Fig 2: APEX2 proteomic labeling scheme and characterization of APEX2 fusion constructs on the ER and outer mitochondrial membranes.(A) Scheme of APEX2-catalyzed biotinylation at the endoplasmic reticulum membrane (ERM, top) and outer mitochondrial membrane (OMM, bottom). APEX2 (green) was targeted to the ERM facing the cytosol, via fusion to the N-terminal 27 amino acids of the ER-resident protein P450 oxidase 2C1 and to the OMM via fusion to the C-terminal 31 amino acids of mitochondrial antiviral-signaling protein MAVS. Live cells were pre-incubated with biotin-phenol (red B = biotin) for 30 min, and hydrogen peroxide (H2O2) was added to initiate the biotinylation reaction for 1 min. Cells were quenched and then lysed, and the biotinylated proteins were enriched with streptavidin beads and analyzed by mass spectrometry. IMS, intermembrane space. IMM, inner mitochondrial membrane. (B) Confocal fluorescence imaging of ERM-APEX2 and APEX2-OMM fusion constructs. Constructs were introduced into COS-7 cells using lentivirus. Two days after transduction, the cells were fixed and stained with anti-V5 to detect ERM-APEX2 (Alexa Fluor 568 (AF568) readout), or anti-Flag to detect APEX2-OMM (AF568 readout). Anti-RCN2 was used to visualize the ER, or anti-Tom20 to visualize mitochondria (AF488 readout). Fluorescence channels are not normalized. Scale bars, 5 µm. The insets show a zoomed in portion of the cell. DIC, differential interference contrast. (C) Electron microscopy (EM) characterization of ERM-APEX2 and APEX2-OMM localization. ERM-APEX2 and APEX2-OMM genes were introduced into human embryonic kidney (HEK) 293T cells using lentivirus. Cells were fixed and overlaid with a solution of diaminobenzidine and H2O2. APEX2 catalyzes the localized polymerization of diaminobenzidine, which then reacts with osmium to provide contrast for EM (Lam et al., 2015; Martell et al., 2012). Dark regions indicate APEX2 activity. ER indicated by arrowheads. M, mitochondria. Untransduced HEK cell shown for comparison. Scale bars, 500 nm. The untransduced and APEX2-OMM images in this figure overlap with data shown in Supplementary Figure 7 of Lam et al. (2015). (D) Analysis of ERM-APEX2 and APEX2-OMM-catalyzed biotinylation by streptavidin blot. HEK 293T cells stably expressing ERM-APEX2, APEX2-OMM, APEX2-NES (NES = nuclear export sequence), or no APEX2 construct (lane 10) were labeled live as in (A). Whole cell lysates were separated by gel electrophoresis and blotted with streptavidin-horseradish peroxidase (streptavidin-HRP) on the left, or stained with Ponceau on the right. BP, biotin-phenol. The red bracket indicates a molecular weight range where the banding patterns produced by these APEX2 constructs differ notably. (E) Analysis of streptavidin-enriched biotinylated proteomes by silver stain. ERM-APEX2, APEX2-OMM, and APEX2-NES stable cells were labeled live as in (A), lysed, and incubated with streptavidin beads to enrich for biotinylated proteins. Eluted proteins were separated by gel electrophoresis and visualized by silver stain.

Fig 3: Serum RCN2 levels of patients with coronary artery disease (CAD) or peripheral artery disease (PAD) compared with healthy individuals. Results are means ± SD of 20 to 34 subjects per group. * p < 0.05 vs. healthy individuals. + p < 0.05 vs. CAD. One-way ANOVA was performed to determine statistical significance in RCN2 levels among the groups.

Fig 4: The complex of PIGX, RCN1, and RCN2 regulates gene expression. (A) Heatmap of upregulated genes by PIGX, RCN1, or RCN2 knockdown in common. Gene expression profiles were analyzed by cDNA microarray using Affymetrix HG-U133 Plus 2.0 Array. EHD2 and ZIC1, putative tumor suppressor genes are highlighted in blue. (B and C) Upregulation of EHD2 and ZIC1 was validated by qRT-PCR. Results are the mean ± SD of three independent experiments and P-values were calculated with Student’s t-test (*P<0.05).

Fig 5: PIGX and RCN proteins interact with each other in ER. (A) Immunocytochemical analysis of HeLa cells transfected with PIGX expression vector. Signal of exogenous PIGX was merged with that of ER marker, KDEL. Scale bar, 20 µm. (B and C) Immunocytochemical analysis of HeLa cells for RCN1 (B) or RCN2 (C). Endogenous RCN1 or RCN2 was detected and merged with ER marker. Scale bar, 20 µm. (D and E) Co-immunoprecipitation analysis. GST-FLAG tagged PIGX was co-overexpressed with HA-tagged RCN1 or RCN2 and pulled down using GST-tag. (F and G) Cell growth of T-47D with knockdown of RCN1 (F) and RCN2 (G) was examined by MTT assay. The knockdown effect was validated by qRT-PCR. Results are the mean ± SD of three independent experiments and P-values were calculated with Student’s t-test (*P<0.05).