Figure 5.

CAPS and Rbcn3β knockdown cause acidification defects. A, TIRF image at 405-nm excitation of PC12 cells loaded with compound FFN202 (left). FFN202 loading is decreased if cells are preincubated with 1 μm reserpine, a VMAT blocker (right). B, the 370:355 nm emission ratio of FFN202 increases with increasing buffer pH. Values shown represent means ± S.E. (n = 3) read from a plate reader. C, 370:355 nm fluorescence ratio of FFN202-loaded PC12 cells transfected with CAPS1, Rbcn3α, or Rbcn3β siRNA pools normalized to determinations with a nontargeting siRNA pool. Values are the mean ± S.E. (n = 3, 10 replicates each), and differences were nonsignificant (n.s.). D, 370:335 ratios of PC12 cells either loaded with FFN202 (untreated) or preincubated with 100 nm bafilomycin prior to FFN202 loading (recovered). Bafilomycin-treated cells were normalized to untreated cells in each condition. Values represent the mean ± S.E. (n = 3, 10 replicates each). *, p < 0.05; **, p < 0.005; ***, p < 0.0005. E, TIRF image of PC12 cells expressing NPY-GFP. F, lower magnification epifluorescence images of PC12 cells expressing NPY-GFP before (left) and after (right) addition of 50 mm NH₄Cl. G, Western blot showing CAPS1 depletion in stable lentivirus shRNA CAPS1 knockdown PC12 cells with GAPDH loading control. H, ratio of NPY-GFP fluorescence before and after NH₄Cl addition in multiple (>900) control and CAPS1 knockdown cells. Mean values ± S.E. are shown for five experiments. I, ratio of NPY-GFP fluorescence (±NH₄Cl) in control or CAPS1 knockdown PC12 cells over 90 min as cells recovered from 1-h treatment with 100 nm bafilomycin. Fluorescence ratios were normalized to 1.0 at zero time. Values shown are means ± S.E. for determinations in >100 cells for each condition. *, p < 0.05; **, p < 0.005; ****, p < 0.00005. J, basal and ionomycin-stimulated NPY-Venus secretion in 10 min from BON cells was determined following transfection of the indicated siRNA pools. Mean values ± S.E. are shown. **, p < 0.01 (n = 3).