Fig. 5

STAT3 enhances V-ATPase activity. a Representative immunoblots (left) and quantification (right) of the indicated V-ATPase subunits from lysates of HeLa CRISPR control clone (C-4) and STAT3-KO clones (KO-1 and −11). TUBA1A served as a loading control. b Numbers of lysosomal genes whose expression analyzed by RNA-Seq was decreased or increased over ≥ 1.5-fold (P ≤ 0.05) in HeLa-STAT3-KO cells as compared to HeLa-C4 control cells. Lysosomal genes were defined as genes whose protein products localize to lysosomes according to either Gene Ontology or Kyoto Encyclopedia of Genes and Genomes databases. See Supplementary information, Fig. S5b for the list of altered genes. c Representative immunoblots of the indicted proteins from lysates of HeLa cells transfected with the indicated siRNAs 72 h earlier. n = 3. d Representative images (left) and quantification (right) of PLA puncta with antibodies against ATP6V1A (V1A) and ATP6V0D1 (V0D1) in HeLa CRISPR control (C-4) and STAT3-KO (STAT3-KO-11) cells, as well as in HeLa cells transfected with the indicated siRNAs 72 h earlier. DNA was stained with DAPI. Images were taken with 60× magnification using Zeiss LSM700 confocal microscope. The optimal slice thickness (∼350 nm) was defined by the Zeiss zen software. Scale bar, 10 µm. e Quantification of PLA puncta with antibodies against STAT3 and V1A in HeLa cells (left) or Flag and V0D1 in HeLa-STAT3-Flag cells (right). Cells were transfected with the indicated siRNAs 72 h earlier. f Activity of V-ATPase in the presence of 30 µg/mL superfolder-GFP (sfGFP; control) or ΔN-STAT3-sfGFP. V-ATPase was immunoprecipitated with anti-HA magnetic beads from lysosomal lysates of HeLa cells transiently transfected with pCDNA3.1-HA-ATP6V1A. When indicated, the samples were treated with 100 nM bafilomycin A1. Right, standard curve for the measurement of the free phosphate ion used to estimate the ATP consumption. Protein blot for recombinant proteins is shown in Supplementary information, Fig. S5c. Error bars, SD of ≥ 3 independent experiments. A minimum of ten cells/sample were analyzed in d, e. P-values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test (a, d), DEseq2 (b), or by two-tailed, homoscedastic Student’s t-test (e, f)