Fig. 5: Prion-induced ferroptosis is facilitated by RAC3-driven mesenchymalization.
A Survival of HT-1080 cells treated with RAC-inhibitor EHop-016 (2 μM) and ferroptosis inducer (IKE) rescued by 10 μM α-tocopherol (aToc). B Viability bar graph of RAC3 expressing cells (RAC3 OE) compared to empty vector control with ferroptosis inducer 1.25 μM IKE and 10 μM αToc as a ferroptosis inhibitor. A Western indicates increased expression of RAC3 protein. C Cell survival of PrPC OE, RAC3 OE, and RAC3+PrPc co-expressing cells was detected in freshly infected cell. Cells were treated subsequently with either 2 μM Ferrostatin-1 or DMSO for 3 days. D A flow cytometry histogram depicts the effect of RAC3 OE on induced ROS level via 0.3 μM RSL3 treatment in HT-1080 cells for 3 h measured by DCFH-DA after the indicated time. E Principal component analysis of all metabolomic and lipidomic mass spectrometry features for RAC3 OE cells and cells treated with IKE, compared to control cells. (PC, principal component). F Bar graph illustrates differential expression of 210 annotated phospholipids and lysophospholipids with varying total numbers of double bonds in RAC3 OE cells compared to control cells with an empty vector. Data are representative means ± SD of n = 5 technical replicates. G Differential expression of 210 annotated phospholipids and lysophospholipids with varying total numbers of double bonds in IKE-treated cells compared to vehicle-treated controls. Data are representative means ± SD of n = 5 technical replicates. H Brightfield images of control, RAC3 OE, TGFbeta-1 treated, and RAC3+PrPc OE co-infected cells indicating various levels of roundness (scale bar = 20 µm). I Aspect ratio depicting the effects of TGFbeta-1 treatment, RAC3 OE cells, and RAC3+PrPc co-expressing HT-1080 cells (mean shape, length/width ratio). J Relative expression of epithelial and mesenchymal markers in RAC3 OE and TGFbeta-1 treated cells (3 days) compared to empty vector or DMSO-treated control detected by qPCR. Relative mRNA expression is shown as mean ± SEM of n = 3 technical replicates. Viability data (A–C) are representative means ± SEM of n = 3 biological replicates for experiments repeated independently at least three times. The aspect ratio (I) are representative means of n = 12 replicates measured by high content microscopy. Significance was determined by two-way ANOVA multiple comparisons with Tukey post-test (A–C, I) or two-tailed t-test (J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.
