and two clones of Stat3-/- cells transfected with a full length form of Stat3 cultured in 2i+LIF. Relative mean intensity is shown below each band. Note that Stat3protein levels in clone A and B are comparable to the endogenous levels of the control. GAPDH was used as a loading control. (C) Gene expression analysis of Stat3+/+ cells, Stat3-/- cells and two Stat3 rescue clones (Stat3.A/B) cultured in absence or presence of LIF. Note that both clones respond to LIF and activate Stat3 direct target Socs3. (D) Proliferation assay of Stat3+/+ cells, Stat3-/- cells and Stat3.A/B rescue clones cultured in the presence of LIF. Cells were seeded and scored for four days. Scores were normalized to day1. Mean and s.e.m. of two independent biological replicates of a representative experiment are shown. See also Appendix Figure S7A. (E) Western blot of total and mitochondrial fractions of Stat3+/+, Stat3-/- cells and two MLS-Stat3 clones cultured in 2i+LIF. The nuclear protein TRIM33 and mitochondrial marker TOM20 confirmed successful mitochondrial isolation. Note that MLS-Stat3 is enriched in the mitochondrial fraction, suggesting correct localization of the fusion protein. See also Appendix Figure S7B. (F) Representative confocal images of Stat3-/- and MLS-Stat3 cells stained with anti-Stat3 and anti-ATAD3 antibodies. Merge image shows co-localization between Stat3 and the nucleoids marker Atatd3 (R=0.72 for MLS-Stat3 cells, R=0.22 in Stat3-/-). DAPI serves as nuclear counterstain. Scale bar, 10 μm. (G) Gene expression analysis of Stat3+/+, Stat3-/- cells and three MLS-Stat3 clones cultured in the presence of LIF. MLS-Stat3 specifically induces expression of mitochondrial markers with negligible effects on the nuclear target Socs3. See also Appendix Figure S7E. (H) Chromatin immunoprecipitation (ChIP) performed using anti-Stat3 or a rabbit control IgG antibody in Stat3-/- and MLS-Stat3 cells cultured in 2i+LIF conditions. ChIP-PCR was performed with primers located on three regions of the D-loop (A, B, C). Note that 2 D-loop regions are significantly enriched in MLS-Stat3 compared to Stat3-/- cells. Mean and s.e.m. of three independent experiments are shown. Unpaired t-test: * P<0.05 ** P<0.01. (I) Left: representative confocal images of Stat3-/- and MLS-Stat3 cells subjected to proximity-ligation-assay (PLA) by using anti-Stat3 and anti-ATAD3 antibodies. DAPI serves as a nuclear counterstain. Red dots indicates spacial proximity between Stat3 and the nucleoids marker Atad3. Right: histogram showing quantification of PLA assay performed on Stat3+/+, Stat3-/- and MLS-Stat3 cells. Number of red dots/cell are plotted. Note that double staining in MLS-Stat3 cells results in increased number or red dots compared to Stat3-/- cells Mean and s.e.m. of >15 cells for each sample are shown. Unpaired t-test: ** P<0.01 *** P<0.001. Scale bar, 10µm. See also Appendix Figure S7D. (J) Proliferation assay of Stat3+/+, Stat3-/- cells and three MLS-Stat3 clones cultured in the presence of LIF. Cells were seeded and scored for four days. Mean and s.e.m. of two technical replicates of a representative experiment are shown. See also Appendix Figure S8A. (K) Proliferation assay of Stat3-/- cells and three MLS-Stat3 clones cultured in the presence of LIF. Cells were seeded and treated with DMSO or with 50nM or 100nM of rotenone for 48hours. MLS-Stat3 clones also proliferated more than Stat3-/- both basally and in the presence of rotenone. Mean and s.e.m. of two technical replicates of a representative experiment are shown. (L) Representative brightfield images of Stat3+/+, Stat3-/- and one MLS-Stat3 clone cultured in 2i+LIF showing similar morphology but note smaller colony size for Stat3-/- cells. Scale bar, 100µm.