PMC

Metabolic pathways of redox signaling by released PRDX2 and TRX1.

PRDX2 in supernatants from LPS-treated cells is glutathionylated. The sample was immunoprecipitated with anti-PRDX2. Lane 1, immunoprecipitate; lane 2, total supernatants before immunoprecipitation. (Left) Western blot with anti-GSH. (Right) Western blot with anti-PRDX2.

LPS induces the release of TRX1 by PBMCs. Cells were stimulated with 100 ng/mL LPS for 24 h under physiological (5%; Left) or atmospheric (20%; Right) oxygen levels. TRX1 was measured by ELISA in triplicate PBMC samples from three donors. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective control (without LPS), Student t test.

Glutathionylated proteins are released by LPS-stimulated RAW264 cells. Supernatants from BioGEE-preloaded cells exposed to 100 ng/mL LPS for 24 h were analyzed by Western blot analysis with streptavidin peroxidase. Left lane, nonreduced sample; right lane, sample reduced with 10 mM DTT before loading on the gel.

hPRDX2 induces TNF-α production. (A) hrPRDX2 was added at 10 µg/mL to RAW264 cells, and TNF-α production was measured 24 h later by ELISA in triplicate samples. Solid bars represent PRDX2 preincubated with polymixin B, as described in the text; open bars represent PRDX2 preincubated without polymyxin B. (B) TNF-α induction by hrPRDX2 in RAW cells pretreated with 100 U/mL IFN-γ or not pretreated. *P < 0.01 vs. control (A) or vs. PRDX2 alone (B). (C) hrPRDX2 was added to human primary macrophage cultures from four donors, and TNF-α production was measured by ELISA 24 h later in triplicate.

LPS induces PRDX2 release. (A) Western blot of supernatants from RAW264.7 cells treated for 24 h with and without 100 ng/mL LPS. PRDX2 was measured in triplicate samples each derived from an independent well. (Upper) Western blot with anti-PRDX2. (Lower) Ponceau red staining. (B) Intracellular PRDX2 in the cell lysates of the experiment in A. (Upper) Western blot with anti-PRDX2. (Lower) Western blot with anti-GAPDH. (C) PRDX2 in supernatant from RAW264 cells treated for 24 h with different concentrations of LPS. (D) PRDX2 in the corresponding lysates. Percentages of viable cells, as mean ± SD of quadruplicate samples, were 98 ± 2% with 0.01 ng/mL LPS, 95 ± 7% with 0.1 ng/mL LPS, 92 ± 4% with 1 ng/mL LPS, 91 ± 3% with 10 ng/mL LPS, and 90 ± 2% with 100 ng/mL LPS. (E) Time course of PRDX2 release in supernatants from cells treated with 100 ng/mL LPS for 8, 16, or 24 h.

PRDX2 release in different experimental conditions. (A) PRDX2 mRNA in RAW264 cells at 4 h or 24 h after stimulation with 100 ng/mL LPS. PRDX2 mRNA expressed in arbitrary units (normalized to GAPDH as described in ), representing the fold change relative to one of the control samples at 4 h. Results are the mean ± SD of triplicate samples analyzed in duplicate. (B) Release of PRDX2 by human macrophages (Left) or PBMCs stimulated with 100 ng/mL LPS for 24 h. PRDX2 was measured by ELISA in triplicate samples of macrophages from four donors or PBMCs from five donors. Mean ± SD values for macrophages were control, 3.22 ± 1.92 ng/mL and LPS, 4.97 ± 2.69 ng/mL. Mean ± SD values for PBMCs were control, 2.90 ± 2.54 ng/mL and LPS, 8.10 ± 6.31 ng/mL. Increases were statistically significant for both macrophages and PBMCs (P < 0.05, t test for paired data). (C) Release of PRDX2 by PBMCs at physiological oxygen concentration. PBMCs were stimulated with 100 ng/mL LPS for 24 h under physiological (5%; Left) or atmospheric (20%; Right) oxygen levels. PRDX2 was measured by ELISA in triplicate PBMC samples from three donors. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective control (without LPS), Student t test. (D) PRDX2 released by HEK 293T cells stimulated with 50 ng/mL TNF-α for 24 h. Each lane represents an independent biological sample.