Fig. 5: Inhibition of spontaneous RyR-mediated Ca2+ release does not affected early the phases of the autophagic pathway. | Nature Communications

Fig. 5: Inhibition of spontaneous RyR-mediated Ca2+ release does not affected early the phases of the autophagic pathway.

From: Inactive ryanodine receptors sustain lysosomal availability for autophagy by promoting ER-lysosomal contact site formation

Fig. 5: Inhibition of spontaneous RyR-mediated Ca2+ release does not affected early the phases of the autophagic pathway.The alternative text for this image may have been generated using AI.

a–d top Representative immunoblots of hiPSC-derived cortical neurons treated for 24 hr with DMSO or 30 µM ryanodine (Rya), stained for (P-)AMPK (a), (P-)p70S6 kinase (b), Beclin 1 and vinculin (c), or (P(Ser355))-ATG13 (d). a–d bottom: Densitometric quantification of the above immunoblots plotted relative to DMSO. Each data point represents an independent experiment (n = 5). Data are shown as mean ± s.d. Two-tailed one sample t-tests were performed to assess significant differences relative to DMSO (for (a) P = 0.6108, t = 0.5513 df = 4, for (b) P = 0.3314, t = 1.104 df = 4 for (c) P = 0.2979 t = 1.196 df = 4 for (d) P = 0.3542, t = 1.047 df = 4). e Representative fluorescence microscopy images of hiPSC-derived cortical neurons immunolabeled for FIP200 and WIPI2, co-stained with Hoechst. Scale bar: 10 µm. f Quantification of FIP200 and WIPI2 puncta per cell. Each data point depicts the average number of puncta per cell of one independent repetition (n = 6). Data shown as mean ± s.d. Two-tailed unpaired t-tests were performed FIP200, P = 0.3217, t = 1.043, df = 10; WIPI2, P = 0.1824, t = 1.433, df = 10. g Representative immunoblot of neurons treated for 3 hr with DMSO or 30 µM Rya ± bortezomib (Bort, 10 µM) and/or bafilomycin A1 (BM, 100 nM), labeled for vinculin and APP. h Densitometric quantification of APP normalized to loading controls and plotted relative to DMSO. Each symbol represents an independent repetition (n = 7). A two-tailed one-sample t-test (DMSO vs Rya) was performed: P = 0.0077, t = 3.932, df = 6. A one-way ANOVA with Tukey multiple comparison testing (with significance level adjusted to p < 0.025 to account for multiple testing) was performed to test for significant differences between the Rya condition and Rya + Bort or Rya + BM condition (P = 0.0006, F = 11.39, df = 20, *,** and ***p < 0.025, **p < 0.01 and p < 0.001 respectively). Molecular weights (kDa) are indicated on all blots. Source data are provided as a Source Data file.

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