Results of markers of kidney injury in the three groups.

<p>(A) serum creatinine; (B) serum BUN; (C) serum CysC; (D) urine NAG, urine N-acetyl-β-D-glucosaminidase. Data are presented as mean±SD, n = 6, *<i>P</i> <0.05 versus Group C, Ψ<i>P</i> <0.05 versus Group S. For graphs pooled estimates for pairwise comparisons derived from Analysis of Covariance with adjustment for baseline serum creatinine at 53.61±9.53μmmol/L, serum BUN 4.31±1.34μmmol/L, serum CysC 24.35±4.2μg/L, were as follows: <b>serum creatinine;</b> 5min post CPB(T2): Group C, 64.24±12.53μmmol/L, Group U, 62.33±11.73μmmol/L. Test for overall treatment effect <i>P</i> = 0.074. 120min post CPB(T3): Group C, 86.62±11.41μmmol/L, Group U, 71.72±12.55μmmol/L. Test for overall treatment effect <i>P</i> = 0.032. <b>serum BUN;</b> 5min post CPB(T2): Group C, 5.43±1.87μmmol/L, Group U, 5.41±1.72μmmol/L. Test for overall treatment effect <i>P</i> = 0.081. 120min post CPB(T3): Group C, 7.37±1.72μmmol/L, Group U, 6.13±1.69μmmol/L. Test for overall treatment effect <i>P</i> = 0.025. <b>serum CysC;</b> 5min post CPB(T2): Group C, 30.47±4.4μg/L, Group U, 26.66±5.7μg/L. Test for overall treatment effect <i>P</i> = 0.069. 120min post CPB(T3): Group C, 40.62±6.5μg/L, Group U, 30.72±6.2μg/L. Test for overall treatment effect <i>P</i> = 0.001.</p

Results of plasma markers of inflammation in the three groups.

<p>(A) serum IL-6, interleukin-6; (B) serum TNF-α, tumor necrosis factor-α. Data are presented as mean±SD, n = 6, *<i>P</i> <0.05 versus Group C. For graphs pooled estimates for pairwise comparisons derived from Analysis of Covariance with adjustment for baseline serum IL-6 at 0.94±0.13μg/L, serum TNF-α 0.32±0.05ng/L, were as follows: <b>IL-6;</b> 5min post CPB(T2): Group C, 1.37±0.16μg/L, Group U, 1.01±0.13μg/L. Test for overall treatment effect <i>p</i> = 0.021. 120min post CPB(T3): Group C, 1.38±0.28μg/L, Group U, 1.13±0.24μg/L. Test for overall treatment effect <i>P</i> = 0.001. <b>TNF-α</b>; 5min post CPB(T2): Group C, 0.37±0.19 ng/L, Group U, 0.33±0.19ng/L. Test for overall treatment effect <i>P</i> = 0.075. 120min post CPB(T3): Group C, 0.35±0.13 ng/L, Group U, 0.32±0.13 ng/L. Test for overall treatment effect <i>P</i> = 0.088.</p

Typical histological examination results in the three groups.

<p>A, Group S, Tubules and glomeruli appear normal (H&E×400); B, Group C, after 2h CPB, kidney histologic changes include tubular dilatation(*), vacuole formation(▲), and glomerular over-filling (arrow) appeared obvious (H&E×400); C, Group U, injury changes of kidney still exist as tubular dilatation(*), vacuole formation(▲), and glomerular over-filling (<i>arrow</i>) but were milder with intervention of ulinastatin(H&E×400).</p

Effects of TRIM38 knockdown on TLR3-induced IFN-β activation.

<p>(A) TRIM38 protein level in 293/TLR3 cell line stably expressing TRIM38-specific or non-targeting shRNA. 293/TLR3 cells were transfected with TRIM38-specific shRNA or non-targeting shRNA (NT) followed by puromycin selection and cloning. Screened cell populations were analyzed by immunoblot with an antibody specific for TRIM38. (B) Effects of TRIM38 knockdown on poly(I:C)-induced IFN-β activation. TRIM38 knockdown or control 293/TLR3 cells were transfected with IFN-β-luc plasmid. Twenty-four hours after transfection, cells were left untreated or stimulated with 100 µg/ml of poly(I:C) for 4 h before luciferase analysis was performed. (C) Effects of TRIM38 knockdown on poly(I:C)-induced IRF3 phosphorylation. TRIM38 knockdown or control 293/TLR3 cells were incubated with 100 µg/ml of poly(I:C) for 4 h before the immunoblot analysis was performed. (D, E) Effects of TRIM38 knockdown on poly(I:C)-induced transcription of IFN-β and ISG56 genes. Indicated cells were stimulated with poly(I:C) for 4 h, then total RNA was extracted for real-time PCR analysis. Similar results were obtained from three independent experiments.</p

TRIM38 induces TRIF degradation through proteasomal pathway.

