Fig. 1: PrPC represses cellular and endoplasmic ROS.
A Cellular free iron content in empty vector control (control OE) and PrPC OE HT-1080 cells measured by 5 nM calcein or 1 μM FerroOrange staining. Wild-type HT-1080 treated with 500 μM ferric ammonium citrate (FAC) and 100 μM deferoxamine (DFO) compared to DMSO as the positive and negative control, respectively, a typical FACS histogram is depicted. Insets show ferritin heavy chain (FTH, 21kD), ferritin light chain (FTL, 20kD) and PrPC levels by Western blot with normalization by beta-actin (ACTB, 42kD). B Relative PRNP mRNA expression levels change up to 21 days post-infection with a lentiviral expression vector-containing PrPC (OE) (encoded by PRNP) and a separate cistron for puromycin resistance, in 1 μg/mL puromycin-containing media. Total RNA was normalized to empty vector control (C). C Cytosolic ROS levels detected by 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) using flow cytometry in indicated HT-1080 cells at 7 dpi (days post-infection). A representative flow cytometry histogram of three independent repetitions is depicted (left panel). Boxplot shows (right panel) fluorescence intensity in control and PrPC OE cells after 0.1 μM RSL3 for 0 h and 2 h, with mean, max and min indicated. D Pearson correlation analysis of GPX8 mRNA expression with PRNP mRNA expression (R = 0.621; two-tailed P < 0.0001; 95% confidence interval 0.587 to 0.651) of 18,575 genes determined for 1393 individual cell lines. GAPDH is included as a reference gene. TPM, transcripts per million. E Subcellular colocalization of PrPC together with the endoplasmic reticulum-specific marker concanavalin A (10 μg/mL). Pictures shown are representative results of at least three independent repetitions performed with similar outcomes (scale bar = 20 µm). F Kinetic analysis of ER ROS using Hyper-3 ER fluorescent reporter (emission ratio 488/405) upon PrPC expression (PRNPtet +dox). Samples were measured by flow cytometry for 1 min to determine baseline intensity before supplementation with DTT (∆ 5 mM) or H2O2 (▲ 50 mM). -dox/+dox are samples treated with vehicle or doxycycline, respectively. DTT, dithiothreitol. G Glutathione peroxidase (GPX) activity in PrPC OE compared to empty vector control showing in bar graph. Insets show PrPC and GPX8 levels by Western in PrPC OE and control cells. H Fluorescence intensity of Hyper-3 ER in HT-1080 cells expressing GPX8 compared to empty vector control cells. Average fluorescence intensity values (emission ratio 488/405) were determined by flow cytometry. Insets show GPX8 level by Western. A, C FACS histograms of at least three independent experiments are depicted. Boxplot (C) is shown with whiskers min to max. Relative mRNA expression (B) is shown as mean ± SEM of n = 3 technical replicates. Boxplot data (C) is plotted as whiskers min to max, showing all points of n = 6 each replicates of duplicate repetitions of the experiment with similar results. Significance was determined by two-tailed t-test (C). Activity data (G) is plotted as representative mean ± SEM of n = 4 biological replicates for independent experiments. Intensity (H) was calculated versus control, shown as mean ± SEM of n = 3 biological replicates. P-values of two-tailed t-test (B, G, H) or ANOVA multiple comparisons with Tukey post-test are shown for comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.
