RILP may play a minor role in axonal autophagosome transport. (A) Time series and kymograph from separate LC3+ autophagosomes demonstrating comigration with RILP. Scale bars, 2 µm. (B) Quantification of LC3+ puncta comigrating with RILP in different subaxonal regions. n = 8–10 neurons; one-way ANOVA (P = 0.9577). (C and D) Example immunoblot and quantification of autophagosomal isolation illustrating enrichment of RILP on the outer membrane. n = 3 preps; one-way ANOVA (P = 0.0039). (E–H) Representative micrographs and quantifications of PLA puncta for Halo-tagged RILP and endogenous LC3 (n = 18 neurons) or endogenous DIC (n = 8 per region) along the axon (dotted gray line). Arrowheads indicate PLA puncta. Scale bar, 10 µm. Dashed gray line indicates negative control (missing primary antibody). One-way ANOVA. (I) Immunoblotting and quantification of PC12 cell lysates show KD efficiency of RILP siRNA. n = 4 repeats. Normalization factor (NF) determined using Revert Total Protein Stain. (J) Quantification of LC3+ puncta motile behavior in the distal, mid-, and proximal axon. n = 10–13 neurons. Two-way ANOVA with Tukey’s multiple comparisons test; proximal (retrograde: mock vs. KD, P = 0.0650; mock vs. rescue, P = 0.0094; KD vs. rescue, P = 0.7205; stationary/bidirectional: mock vs. KD, P = 0.0067; mock vs. rescue, P = 0.0015; KD vs. rescue, P = 0.8641). Symbols indicate comparison to mock. (K) Example kymographs from the proximal axon. Corresponding micrographs below. Scale bar, 5 µm. Bars throughout show mean ± SEM. **, P < 0.01.
Figure S4.

RILP may play a minor role in axonal autophagosome transport. (A) Time series and kymograph from separate LC3+ autophagosomes demonstrating comigration with RILP. Scale bars, 2 µm. (B) Quantification of LC3+ puncta comigrating with RILP in different subaxonal regions. n = 8–10 neurons; one-way ANOVA (P = 0.9577). (C and D) Example immunoblot and quantification of autophagosomal isolation illustrating enrichment of RILP on the outer membrane. n = 3 preps; one-way ANOVA (P = 0.0039). (E–H) Representative micrographs and quantifications of PLA puncta for Halo-tagged RILP and endogenous LC3 (n = 18 neurons) or endogenous DIC (n = 8 per region) along the axon (dotted gray line). Arrowheads indicate PLA puncta. Scale bar, 10 µm. Dashed gray line indicates negative control (missing primary antibody). One-way ANOVA. (I) Immunoblotting and quantification of PC12 cell lysates show KD efficiency of RILP siRNA. n = 4 repeats. Normalization factor (NF) determined using Revert Total Protein Stain. (J) Quantification of LC3+ puncta motile behavior in the distal, mid-, and proximal axon. n = 10–13 neurons. Two-way ANOVA with Tukey’s multiple comparisons test; proximal (retrograde: mock vs. KD, P = 0.0650; mock vs. rescue, P = 0.0094; KD vs. rescue, P = 0.7205; stationary/bidirectional: mock vs. KD, P = 0.0067; mock vs. rescue, P = 0.0015; KD vs. rescue, P = 0.8641). Symbols indicate comparison to mock. (K) Example kymographs from the proximal axon. Corresponding micrographs below. Scale bar, 5 µm. Bars throughout show mean ± SEM. **, P < 0.01.

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