Fig. 4

STAT3 regulates lysosomal pH and activity. a Lysosomal pH determined by FITC/TMR ratio in a HeLa CRISPR control cell clone (C-4), STAT3-KO clones (KO-1 and −11), and KO-11 clone reconstituted with wild-type (WT) or mutated (Y705F, DBM, S727A) STAT3 constructs. Representative immunoblots show STAT3 and ACTB (loading control) protein levels in the clones. Standard curve for pH measurements is shown in Supplementary information, Fig. S4a. b Lysosomal pH determined as in a in CRISPR control and STAT3-KO HMF3 and H6C7 cells. Standard curve for pH measurements is shown in Supplementary information, Fig. S4a. c Volume of acidic compartment (VAC) in HeLa cell clones described in a analyzed by flow cytometer after 5 min staining with 75 nM Lysotracker Green. Relative fluorescence intensities are shown on the left. A representative flow cytometry profile is shown on the right. For other flow cytometry profiles and gating of the cells, see Supplementary information, Fig. S4c and d. d Representative immunoblots of LAMP1, STAT3, and cathepsin B (CTSB) in lysosomal lysates of the indicated HeLa cell clones. The histogram shows ratios between the active (25 kDa) and inactive (31 kDa) CTSB as percentages of the value in C4 control clone. e AlexaFluor 488-Dextran degradation in the indicated HeLa clones loaded with 0.4 mg/ml AlexaFluor 488-dextran for 20 min, washed, and fixed with or without a 4 h chase period. Representative images taken with 60× magnification using Zeiss LSM700 confocal microscope are shown on the right. Error bars, SD of ≥ 3 independent experiments. A minimum of 10 cells/sample were analyzed in a, b, and e. P-values were calculated by one-way ANOVA combined with Dunnett’s multiple comparisons test (a, c) or two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (b, d) for multiple comparisons, or by two-tailed, homoscedastic Student’s t-test (e)