Figure EV3. Effects of PDIA3^C57Y expression on actin cytoskeleton and neuritogenesis.

1. Mouse embryonic fibroblast (MEF) knock‐out for Pdia3 (Pdia3 ^KO) and wild‐type MEF (Pdia3 ^WT) were transfected with construct for expression of EGFP‐LifeAct. Representative micrographs of live cell imaging performed at 48 h after transfection are shown. Segmentation of time‐lapse images was used to obtain protruding (green) and retracting (red) areas. Zoom of representative cell areas is shown. Lamellipodia number and protrusion/retraction movement were quantified using Fiji software. Scale bar 50 μm. n = 4 independent experiments. A total of 9 movies per group with 1 or 2 cells per movie were quantified. Data are shown as mean ± s.e.m. and statistical analysis performed using two‐tailed Student's t‐test. Pdia3 ^KO MEF was checked by Western blot analysis. β‐actin was employed as loading control.
2. Pdia3 ^KO MEF was co‐transfected with constructs for expression of wild‐type PDIA3‐V5 or PDIA3^C57Y‐V5, or empty vector (Mock), and EGFP‐LifeAct. n = 3 independent experiments. A total of 10 movies per group with 1 or 2 cells per movie were quantified. Graphs show quantification of lamellipodia number and protruding/retracting velocity. Data are shown as mean ± s.e.m. and statistical analysis performed using one‐way ANOVA with Tukey's post hoc test. Wild‐type PDIA3‐V5 or PDIA3^C57Y‐V5 levels were checked by Western blot analysis. β‐actin was employed as loading control.
3. NSC‐34 neuronal cell lines stably expressing wild‐type PDIA3‐V5 or PDIA3^C57Y‐V5, or empty vector (Mock) were transfected with constructs for overexpression of α5‐integrin (α5‐int) fused to GFP, β2‐integrin (β2‐int) fused to YFP, β3‐integrin (β3‐int) fused to YFP, and β5‐integrin (β5‐int) fused to 2xGFP and analyzed by filter trap under non‐reducing (−DTT, dithiothreitol) or reducing (+DTT) conditions as described in Fig 5I. The graphs show quantification of aggregates of each integrin paralog relative to mock control detected under non‐reducing conditions. Lines connect aggregates quantified in the same experiment. n = 3 independent experiments.
4. Pdia3 ^KO MEF was co‐transfected with constructs for expression of wild‐type PDIA3‐V5 or PDIA3^C57Y‐V5, or empty vector (Mock), and β5‐integrin (β5‐int) fused to 2xGFP and analyzed by Western blot and filter‐trap under non‐reducing (−DTT, dithiothreitol) or reducing (+DTT) conditions. 1, Mock, 2, PDIA3‐V5, and 3, PDIA3^C57Y‐V5. Representative image of five independent experiments. The graph shows quantification of integrin aggregates relative to mock control detected under non‐reducing conditions. Lines connect aggregates quantification in the same experiment.

Source data are available online for this figure.