Fig. 1

STAT3 localizes to the lysosomal membrane. a Representative images of A549-RFP-STAT3 cells labeled with the indicated organelle markers (blue). Values, mean percentage of RFP-STAT3 puncta colocalizing with the indicated organelle marker ± SD of three independent experiments with ≥ 10 cells/sample analyzed in each. Colocalization analysis was not applicable (NA) in KDEL-BFP-labeled cells due to the diffuse staining. The areas marked with white squares are magnified in upper right corners. Images of live cells were taken with 60× magnification using Zeiss LSM700 confocal microscope. See Supplementary information, Fig. S1 for colocalization of STAT3 and lysosomes in other cells. b Representative immunoblots of STAT3 and the indicated organelle markers in total cell lysates or the indicated flow throughs (FT) and immunoprecipitates (eluate) from HeLa cells. n = 3. c Representative immunoblots of the indicated proteins in total cell lysates or the indicated fractions of HeLa cells (left) and quantification of P-Y705-STAT3 and P-S727-STAT3 levels relative to total STAT3. d Representative immunoblots (top) and quantification (bottom) of the indicated proteins in lysates of HeLa cell lysosomes purified by iron-dextran method and left untreated or treated with 10 µg/ml proteinase K and 100 µg/ml digitonin for 10 min at 25 °C when indicated. CTSD, cathepsin D. e Representative images (left) and quantification of cytosolic RFP-STAT3 puncta (right) in A549-RFP-STAT3 cells left untreated or treated with 100 ng/mL IL6 for 30 min and stained with Hoechst. Error bars, SD of three independent experiments, with ≥ 10 cells analyzed/sample. P- values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test (c) or two-tailed, homoscedastic Student’s t-test (e). The optimal slice thickness (∼350 nm) of confocal images (a, e) was defined by the Zeiss zen software. Scale bar, 10 µm