Figure S1 is related to Figure 1B, showing more details of outer segment of

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1 Supplemental Information Supplementary Figure legends and Figures Figure S1. Electron microscopic images in Sema4A +/+ and Sema4A / retinas Figure S1 is related to Figure 1B, showing more details of outer segment of photoreceptors in Sema4A retina. (A) Electron microscopic images shown in Figure 1B are enlarged. (B) Magnified images of outer segment of photoreceptors showing apical microvilli in both mice (red arrowheads). Close contacts of apical microvilli of with stacked discs of outer segments were apparent in Sema4A +/+ retina. while apical microvilli were adjacent to whorls of outer segments in Sema4A -/- retina. Of note, the appearance of stalk of photoreceptors, which link outer segment and cell body, adjacent to (yellow arrowheads) indicated the almost complete loss of outer segment. Scale bars, 2.5 μm. Figure S2. Localization of Sema4A in cells and effects of Sema4A-Fc on photoreceptor degeneration in Sema4A / retinas Figure S2 is related to Figure 1, which shows Sema4A expression in cells and the failure of Sema4A-Fc injected into Sema4A / retinas to rescue the photoreceptor 1

2 degeneration phenotype. (A) Immunofluorescent images of Sema4A +/+ retinas at P28. Higher magnification images of the boxes in the images on the left are shown on the right. Sema4A (green) was localized in cells. Scale bars, 20 mm. (B) A sectioned image of a P14 mouse retina injected with FITC-labeled control IgG-Fc. The fluorescent outer photoreceptor layer (bracket) was evaluated for photoreceptor damage. Scale bars, 20 mm. (C) DAPI-stained nuclei (blue) in the area (square) denoted in (B). Injection of Sema4A-Fc into Sema4A / retinas did not rescue the photoreceptor phenotype. Scale bars, 20 mm. Figure S3. Prosaposin localization in cells and binding to the Sema domain of Sema4A Figure S3 is related to Figure 2, showing prosaposin expression in cells and schematic models of the Sema4A constructs and the binding of prosaposin to the Sema domain of Sema4A. (A) Immunofluorescent images of Sema4A +/+ retinas at P28. Higher magnification images of the boxes in the images on the left are shown on the right. Prosaposin (green) was detected in cells and retinal ganglion cells. Scale bars, 20 mm. (B) Schematic models of the Sema4A constructs. Sema4A-FLAG was composed of full-length 2

3 Sema4A fused with a FLAG epitope at the C terminus. Sema4A-EC-FLAG was composed of the extracellular region, transmembrane domain, and a short stretch of the intracellular region fused with a FLAG epitope at the C terminus. Sema4A-IC-FLAG was composed of a short stretch of the extracellular region, the transmembrane domain, and the intracellular region fused with a signal sequence and FLAG epitope at the N terminus. Sema4A-Sema-FLAG and Sema4A-PSI-FLAG contained the Sema domain and PSI domain, respectively, and were each fused with a signal sequence and FLAG epitope at the N terminus. Sema4A-Fc was composed of the extracellular region of Sema4A fused with human IgG Fc. (C) Immunoprecipitation (IP) and immunoblot (Blot) analyses showed an association between prosaposin and the Sema domain of Sema4A. Figure S4. Photoreceptor development and light-induced apoptosis in retinal explant cultures Figure S4 is related to Figure 2. In this supplemental figure, we show maturation of embryonic retinal explants without and the protective effects of prosaposin but not Sema4A-Fc against light-induced photoreceptor apoptosis in these explants. (A) Immunofluorescent images of retinal explants cultured in the dark for 3, 10, or 21 3

4 days. Scale bars, 50 μm. Histograms show average ratios (± SEM; n = 10) of the thickness of the rhodopsin-positive photoreceptor layer (P) relative to the total thickness of the retina (T) (ns, not significant). Maturation was observed in retinal explants lacking from Sema4A +/ and Sema4A / retinas. (B) TUNEL assays of retinal explants with or without in the dark for 21 days or in the dark for 14 days followed by 7 days in the light. Scale bars, 50 μm. Histograms show the average number of TUNEL-positive cells (± SEM; n = 10) in the retinas (*P < 0.01; ns, not significant). Retinal explants lacking or Sema4A show light-induced photoreceptor apoptosis. (C) TUNEL assays of retinal explants treated with recombinant proteins (50 nm) for 14 days in the dark followed by 7 days in the light. Scale bars, 50 μm. Histograms show the average number of TUNEL-positive cells (± SEM; n = 10) in the retinas (*P < 0.01; ns, not significant). Prosaposin but not Sema4A-Fc rescued photoreceptors from light-induced apoptosis in retinal explants lacking or Sema4A. Figure S5. Photoreceptor damage in prosaposin / retinas Figure S5 is related to Figure 2, showing degeneration of the outer segment in the retinal outer nuclear layer and light-induced photoreceptor apoptosis based on TUNEL staining in prosaposin / retinas. 4

