Fig 2: 6-Bio enhances MTOR-dependent autophagy and confers neuroprotection in a mouse MPTP toxicity model. (A) Representative western blots indicating dose-dependent modulation of autophagy related proteins (LC3B, RPS6KB1and EIF4EBP1) by 6-Bio in HeLa cells. TUBB was used as a loading control. (B) to (D) Representative photomicrographs of TH+ immunostained DAergic neurons (B) in SNpc (arrow) of mouse midbrain. Placebo control cohort, MPTP-treated (23.4 mg/kg body weight), 6-Bio (5 mg/kg body weight) or both [Prophylaxis (MPTP+Pro)/Coadministration (MPTP+Co)], scale bar: 300 µm. Stereological (C) and densitometric quantification (D) indicating the number of TH+ DAergic neurons and their intensity in SNpc neurons. Statistical analysis was performed using the Student unpaired t test, one-way ANOVA and the post-hoc Bonferroni test. Scale bar: 50 µm. Error bars, mean ± SEM ns-nonsignificant, **-P < 0.01, ***-P < 0.001.

Fig 3: UPP1, UPP2 and MITF expression across the CCLE collection and in melanoma.(a) UPP1, UPP2 and MITF expression across the complete CCLE collection (n = 30 lineages). (b) Protein immunoblot of a panel of melanoma (n = 9) and non-melanoma (n = 3, 293T, K562 and HeLa) cell lines grown and showing expression of UPP1 as well as MITF, TYR and MLANA, three melanoma markers. MDA: MDA-MB-435S. (c) Top: Immunoblot of UACC-257 melanoma cells with wild-type (UPP1WT) and knock-out (UPP1KO). Bottom: Immunoblot of MDA-MB-435S melanoma cells in wild-type (UPP1WT), knock-out (UPP1KO) and hypomorphic (UPP1hypo) clones (see methods). (d) Cell growth assay of MDA-MB-435S clones in sugar-free media containing dialyzed FBS complemented with 10 mM of either glucose or uridine and dialyzed FBS. Negative doublings indicate cell death. Data are shown as mean ± SEM with two-sided t-test relative to UPP1WT cells in the same media (n = 3 replicate wells, P < 2.9 × 10-6, P < 1.3 × 10-5, P < 3.1 × 10-7). (e) Cell growth assay of three melanoma cell lines with high UPP1 expression in sugar-free media complemented with 10 mM of glucose or 0.5 mg/mL of RNA. Data are shown as mean ± SEM with two-sided t-test relative to UPP1WT and were not corrected for multiple comparison (n = 3 replicate wells, P < 4.5 × 10-3, P < 1.3 × 10-4, P < 1.5 × 10-3. TUBB and Actin loading controls were performed on the same gels. All growth assays and screens included 4mM L-glutamine and 10% dialyzed FBS. Source data

Fig 4: Gpc4 KO Mice Have Increased Overlap of NP1 with Presynaptic Terminals in the Developing Visual System In Vivo(A–D) Increased colocalization between NP1 and VGlut2 in Gpc4 KO mice in the SC at P6 suggests reduced presynaptic release of NP1 in the absence of Gpc4. (A) Example images from the SC of WT (top) and Gpc4 KO (bottom), VGlut2 in cyan and NP1 in red. (B–D) Quantification of VGlut2, NP1, and colocalized puncta, respectively, normalized to WT.(E–H) Increased colocalization between NP1 and VGlut2 in Gpc4 KO mice in the VC at P6. (E) Example images from the VC of WT (top) and Gpc4 KO (bottom), VGlut2 in cyan and NP1 in red. (F–H) Quantification of VGlut2, NP1, and colocalized puncta, respectively, normalized to WT. In (A) and (E), arrowheads mark representative colocalized puncta. Scale bar, 5 µm.(I) Triple fluorescent ISH for Gpc4- and cell-specific markers in the SC of astrocyte specific Gpc4 KO. Gpc4 (red) colocalizes with Aldh1l1 (green) and rarely with Tubb3 (blue) in the SC at P6 in Gpc4f/y;cre -ve (top), decrease of Gpc4 signal in Gpc4f/y;Aldh1l1cre +ve (bottom). Left panels merge plus DAPI (inset shows zoom in of boxed region), followed by merge minus DAPI, then single-channel panels as labeled: Gpc4, Aldh1l1, and Tubb3. Arrowheads mark example cells positive for Gpc4 and Aldh1l1. Scale bar, 50 µm, zoomed-in box scale bar, 5 µm.(J–M) Increased colocalization between NP1 and VGlut2 in Gpc4f/y;Aldh1l1cre +ve mice in SC at P6, suggests reduced presynaptic release of NP1 in the absence of astrocyte Gpc4. (J) Example images from the SC of Gpc4f/y;cre -ve (top) and Gpc4f/y;cre +ve (bottom), VGlut2 in cyan and NP1 in red. Arrowheads mark representative colocalized puncta. Scale bar, 5 µm. (K–M) Quantification of VGlut2, NP1, and colocalized puncta, respectively, normalized to Gpc4f/y;cre -ve. Graphs show mean ± SEM, number of mice inside the bar. Statistical analysis by t test, p value on graph.See also Figures S5 and S6.

Fig 5: Gpc4 Is Expressed by Astrocytes throughout the Developing Visual System(A and B) In situ hybridization (ISH) in the developing visual system at P6 shows presence of astrocytes and expression of Gpc4 mRNA. (A) Left: ISH for astrocyte marker Aldh1l1 in the SC at P6. Right: negative control probe, showing absence of signal. (B) ISH for Gpc4 in the visual system at P6 in Gpc4 WT (left) and KO (right)—SC (top), dLGN (middle), and VC (bottom). Gpc4 is expressed in all regions, and there is no signal in the Gpc4 KO. In each set, left, DAPI to mark cell nuclei; middle, low-power image of mRNA probe; and right high-power image of region outlined by red box in low-power image, arrowheads mark positive cells. Note the different image size for VC versus SC and dLGN. Scale bars for (A) and (B), 50 µm.(C–F) Triple fluorescent ISH for Gpc4 and cell-specific markers in the SC (C and D) and VC (E and F) at P6 shows colocalization of Gpc4 with Aldh1l1 and limited overlap with the neuronal marker Tubb3. (C) Gpc4 mRNA (red) colocalizes with Aldh1l1 mRNA (green) and rarely with Tubb3 (blue) in the superficial layers of the SC at P6 in WT (top), absence of Gpc4 signal in Gpc4 KO (bottom). (D) Quantification of (C), fraction of Gpc4+Aldh1l1-positive cells and Gpc4+Tubb3-positive cells. n = 3 mice. (E) Gpc4 (red) mainly colocalizes with Aldh1l1 (green) with limited overlap with Tubb3 (blue) in layer 4 of VC at P6 in WT (top); absence of Gpc4 signal in Gpc4 KO (bottom). In (C) and (E), left panels show merge plus DAPI (inset shows zoom in of boxed region), followed by merge minus DAPI, then single-channel panels as labeled: Gpc4, Aldh1l1, and Tubb3. Arrowheads mark example cells positive for Gpc4 and Aldh1l1. (F) Quantification of (E), fraction of Gpc4+Aldh1l1-positive cells and Gpc4+Tubb3-positive cells. n = 3 mice. Scale bars for (C) and (E), 50 µm, zoomed-in box scale bar, 5 µm. Graphs show mean ± SEM, statistical analysis by t test, p value on graph.See also Figure S4.