Acidification inhibitors neutralize endosomal pH.

A, B: pH calibration in AGS cells. AGS cells pulsed with pH-sensitive (FITC) and pH-insensitive (TMR) dextran were incubated in buffers of different pH with 5μg/ml of Nigericin and imaged live. A steady increase in the endosomal ratio of FITC/TMR with increasing pH is observed. The observed ratio vs clamped pH is fit to a sigmoidal curve (red curve) which is used as a calibration curve to estimate the pH of endosomes. Numbers of cells in each condition is >100 cells. The data in B is represented as mean +/- SD. C, D: For the experiment described in Fig 2F–2H, images including pH maps are shown in Fig 2F, Fig 2H, S4C and quantification in Fig 2G, S4D. FITC and TMR endosomal intensities, numbers of endosomes and FITC/TMR endosomal ratio are quantified in S4D. BafA1 200nM/400nM and NH₄Cl increases FITC intensity and reduces numbers of endosomes. NH₄Cl also affects trafficking as seen with an increase of TMR intensity. Endosomal ratio (as a proxy for endosomal pH) also shows an increase with all the acidification inhibitors. Control₁ is 0.2% DMSO, Control₂ is 0.4% DMSO and Control₃ is 0% DMSO. Number of repeats ≥ 3 for each treatment and each repeat has >80 cells. p-value table is indicated in S1 Table. E, F: Estimation of FITC/TMR ratio of early endosomes. AGS cells were pulsed with FITC and TMR dextran for 20 minutes, chased for 10 minutes and imaged live. Throughout the pulse and chase duration, the cells were incubated with Control (0.2%DMSO) or BafA1 400nM or Niclosamide 10μM. Dextran uptake and TMR endosomal intensity are marginally reduced with Niclosamide while unaffected with BafA1. An increase in FITC endosomal intensity as well FITC/TMR endosomal ratio is observed with both inhibitors. Number of cells > 35 for each condition. p-value table is indicated in S1 Table. Data representation in D and F is as described in Figs 2 and 1, respectively. Scale bar: 40μm (A, C, E).

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