5 (A) Hematoxylin and eosin staining showing a smaller outer nuclear layer in a prosaposin / retina (arrows). Scale bars, 50 μm. (B) TUNEL staining (green) and DAPI-stained nuclei (blue) in P14 mouse retinas 60 min after exposure to light. Scale bars, 50 μm. Histograms show the average number of TUNEL-positive cells (± SEM; n = 10) in the retinas (*P < 0.01). Light-induced photoreceptor apoptosis significantly increased after 60 min in prosaposin / retinas. Figure S6. Colocalization of prosaposin and Sema4A with organelle-specific markers Figure S6 is related to Figure 2; in this supplementary figure, we show that Sema4A and prosaposin colocalized with such endosomal markers as Rab family member based on immunohistochemistry and immunoblotting of subcellular fractions. (A) Immunohistochemistry for prosaposin (green) with a Golgi marker (red: Bodipy TR ceramide) in cells. Scale bar, 20 µm. (B) cells in the presence of H 2 O 2 were incubated with biotinylated transferin (b-tfn) for 3 min with or without a 10-min chase period to label sorting or recycling endosomes, respectively. After homogenization, fractions enriched for early endosomes were prepared using a step-floatation gradient. Fractions were analyzed using immunoblotting. Std, Standard control; Fraction 1, top of 5

6 the gradient; Fractions 1 & 2, homogenization buffer (8% sucrose); Fraction 3, 25% sucrose/homogenization buffer interphase; Fraction 4, 25% sucrose; Fraction 5, 35%/25% sucrose interphase; Fraction 6, 35% sucrose; Fraction 7, 40.6%/35% sucrose interphase; Fraction 8, load (40.6% sucrose); Fraction 9, pelleted material. (C, D) Immunohistochemistry for Sema4A-FLAG (green) and either Rab5 or Rab7 (red) in cells treated with or without H 2 O 2 (250 μm) for 60 min. Scale bars, 20 μm. Figure S7. Sema4A binds to the Rab11 FIP2 complex through the cytoplasmic region. Figure S7 is related to Figure 2D, showing the binding of Rab11 and FIP2 to the intracellular region of Sema4A. Immunoprecipitation (IP) and immunoblot (Blot) analyses show an association between the intracellular region of Sema4A and Rab11/FIP2. Figure S8. Apical and basolateral distributions of Sema4A in cells Figure S8 is related to Figure 3; we quantified the relative levels of Sema4A on the apical and basal sides of cells. (A) GFP-labeled Sema4A was examined in two domains: an apical domain (upper 6

7 one-third of each cell) and a basolateral domain (lower two-thirds of each cell). (B) Quantification of GFP signals reflecting Sema4A localization in two domains of cells. Data were obtained from 30 cells for each domain and are summarized as the ratios relative to total Sema4A-associated GFP signals. Vertical bar indicates ± SEM. Figure S9. Exosomal release of prosaposin is dependent on binding to Sema4A Figure S9 is related to Figure 4, showing decreased exosomal release of prosaposin and procathepsin D in cells expressing Sema4A-EC or Sema4A-IC and no effect from mannose-6p receptor (mannose 6-PR) on binding between prosaposin and Sema4A. (A) Immunoblotting (Blot) shows reduced prosaposin and procathepsin D levels in exosomes from cells expressing Sema4A deletion constructs. (B) The indicated amounts of affinity-purified prosaposin were combined with Sema4A and mannose 6-PR and molecules bound to prosaposin were immunoprecipitated and immunoblotted. The amounts of Sema4A bound to prosaposin did not depend on the level of mannose 6-PR, which does not bind to prosaposin. Figure S10. Colocalization of CRALBP and CRBP1 with Rab11 Figure S10 is related to Figure 6, showing the binding of CRALBP to the Sema domain 7

