Fig. 2

STAT3 interacts with V-ATPase on the lysosomal membrane. a V-ATPase subunits detected by high-accuracy mass spectrometry in anti-Flag immunoprecipitates of lysosomal lysates from HeLa cells transiently transfected with pBCMV-STAT3-Flag-puro. Protein amount in relation to STAT3 was estimated from summed peptide intensities (iBAQ algorithm in Maxquant software). The criteria for false discovery rate (FDR) of peptide spectrum match was set to 0.05 and protein FDR to 0.01. Numbers of peptides with identification P-values < 0.01 are indicated. Experiment was repeated once with similar results. b Representative immunoblots of endogenous STAT3 and ATP6V1A (V1A) in anti-STAT3 immunoprecipitates (IP) and lysates of crude lysosome fractions of HeLa cells. n = 3. c Representative immunoblots of the indicated proteins in anti-HA immunoprecipitates (IP) and lysates of crude lysosome fractions of HeLa cells transiently transfected with pCDNA3.1-HA-ATP6V1A. n = 3. d A representative SR-SIM image of a HeLa cell stained with anti-STAT3 and anti-ATP6V0D1 antibodies. The areas marked with white squares are magnified in both sides. Scale bars, 5 µm (middle) and 200 nm (left and right). e Representative images of the indicated SR-SIM projections (left) and colocalization analysis (right) of a punctum staining with anti-STAT3 and anti-ATP6V0D1 (V0D1) antibodies in HeLa cells. Scale bars, 145 nm (yellow) and 200 nm (white). f Representative images (left) and quantification (right) of PLA puncta with antibodies against STAT3 and ATP6V1A in HeLa CRISPR control cell clone (C-4) and STAT3-KO clone (STAT3-KO-11). Lysosomes were visualized by loading with cascade blue dextran. See Fig. 4a for the immunoblot verifying STAT3 depletion. g Representative images of PLAs using antibodies against STAT3 and ATP6V1A in CRISPR control and STAT3-KO human pancreatic duct epithelial cells (H6C7) and human mammary fibroblasts (HMF3) counterstained with DAPI. See Supplementary information, Fig. S2c for the immunoblot verifying STAT3 depletion. h Representative images (left) and quantification (right) of PLA puncta with antibodies against STAT3 and LAMP1 or CD63 in HeLa-C-4 clone. Nuclei were counterstained with DAPI. Cell images in f–h were taken with 60×  magnification using Zeiss LSM700 confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. The images of H6C7 cells in g are integrated stacks. Scale bar, 10 µm. Error bars, SD of three independent experiments with ≥ 10 cells analyzed/sample. P-values were calculated by two-tailed, homoscedastic Student’s t-test (f) or one-way ANOVA combined with Dunnett’s multiple comparisons test (h)