Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity

doi: 10.1073/pnas.1920122117

Figure Lengend Snippet: TPC1 affects IgE-dependent Ca 2+ signaling via regulation of the Ca 2+ homeostasis between different intracellular stores. ( A ) Ratiometric Fura-2 AM-dependent measurements of cytosolic Ca 2+ concentrations in the presence of 2 mM [Ca 2+ ] e . Tpc1 +/+ (black, n = 19) and Tpc1 −/− (red, n = 23) mast cells were pretreated with DNP-specific IgE antibodies (100 ng/mL) overnight and stimulated with 100 ng/mL DNP-BSA at 50 s (arrow). Data were normalized to the initial F 340 /F 380 ratio, averaged, and blotted versus time as mean ± SEM ( Left ). Bar graphs show mean peak cytosolic Ca 2+ concentrations at 90 s extracted from A ± SEM ( Right ). * P < 0,05 (two-tailed Student’s t test). ( B ) Quantification of the AUC was used as an estimate of the total increase in the cytosolic Ca 2+ concentration over time. Data are shown as mean ± SEM. *** P < 0,001 (two-tailed Student’s t test). ( C ) Ratiometric Fura-2 AM-dependent measurements of cytosolic Ca 2+ concentrations in the absence of [Ca 2+ ] e (0.5 mM EGTA) ( Left ). Tpc1 +/+ (black, n = 36) and Tpc1 −/− (red, n = 27) mast cells were treated as in A . Data were normalized to the initial F 340 /F 380 ratio, averaged, and blotted versus time as mean ± SEM. Bar graphs show mean peak cytosolic Ca 2+ concentrations at 125 s extracted from C ± SEM ( Right ). ** P < 0,01 (two-tailed Student’s t test). ( D ) Quantification of the AUC was used as an estimate of the total increase in the cytosolic Ca 2+ concentration over time. Data are shown as mean ± SEM. *** P < 0,001 (two-tailed Student’s t test). ( E ) Tpc1 +/+ mast cells were preincubated with Tet for 15 min prior to DNP-BSA stimulation; cytosolic Ca 2+ signals were acquired and analyzed as in A . Traces from C are depicted as dashed lines, for better comparison ( Left ). Mean peak cytosolic Ca 2+ concentrations at 125 s extracted from Left (gray, solid bar) and as comparison peak levels from C (open bars) ( Right ). ( F ) Average AUC calculated from E (gray, solid bar) and as comparison from D (open bars) ± SEM. * P < 0.05, ** P < 0.01 (one-way ANOVA). ( G ) Tpc1 +/+ mast cells were preincubated with trans -Ned-19 for 15 min prior to DNP-BSA stimulation; cytosolic Ca 2+ signals were acquired and analyzed as in C . Traces from C are depicted as dashed lines, for better comparison ( Left ). Mean peak cytosolic Ca 2+ concentrations at 125 s extracted from Left (gray, solid bar) and as comparison peak levels from C (open bars) ( Right ). ( H ) Average AUC calculated from G (gray, solid bar) and as comparison from D (open bars) ± SEM. *** P < 0.001 (one-way ANOVA).

Article Snippet: For experiments with the low-affinity Ca 2+ fluorochrome Fura-2FF AM (Biomol) , cells were loaded for 45 min using the same supplemented Na + Ringer.

Techniques: Two Tailed Test, Concentration Assay, Comparison