Fig. 4: Restoration of PDIA1 to shPDIA1 EVs rescues their ability to increase cellular stress and anchorage-independent growth.

A Western blot showing PDIA1 levels in TCCSUP EVs following extrusion with recombinant PDIA1. CD9 was used as an EV marker and normalization control for EV proteins. B ROS level in SV-HUC cells following treatment with EVs derived from scramble TCCSUP control cells, or from shPDIA1 TCCSUP EVs extruded with rPDIA1 or PBS control, as analyzed via DCFDA and flow cytometry assays. Data represent the fold change in the DCFDA histogram geometric mean relative to PBS control. C NFE2L2 gene expression in SV-HUC cells treated with EVs derived from scramble TCCSUP control cells, or from shPDIA1 TCCSUP EVs extruded with rPDIA1 or PBS control. D Representative images and quantification of DNA damage in SV-HUC cells following an 18-hour treatment with EVs derived from scramble TCCSUP control cells or from shPDIA1 TCCSUP EVs extruded with rPDIA1 or PBS control, as assessed by γH2AX immunofluorescence staining. Scale bars: 10 µm. E Representative images and quantification of the number of colonies (top right panel), percent colony area normalized to that in PBS-treated control (bottom left panel), and the product of percent colony area and corresponding intensitites normalized to that in PBS-treated control (bottom right panel) in soft agar formed by SV-HUC cells following a 13-week treatment with EVs derived from scramble TCCSUP control cells, or from shPDIA1 TCCSUP EVs extruded with rPDIA1 or PBS control. The number of colonies was obtained with the Colony Counter plugin of ImageJ whereas the percent colony area and their intensities were derived using the ColonyArea plugin of ImageJ. F Representative images and quantification of the colonies in soft agar formed by transformed SV-HUC established by long-term cancer EV stimulation (parental), and by transformed SV-HUC transduced with scramble control (scramble) and PDIA1-targeting lentiviral shRNA (shPDIA1). For (B–E), one-way ANOVAs were performed with Fisher’s LSD multiple comparison test; n = 4, 3, 5, and 7, respectively; error bars indicate means ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EV extracellular vesicle, PDIA1 protein disulfide isomerase A1, shPDIA1 short hairpin RNA targeting PDIA1, rPDIA1 recombinant PDIA1, NFE2L2 NF-E2-Related Factor 2, DCFDA 2’,7’-dichlorofluorescein diacetate, PBS phosphate-buffered saline, ANOVA analysis of variance, γH2AX γ histone 2a family member X, CD9 cluster of differnetiation 9.