Fig 2: Validation of markers identified in our proteomics and transcriptomics screen in mouse and human models of ependymoma.a Western blots and quantification of proteins levels comparing control and nlsYAP5SA animals (control = NEX+/+ YAP1nlsYAP5SA/+; nlsYAP5SA = NEXCre/+ YAP1nlsYAP5SA/+) (n = 3 mice, each). Significant increase in YAP1 (p = 0.0042), Vimentin (p = 0.0017), GFAP (p < 0.0001) and HOPX (p = 0.0002) are observed in nlsYAP5SA (two-tailed unpaired t tests). No significant difference in protein level was observed for TAZ nor CTGF. Error bars = SEM. b Immunofluorescence stainings for selected YAP1 downstream genes in LATS1/2 cKO tumours at P20. Strong staining of ANKRD1, AMOTL2 and AXL are observed in the tumour. ANKRD1 was uniformly expressed in throughout all tumour cells, AMOTL2 and AXL exhibited higher levels in tumour near its border. Scale bar = 100 µm. c Immunofluorescence of HOPX and YAP1 in a LATS1/2 cKO tumour at P20, showing higher staining at tumour edge, HOPX co-localises with YAP1 in this area (white shows colocalisation, Scale bar = 100 µm). d A subset of HOPX expressing cells have clear, nuclear YAP1 staining, example cell shown from a different LATS1/2 cKO tumour (Scale bar = 10 µm). e Two YAP1-MAMLD1-fused ependymoma characterised by uniform cells forming pseudo-rosettes in a fibrillary background (H&E, scale bar = 100 µm (Patient 1), 200 µm (Patient 2)); tumour cells show YAP1 nuclear expression (immunoperoxidase, scale bar = 50 µm); there is cytoplasmic and nuclear expression of HOPX (immunoperoxidase, scale bar = 100 µm), whereas p65 is negative in tumour cells (immunoperoxidase, scale bar = 100 µm).

Fig 3: Sec22b and Ykt6 play key roles in autophagosome retrograde trafficking in neurons exposed to I/R. A, Western blot analysis of Sec22b in penumbra tissue of male and female mice 6 h after MCAO/R. B, The relative protein level of Sec22b was quantified. Data are presented as the mean ± SD. ***p < 0.0001 for sham versus MCAO/R in males; ###p < 0.0001 for sham versus MCAO/R in females (F(1,20) = 0.2836, p = 0.6002, one-way ANOVA). N = 6 mice per group. C, Immunofluorescence analysis was performed in the penumbra tissue of mice at 6 h after MCAO/R. Sec22b antibody (red), NeuN antibody (white) and GFAP antibody (green) were used, and nuclei were fluorescently labeled with DAPI (blue). Scale bar: 50 µm. D, Double immunofluorescence analysis was performed with antibodies against Sec22b (green) and LC3 (red) in neurons at 6 h after OGD/R. Nuclei were fluorescently labeled with DAPI (blue). Representative images of reproducible triplicate experiments. Arrows indicate the colocalization of Sec22b and LC3 in neurites. Scale bar: 20 µm. E, Western blot analysis of Ykt6 in penumbra tissue of male and female mice 6 h after MCAO/R. F, The relative protein level of Ykt6 was quantified. Data are presented as the mean ± SD. *p = 0.0165 for sham versus MCAO/R in males; ##p = 0.0039 for sham versus MCAO/R in females (F(1,20) = 0.06,209, p = 0.8058, one-way ANOVA). N = 6 mice per group. G, Western blot assay of Ykt6 in cultured neurons 6 h after OGD/R. H, The relative protein level of Ykt6 was quantified. Data are presented as the mean ± SD. **p = 0.0073 for Nor versus OGD/R group by unpaired t test. N = 3 independent replicates. I, Immunofluorescence analysis was performed with an antibody against LC3 (red) in neurons overexpressing GFP-Ykt6 at 6 h after OGD/R. Nuclei were fluorescently labeled with DAPI (blue). Representative images of reproducible triplicate experiments. Arrows indicate the colocalization of GFP-Ykt6 and LC3 in the proximal neurite. Scale bar: 20 µm. J, Western blotting was performed to detect the response of Sec22b knock-down and Ykt6 overexpression to baf A1 in cultured neurons under non-OGD conditions. Rapamycin treatment was used as a positive control. K, The LC3-II/I ratio was calculated. Data are presented as the mean ± SD. **p = 0.0062 for baf A1– versus baf+ in the Rapa group (F(1,20) = 13.97, p = 0.0013, two-way ANOVA). N = 3 independent replicates. L, Western blot analysis of LC3 in neurons exposed to the indicated treatments 6 h after OGD/R. M, The LC3-II/LC3-I ratio was calculated. **p = 0.0039 for baf A1– versus baf A1+ in Sec22b-si group; Data are presented as the mean ± SD. **p = 0.0054 for baf A1– versus baf A1+ in Ykt6-OE group (F(1,12) = 23.73, p = 0.0004, two-way ANOVA). N = 3 independent replicates. Original images of Western blottings for this figure are shown in Extended Data Figure 3-1.

Fig 4: GC-C mRNA is localised in peri-ischaemic cortical astrocytes but not in the contralateral hemisphere of wild-type (WT) mice 48 h after MCAO. RNAScope® assay with a GC-C probe was performed along with immunohistochemical staining with a GFAP antibody in both ipsilateral and contralateral hemispheres. GC-C mRNA was expressed in peri-ischaemic astrocytes (Merged3, white arrows), whereas GC-C was not present in astrocytes of the contralateral hemisphere (Merged2, white arrows). Merged1: merged images of GC-C, GFAP and DAPI staining; bar represents 50 µm. Merged2 and Merged3: the enlarged images of astrocytes in the contralateral cortex and a peri-ischaemic area, respectively, stained with DAPI, GC-C and GFAP; bar represents 20 µm. DAPI, 4',6-diamidino-2-phenylindole; GC-C, guanylate cyclase C; GFAP, glial fibrillary acidic protein

Fig 5: vesicular localization of BDNFpro.a z-stack reconstruction shows astrocytes labeled by GFP. Cortical slices from control mice injected with AAV-GFAP-GFP virus were fixed 10 min after TBS and processed for immunostaining and confocal analysis. Scale bar: 10 µm. Magnification of a ROI shows one GFP-astrocyte delimited by an approximate territory (white dashed). Scale bar: 10 µm. BDNFpro/GFP and Vamp2/GFP co-localizations signals are shown. Magnification shows representative areas (dashed squares 1 to 4) in which BDNFpro/GFP and Vamp2/GFP signals overlap. Scale bars: 1 µm. b 3D-SIM image of a GFP-labeled astrocyte in a TBS-slice from control mice. Scale bar: 10 µm. Magnification of a ROI shows BDNFpro/Vamp2 colocalization signal. Scale bar: 500 nm. Magnification shows BDNFpro/Vamp2 colocalization signal in fine membrane extensions of the cell periphery (dashed squares 1 to 4). Scale bars: 50 nm. c EM image depicts BDNFpro-gold at astrocytic microdomains (light blue) surrounding an axon bouton. Scale bar: 100 nm. Magnification of the ROI shows gold particles (red arrowheads) in vesicular-like structures. Scale bar: 20 nm. d Digital reconstruction of the image in (c). Astrocytic vesicles (black boundary) are shown.