Fig. 6

STAT3 regulates cytosolic pH. a Intensity of lysosomal RFP-STAT3 in A549-RFP-STAT3 cells loaded with 0.4 mg/ml cascade blue dextran for 1 h, chased for 5 h, and treated with EBSS for 4 h, 0.1 µM bafilomycin A1 or 10 µM niclosamide for 1 h, 25 µM EIPA in the absence of NaHCO₃ or the presence of 50 mM propionate for 0.5 h, or with 1 mM LLOMe for 15 min. Histograms show mean lysosomal RFP intensities/cell (top) and distribution of lysosomal RFP intensities in a cell population (bottom). Representative images of live cells taken with 60× magnification using Zeiss LSM700 confocal microscope are shown on the right. Scale bar, 10 µm. b Representative immunoblots of LAMP1 and STAT3 in lysosomal lysates of HeLa cells left untreated or treated as in a, except for LLOMe treatment that was for 1 h. The histogram shows relative ratios of STAT3/LAMP1. Cytosolic acidification caused by these treatments is shown in Supplementary information, Fig. S6a. c Representative immunoblots of the indicated proteins in lysates of HeLa cells treated as in b. The histogram shows relative ratios of P-Y705-STAT3 and P-S727-STAT3. d Representative immunoblots of the indicated proteins in lysates of HeLa cells treated with 25 µM EIPA + 50 mM propionate for the indicated times. CCND1, cyclin D1; BIRC5, survivin. n = 3. e Quantitative PCR analysis of CCND1 mRNA levels in HeLa cells left untreated or treated with 25 µM EIPA + 50 mM propionate for 4 h. ACTA1 mRNA served as an internal control. f Quantification of STAT3/LAMP2 ratios in immunoblots of proteins from lysosomes immunopurified with anti-LAMP1. See Supplementary information, Fig. S6d for a representative blot. g Quantification of PLA (anti-STAT3 and anti-ATP6V1A) puncta in MCF7-vector and MCF7-p95DNErbB2 cells. Error bars, SD of ≥ 3 independent experiments. P-values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test (b, c, d, f and g) or by two-tailed, homoscedastic Student’s t-test (e) in comparison with the untreated cells