Fig 2: Investigation of PDIA1 and RhoGDIα co-regulation in different systems. (A) RhoGDIα protein levels following PDIA1 overexpression in VSMC using a doxycyclin-inducible lentiviral vector carrying a myc-tag; (B) Effects of PDIA1 overexpression upon RhoGDIα levels in a transgenic mouse model. Tissue from transgenic PDIA1-overexpressing mice: brain, kidney, liver, aortae and heart. Data representative of n ≥ 3, 2 (heart) or 1 (brain). (C) RhoGDIα protein and mRNA levels in VSMC from transgenic PDIA1-overexpressing mice. Representative of 3 independent experiments. Uncropped western blots are shown in Supplementary Figs S8 and S9.

Fig 3: Viral inclusions are associated with ER exit sites. a Sec61β-Emerald cells were transfected with mCherry-NP and infected with PR8 virus, at an MOI of 10, for 16 h. b A549 cells were co-transfected with plasmids encoding mCherry-NP and ER-GFP and infected with PR8 virus, at an MOI of 10, for 16 h. a, b Cells were imaged under time-lapse conditions. Individual frames with single moving particles highlighted with yellow arrows are shown. Images were extracted from Supplementary Movies 17 and 18. More than 15 cells from 3 independent experiments were analyzed in each condition. c A549 cells were infected or mock-infected (M) with PR8 virus, at an MOI of 3, and fixed at the indicated times. Cells were stained for the ER proteins Sec31A (in green) and PDI (in gray) and the viral NP protein (in red). Areas highlighted by the white box are shown on the right of each panel. Yellow arrowheads indicate co-localization between Sec31A and NP. Bar = 10 μm. More than 30 cells from 2 independent experiments were analyzed. d A549 cells were co-transfected with plasmids encoding mCherry-NP and GFP-Sec16 and infected or mock-infected (M) with PR8 virus for 16 h. Cells were imaged under time-lapse conditions. Representative cells are shown in the left large images. Individual frames with single moving particles highlighted with yellow arrows are shown in the small panels. Two examples are provided for the infected cell (16 h.1 and 16 h.2). Bar = 7.5 μm. Images were extracted from Supplementary Movies 19 and 20. Images are representative of at least 15 cells, from 2 independent experiments

Fig 4: Ces1d localizes on both ER and lipid droplets in adipose tissue.(A) Co-immunofluorescence (Co-IF) staining with α-Ces1d (green) and α-Perilipin-1 (PLIN1, red) (lipid droplet marker protein) antibodies on the sWAT from WT mice fed by regular chow or HFD (representative of six fields, experiments were repeated for three times). Scale bars: 25 μm. (B) WB analysis of Ces1d in the ER fractions isolated from the liver and WAT from WT mice fed by regular chow or HFD. BIP was used as the loading control for the ER fraction (n = 3 per group, representative of three repeats). (B, C) Quantification of the band intensity for Ces1d in (B) (n = 3 per group, each point represents a biology replicate). Data are represented as mean ± SEM, t test, **P < 0.01. (D) WB analysis of Ces1d in different cell fractions, including whole cell lysates (WCL), cytoplasm (Cyto), ER, and lipid droplets of the liver and sWAT from the wild-type mice. ERK was used as the loading control for cytosolic proteins, whereas PDI and Rtn4a were used as the loading controls for the ER proteins. 0.5% of the total WCL, 1% of the Cyto, ER, and lipid droplets extracts were loaded (representative of three repeats). (D, E) Quantification of the band intensity for Ces1d in (D) (n = 4 per group, each point represents a biology replicate). Data are represented as mean ± SEM. (F) WB analysis of Ces1d in the lysates from the liver, sWAT, and BAT from WT mice. The samples were pretreated with de-glycosylation enzymes to remove both O- and N-glycans before the analysis.

Fig 5: Rac1 and regulation of PDIA1 – Nox1 axis.a Superoxide production after 72 h of PDIA1 silencing in HKE3 transfected with Rac1G12V, treated or not with 10 µM of NoxA1ds (Nox1 peptide inhibitor), measured by DHE oxidation detected by HPLC. **p < 0.01, ANOVA plus Tukey's multiple comparison test (n = 3). b Superoxide production after 72 h of PDIA1 silencing in HCT116 cells treated or not with 50 µM of W56 Rac1 peptide inhibitor. *p < 0.05, ANOVA plus Tukey's multiple comparison test (n = 4). c PDIA1 co-immunoprecipitation, IP: PDIA1 immunoprecipitation in Caco2, HKE3, and HCT116 cells, IgG: Immunoglobulin control, Input: 1% of total protein lysate, IB: immunoblot against PDIA1, KRas, Rac1, GAPDH, and tubulin (n = 2). EOH: 2-hidroxyethidiium relative superoxide production