Fig 2: ARF1-dependent cargo loading into secretory vesicles is PAQR11 dependent.(A) Western blot analysis of whole cell lysate (WCL) and vesicle- and Golgi-fractionated H1299 cell lysates to confirm enrichment of fractionated lysates in organelle-specific markers. ER, endoplasmic reticulum. (B and C) Western blot analysis of FAM3C and PLAU in WCL and Golgi- and vesicle-enriched fractions of siRNA-transfected H1299 cells (B). Protein levels relative to siCTL controls quantified densitometrically (C). (D) FAM3C and PLAU expression constructs. (E) Confocal micrographs of H1299 cells transfected with expression constructs. Vesicles containing ectopic FAM3C and PLAU were detected using fluorescent tags (EGFP-FAM3C) or antibodies against Flag (PLAU). Golgi localization determined on the basis of colocalization with the trans-Golgi marker Golgin-97. Nuclei were counterstained with DAPI (blue). Scale bar, 8 µm. (F) Contrast-adjusted confocal micrographs of H1299 cells cotransfected with Flag-PLAU and indicated siRNAs and stained with anti-Flag antibodies. Arrows indicate Flag-PLAU+ vesicles. Dotted lines indicate cell boundary. Scale bar, 10 µm. Vesicle numbers per cell (dots) were quantified. (G) Schematic illustration of a working model in which p53 loss leads to PAQR11 up-regulation, initiating an ARF1-dependent secretory process that is amplified through an autocrine PLAUR-dependent signaling pathway. Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig 3: Lysosomal V-ATPase increases in response to mTORC1 inactivation.a Magnetic enrichment of lysosomes from MEFs, analysed by western blot. PNS: post nuclear supernatant; FT: column flow through; Elu: lysosomal eluate. Contaminating organelle markers are Golga1 (Golgi), Calreticulin (ER), Vdac (mitochondria), Pex19 (peroxisomes). b Enriched proteins in lysosomal fractions, quantified by label-free mass spectrometry. The dashed line (log2 fold change >2) demarcates the cut-off for lysosomal proteins. c Changes in lysosomal proteins after 1 h aa starvation + 30 min aa restimulation (+aa) versus 1 h aa starvation (-aa) in MEFs, quantified by label-free mass spectrometry. d V-ATPase subunits in membrane fractions of dextran-loaded lysosomes from MEFs after 1 h ± torin 1 [400 nM], or after 1 h amino acid starvation ± 30 min aa restimulation (±aa), analysed by western blot. e V-ATPase subunit abundance changes in membrane fractions, quantified by western blot for torin 1/control and +aa/-aa as in d. Data are normalized replicate mean ± SEM. Dashed lines indicate protein abundance in the control/-aa groups. a One representative of n = 5 biologically independent experiments. b, c n = 5, e n = 5 (V1A, V1E, Vod1 + torin 1, V1E + aa) or n = 9 (V1A, Vod1 +aa) biologically independent experiments. In b, c, adjusted p-values were calculated using limma moderated t-statistic and adjusted with the Benjamini–Hochberg method for multiple testing. In e, p-values were calculated using a one sample t-test with a hypothetical mean of 1. Source data are provided as a Source data file.

Fig 4: Internalization of exogenous fibulin-4 and its function in LOX activation, and a model for the role of fibulin-4 in LOX activation.(A) Western blot analysis of LOX secreted from WT and Fbln4-/- MEFs, with or without addition of recombinant fibulin-4 (rFBLN4) in the medium. (B to E) Internalization of FLAG-rFBLN4 by Fbln4-/- MEFs. Cell surface–bound FLAG-rFBLN4 after 4°C incubation (B) could be removed by acid wash (D). The cells were warmed at 37°C followed by acid wash, and internalized FLAG-rFBLN4 was visualized (E). (F) Quantification of internalized FLAG-rFBLN4 with or without Dynasore (100 µM) and Filipin (2.5 µg/ml) treatment. Bar graphs show mean ± SEM (n = 6 to 8). **P < 0.01 and ***P < 0.001 compared with dimethyl sulfoxide (DMSO). (G) Inhibition of rFBLN4-induced LTQ formation in LOX secreted from Fbln4-/- MEFs by Dynasore (100 µM). The ratio of the quantified intensity of lower and upper bands of LOX is shown. Bar graphs show mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with DMSO. (H) Immunofluorescent staining showing colocalization of internalized FLAG-rFBLN4 and Golgin-97, a TGN marker. Scale bars, 10 µm. (I) A proposed model for the role of fibulin-4 in LOX activation. ER, endoplasmic reticulum.

Fig 5: Intracellular localization of GnT-IVa and GnT-IVb. DKO cells transfected with a plasmid for expression of GnT-IVa or GnT-IVb were stained with anti-myc (green), antibodies for marker proteins (Golgin-97 for the Golgi, calnexin for the ER, or N-cadherin for the plasma membrane) (red), and DAPI (blue). The scale bar represents 10 µm. DAPI, 4',6-diamidino-2-phenylindole; DKO, double KO; ER, endoplasmic reticulum; GnT, N-acetylglucosaminyltransferase.