8 of Sema4A, colocalization of CRALBP and CRBP1 with Rab11 based on immunoblotting of subcellular fractions and immunohistochemistry. (A) Immunoprecipitation (IP) and immunoblot (Blot) analyses showed an association between CRALBP and the Sema domain of Sema4A. (B) cells were incubated with biotinylated transferrin for 3 min followed by a 10-min chase period to label recycling endosomes. Subcellular fractions were isolated as described in Figure S4B. (C, D) Immunofluorescent images of cells. Higher magnification images of the boxes in the images on the left are shown on the right. Scale bars, 20 μm. CRALBP (green) and CRBP1 (green) colocalized with Rab11 (red). 8

9 Toyofuku et al., Fig. S1 A B Sema4A+/+ P5 P14 Sema4A+/+ P28 P28 Sema4A / P5 Sema4A / P14 P28 P28

10 Toyofuku et al., Fig. S2 A B C Control Fc Trans Sema4A Trans Sema4A+/+ Control Fc Sema4A+/+ Sema4A / INL Sema4A / DAPI INL Sema4A Fc GCL GCL Sema4A+/+ Sema4A /

11 Toyofuku et al., Fig. S3 A B C Trans Prosaposin Trans Sema4A FLAG Extracellular TM Intracellular region region Sema4A EC FLAG Prosaposin / INL DAPI Sema4A IC FLAG GCL Sema4A Sema FLAG FLAG Prosaposin Myc Sema4A FLAG Sema4A Sema FLAG + Sema4A PSI FLAG + + IP: Anti FLAG IP: Anti Myc Blot: Anti FLAG Anti Myc Anti FLAG Anti Myc Sema4A PSI FLAG Sema4A Fc Fc FLAG Sema domain PSI domain

12 3 d 10 d 21 d (P) / total thickness (T) (%) A minus T P ns ns Toyofuku et al., Fig. S4 S ema4a+/+ P 20 T 0 Sema4A+/+ Sema4A / Sema4A / receptor layer ( Days (d) Photo B P ns 30 P C ns ns TUNEL positive cells 50 Sema4A+/+ Light plus minus Sema4A / Sema4A Prosaposin Dark Light Dark Light Ig Fc 40 Fc FC Sema4A+/+ Sema4A / DAPI / TUNEL Sema4A / Sema4A+/+ DAPI / TUNEL Sema4A / Sema4A+/+ TUNEL positive cells P ns ns P IgG Fc Dark Light Dark Light Sema4A Fc Prosaposin Fc (+) ( ) ( )

13 A P14 B Light (60 min) INL 60 Toyofuku et al., Fig. S5 Prosaposin +/ Prosaposin / / Prosaposin +/ TUNEL INL Prosaposin +/ ls INL DAPI TUNEL positive cell GCL INL Prosaposin / DAPI TUNEL INL GCL INL 10 0 Light(60 min) Prosaposin /

14 Toyofuku et al., Fig. S6 A B C Cont Rab5 Prosapo osin Rab11 Sema4A Prosaposin 5 Sem ma4a / Rab C H2O2 3 min b Tfn Go olgi 3 min b Tfn & 10 min chase D Cont Std fraction % of total protein Merge Sema4 4A / Rab 7 D H2O2

15 Toyofuku et al., Fig. S7 Sema4A FLAG + Sema4A EC FLAG + Sema4A IC FLAG FIP2 myc + + Rab11 Xpress Blot: Anti FLAG IP: Anti FLAG Anti Myc Anti Xpress

16 Toyofuku et al., Fig. S8 A Upper one third region: Apical side Lower two thirds region: Baso lateral side B Apical Baso lateral Apical Baso lateral Apical Baso lateral Apical Baso lateral Ratio 0.5 Ratio 0.5 Ratio 0.5 Ratio

17 Toyofuku et al., Fig. S9 A Sema4A FLAG Sema4A EC FLAG Sema4A IC FLAG H 2O 2 Exosome Blot: Hrs B Prosaposin (ng/ml) Sema4A (ng/ml) Mannose 6PR (ng/ml) Blot: Prosaposin Tsg101 Input Sema4A Sema4A Mannose 6PR Prosaposin Procathepsin D IP: prosaposin Sema4A Mannose 6PR

18 Toyofuku et al., Fig. S10 A CRALBP V5 Sema4A FLAG Sema4A Sema FLAG Sema4A PSI FLAG Blot: IP: Anti FLAG Anti FLAG Anti V5 Rab11 C Cont IP: Anti V5 Anti FLAG Anti V5 CRALBP / B D Cont Rab11 CRALBP CRBP1 CRBP1 / Rab b11 3 min b Tfn & 10 min chase Std fraction % of total protein