Fig 2: Impairment of mitochondrial reductase systems linked to glutathione and thioredoxin exacerbates LPS-induced PDCE2 glutathionylation, ROS production and cell death.(A) BioGEE incorporation assays as conducted in Fig. 1 were used to assess glutathionylation of PDCE2 in THP-1 promonocytes after 6 h treatment with 10 µM MitoCDNB, 1 µg/mL LPS, or a combination of the two (n=5). Using raw densitometry values, a significant increase in glutathionylated PDCE2 was seen with the combined treatments relative to either treatment alone (* p<0.05). (B) MitoSox Red was used to assess ROS in WT and PDCE2 KO THP-1 promonocytes. A significant decrease in mitochondrial ROS generation at 4.5 h (shown here) as well as other time points (also shown in Fig. S5) was seen with the combined treatments in PDCE2 CRISPR cells relative to their Wild Type Counterparts (n=14, * p<0.05, ** p<0.01, *** p<0.001). (C) Promega Multi-Tox Fluor assay was used to assess cell death in in WT and PDCE2 KO THP-1 cells. The increase in cell death observed with the combined treatments in WT THP-1 cells was significantly less in PDCE2 KO cells (n=12). (D) LDH release was assessed as an independent measure of cell death using Promega Homogenous Membrane Integrity assay. Again the LPS-induced increase in LDH release observed with the combined treatments for WT cells was greater that for PDCE2 KO cells (n=12). (A-D) All statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons post-test; * p<0.05, ** p<0.01, *** p<0.001.

Fig 3: PDCE2 knockout lowers LPS-induced ROS production and cell death.(A) Mitochondrial ROS production was assessed in LPS-treated wild type (WT) THP-1 cells in comparison with CRISPR-mediated PDCE2 knockout (KO) THP-1 cells using 5 µM MitoSox Red assessed 6 h after LPS or buffer addition. The strong ROS response to LPS exhibited by WT THP-1 cells was blunted in PDCE2 KO cells (n=8, conducted in triplicate on different days). (B) H2O2 release from cells with and without LPS stimulation was assessed in THP-1 WT and KO cells similar to (A) using an Amplex Red/HRP assay to detect extracellular H2O2. The PDCE2 KO cell samples exhibited lower values of extracellular H2O2 than WT cells whether or not they were treated with LPS, and there was no significant response to LPS by the KO cells, unlike WT THP-1 cells (n=12). (C) Cell death was assessed in WT and PDCE2 KO THP-1 cells using Promega Multi-Tox Fluor assay. A significant increase in Dead Cell/Live Cell fluorescence ratio was observed in response to 6 h LPS treatment of WT cells, but this response was dampened in PDCE2 KO cells (n=15). (D) LDH release as a measure of cell death was assessed in WT and PDCE2 KO THP-1 cells using Promega Homogenous Membrane Integrity assay. A significant increase in percent cytotoxicity was observed in response to 18 h LPS treatment of WT cells but not PDCE2 KO cells (n=14). (A-D) All statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons post-test; * p<0.05, ** p<0.01, *** p<0.001.

Fig 4: LPS and MitoCDNB synergize to generate enhanced levels of glutathionylated PDCE2, promoting THP-1 cell death through proinflammatory signaling through cytokines.

Fig 5: Mutation of PDCE2 Lys residues to prevent lipoylation dampens ROS production and cell death. (A) Mitochondrial ROS production was assessed in PDCE2 KO THP-1 cells transfected with Empty Vector or PDCE2 expression plasmids with and without LPS stimulation using MitoSox Red (n = 12, *p < 0.05, n. s. = not significant). (B) Cell death was assessed in transfected PDCE2 KO THP-1 cells as in (A), in this case using Promega Multi-Tox Fluor assay (n = 14). (A and B) Statistically-significant increases in mitochondrial ROS production and cell death observed upon treatment of WT-expressing cells with LPS were not observed in the cells expressing PDCE2 lacking the lipoyl-linking Lys residues (K132R/K259R). All statistical analysis was conducted using one-way ANOVA with Tukey's multiple comparisons post-test; *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)