Electron flux via Prx3 and Trx2 during hyperoxic injury.

<p>Schematic demonstrating mitochondrial electron flux via the Prx3 and Trx2 under control and hyperoxic conditions.</p

Hyperoxia does not alter mitochondrial mass.

<p>Ratio of mitochondrial:nuclear Ct values for (A) <i>D-Loop</i> and (B) <i>COX1</i> after normalization to <i>β2M</i> quantified by qPCR in A549 during hyperoxic culture. Data are expressed as mean ± standard deviation of 3 biological replicates analyzed by one-way ANOVA.</p

Mitochondrial oxidants promote hyperoxic cell death.

<p>MitoSOX was used to quantify mitochondrial oxidants in A549 cells cultured in hyperoxia. (A) MitoSOX red fluorescence intensity and (B) fold-change after 2 days of hyperoxic culture and during a hyperoxic time course. (C) Hyperoxic cell death measured via flow cytometry using TOPRO-3. (D) Fold-change in MitoSOX red fluorescence intensity of A549 cells after 2 days of hyperoxic culture with increased concentrations of MitoTEMPO supplemented in the media. (E) Death of A549 cells cultured concurrently with 3 days hyperoxia and MitoTEMPO supplementation. Data are expressed as mean ± standard deviation of 3 biological replicates analyzed by one-way ANOVA. Statistical significance was defined as *p<0.05, **p<0.01, and †p<0.001.</p

Protein expression of mitochondrial redoxins Prx3, Trx2, and TrxR2 during hyperoxia.

<p>Representative SDS-PAGE/immunoblots of cell lysates probed for Prx3, Trx2, and TrxR2 with β-actin as a loading control in (A) A549 and (B) H1299 human lung adenocarcinoma cell lines cultured for increasing days in hyperoxia. Images are representative of 3 independent biological replicates.</p

Trx2 and Prx3 oxidation during hyperoxia.

<p>(A,C) Differential Trx2 and Prx3 thiol labeling by AMS and NEM respectively, and (B,D) representative detection by SDS-PAGE/immunoblot in A549 cells after treatment with hyperoxia. Images are representative of 3–4 independent biological replicates.</p

Trx2 inhibition sensitizes cells to hyperoxic cell death.

<p>(A) Inhibition of TrxR activity in A549 cells cultured for 24 hours with 1 or 2.5 μM AFN. (B) Fold-change in MitoSOX red fluorescence intensity after 2 days hyperoxic culture and (C) TO-PRO-3 labeling after 3 days of hyperoxic culture with 1 or 2.5 μM AFN. (D) SDS-PAGE/immunoblot of A549 cell lysates for Trx2 and Prx3 protein expression 2 days following lentiviral delivery of non-targeting (NT) and Trx2- or Prx3-targeting shRNAs. (E) Fold-change MitoSOX red fluorescence intensity after 2 days of hyperoxia and (F) TO-PRO-3 labeling after 3 days hyperoxic culture following lentiviral transduction of NT, Trx2, or Prx3 shRNAs in A549 cells. Data are expressed as mean ± standard deviation of 3 biological replicates analyzed by one-way ANOVA. Statistical significance was defined as *p<0.05, **p<0.01, and †p<0.001 (n = 3).</p

Oxygen-dependent gene expression of Prx3, Trx2, and TrxR2.

<p>qPCR analysis of (A) Prx3, (B) Trx2, and (C) TrxR2 expression in A549 and H1299 cells during hyperoxic culture using GAPDH as a loading control. Data are expressed as mean ± standard deviation of 3–4 biological replicates analyzed by one-way ANOVA. Statistical significance was defined as *p<0.05, **p<0.01, and †p<0.001.</p

Trx2 inhibits hyperoxic phosphorylation of ASK1.

<p>Expression of pro-apoptotic proteins by SDS-PAGE/immunoblot in A549 cells during (A) hyperoxic culture. (B) Immunoblots of lysates from A549 cultured in hyperoxia for two days following lentiviral delivery of NT, Trx2, or Prx3 shRNAs. Images are representative of 3 independent biological replicates.</p