Fig 2: Rod-specific Tsc2 knockout does not change autophagy flux. (A and B) Western blots (Bottom) and quantification plots (Top) of comparative changes in autophagy markers caused by CQ injections 4 h before the retina collection. Data are normalized on the average density of LC3-II or SQSTM1 levels of the untreated group for RhoP23H/WT or WT (Tsc2fl/fl) littermate mice. All animals are 1 mo old. Representative results from three independent experiments for each mouse line are shown. The infrared (HSC70) and ECL (LC3 and SQSTM1) Western blotting systems were used for protein detection. The bands (indicated with M) between CQ-treated and -untreated groups on HSC70 blots correspond to the prestained protein markers detected with Odyssey Image System. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns for P > 0.05.

Fig 3: Serum EVs contain high levels of platelet proteins in human blood.(A) Western blot analysis of the sEV fractions derived from plasma and serum of 5 individuals, using antibodies against EV markers and platelet-specific proteins. Equal amounts of proteins were loaded onto each lane and analyzed. (B) Bar graph showing relative protein levels of CD63 that were calculated from the band intensity of the Western blot image in (A). (C-D) Bar graphs showing the levels of the EV marker CD9 (C), and platelet-specific markers GPIIb, GPIIIa and PF4 (D), relative to that of CD63. Whereas the level of CD9 was comparable between the sEV fractions from plasma and serum, all the platelet markers showed a clear tendency of increased levels in serum EVs. (E) NTA results showing the numbers (left) and median diameters (right) of the particles in the sEV fractions derived from human plasma and serum. (F) Comparison of the sEV fractions derived from mouse and human blood by Western blot analysis using antibodies against EV markers and lipoprotein ApoA1. The sEV fractions isolated from human blood contain very low levels of the EV markers CD63 and Hsc70, but a high level of ApoA1, implying a large difference in particle population between human and mouse blood. Data are shown as the average ± SEM. Student t-test. n.s., not significant.

Fig 4: Subunits of the WAVE complex, including WASF3, are highly expressed in photoreceptor cells. (A) mRNA levels of all known nucleation promoting factors expressed in mouse photoreceptor cells. The values are quantified as fragments per kilobase million (FPKM). The data were obtained from a published RNAseq dataset of flow-sorted rod cells isolated at various time points between P2 and P28 (24). Raw data were acquired from NCBI Gene Expression Omnibus series GSE74660. (B) A comparison of the mRNA expression profiles for Wasf3, three outer segment proteins (Cnga1, Cngb1 and Rom1), actin (Actb) and a housekeeping gene (Hsc70). For each gene, the data were normalized to their maximal expression level at any time point. The data in both panels are shown as mean ± SD.

Fig 5: WASF3 knockout results in normal protein trafficking but disrupted outer segment morphology. (A) Retinal cross sections of P21 WT and Wasf3−/− mice stained with antibodies against indicated outer segment proteins (green). Nuclei were stained with Hoechst (blue). (B) Expanded view of the outer segment layer in retinal cross sections of P21 WT and Wasf3−/− mice stained with anti-ROM1 antibodies. (C) Expanded view of the outer segment layer in retinal cross sections of P21 WT and Wasf3−/− mice stained with fluorophore-conjugated PNA. (D) Western blot for three representative outer segment proteins from whole retinal lysates of WT and Wasf3−/− mice at P21. Three mice of each genotype were analyzed. Each lane was loaded with 20 µg total protein. HSC70 serves as a loading control. Scale bars in all panels are 10 µm.