<p>(A) Overexpression of TRIM38 does not affect endogenous TRIF mRNA expression. HeLa cells were transfected with increasing amounts of a TRIM38-Flag plasmid (0, 0.5, and 2 µg). After 48 h, total RNA was prepared and used for RT-PCR analysis of the indicated genes. GAPDH was used as an internal control. (B) Overexpression of TRIM38 reduces TRIF protein. 293T cells were transfected with plasmids expressing Flag-TRIF or TRAF3-Flag, together with increasing amounts of TRIM38-Flag plasmid (0, 50, 100, and 200 ng). After 24 h, immunoblot analysis was performed with indicated antibodies. (C) Overexpression of TRIM38 reduces endogenous TRIF. HeLa cells were transfected with increasing amounts of TRIM38-Flag plasmid (0, 0.5, and 2 µg). Fourty-eight hours after transfection, immunoblot analysis was performed using indicated antibodies. (D) Caspase inhibitor does not inhibit TRIM38-mediated TRIF degradation. 293T cells were transfected with Flag-TRIF plasmid and increasing amounts of TRIM38-Flag plasmid (0, 10, 50, 100, 150, and 200 ng) for 6 h. Then cells were treated with DMSO (negative control) or 5 µM Z-VAD-FMK for 16 h before immunoblot analysis was performed. (E) Overexpression of TRIM38 promotes degradation of caspase-resistant TRIF. 293T cells were transfected with plasmids expressing Flag-TRIF or TRIF mutant carrying D284E and D289E substitutions, together with increasing amounts of TRIM38-Flag plasmid (0, 50, 100, and 200 ng). After 24 h, immunoblot analysis was performed. (F) TRIM38 promotes proteasomal degradation of TRIF. 293T cells were transfected with a Flag-TRIF plasmid and increasing amounts of TRIM38-Flag plasmid (0, 10, 50, 100, 150, and 200 ng) for 6 h. Then cells were treated with DMSO, 0.1 µM MG132, or 10 mM NH₄Cl for 16 h before immunoblot analysis was performed.</p

Effects of TRIM38 on TLR3-induced IFN-β signaling.

<p>(A) Expression of TRIM38 mRNA in HeLa cells treated with 100 µg/ml poly(I:C). At indicated time points, cells were harvested, and total RNA was prepared and analyzed by RT-PCR. GAPDH mRNA expression was assessed as an internal control. (B) Expression of TRIM38 protein in HeLa cells treated with 100 µg/ml poly(I:C). At indicated time points, cells were harvested and analyzed by immunoblot using the indicated antibodies. (C) Effect of TRIM38 overexpression on poly(I:C)-induced activation of IFN-β. 293T/TLR3 cells were transfected with an IFN-β-luc plasmid and TRIM38 plasmid (0, 50, 200, and 500 ng). Twenty-four hours after transfection, cells were incubated with 100 µg/ml of poly(I:C) for 4 h, and then luciferase assays were performed. (D) Effect of TRIM38 overexpression on IRF3 phosphorylation. HeLa cells were transfected with TRIM38 plasmid (0, 0.5 and 2 µg). Twenty-four hours after transfection, cells were left untreated or incubated with 100 µg/ml poly(I:C) for 4 h. Immunoblot analysis was performed using the indicated antibodies. (E, F) Effect of TRIM38 overexpression on poly(I:C)-induced transcription of IFN-β and ISG56. HeLa cells were transfected with TRIM38 plasmid (0, 0.5, and 2 µg). Twenty-four hours after transfection, cells were left untreated or incubated with 100 µg/ml of poly(I:C) for 4 h, then total RNA was extracted and quantitative real-time PCR were performed to analyze gene expression.</p

TRIM38 targets TRIF.

<p>(A–C) Effects of TRIM38 on TRIF, TBK1, or IKKi-induced IFN-β activation. 293T cells were transfected with an IFN-β-luc plasmid, together with a plasmid expressing TRIF (A), TBK1 (B), or IKKi (C), and a TRIM38 plasmid (0, 50, 100, and 200 ng), respectively. Luciferase assays were performed after 24 h post-transfection. (D) Effect of TRIM38 on TRIF-mediated IRF3 phosphorylation. 293T cells were transfected with a TRIF plasmid and a TRIM38 plasmid (0, 0.5, and 2 µg). Twenty-four hours post transfection, cell lysates were analyzed by immunoblot with indicated antibodies.</p

RING/B-box of TRIM38 is critical for TRIF degradation.

<p>(A) TRIM38 catalyzes K48-linked ubiquitination of TRIF. 293T cells were transfected with plasmids expressing Myc-tagged full-length or RING/B-box domain deleted (ΔRING/B-box) TRIM38, Flag-TRIF, and HA-ubiquitin plasmids. At 24 h post-transfection, cell lysates were denatured and immunoprecipitated using anti-Flag agrose beads. Immunoblot analysis was performed using an antibody specific against K48-linkage polyubiquitin. (B) Effect of TRIM38ΔRING/B-box mutant on TRIF degradation. 293T cells were transfected with Flag-TRIF plasmid and Flag-tagged full length TRIM38 or TRIM38ΔRING/B-box mutant plasmid (0, 50, and 100 ng). Twenty-four hours after transfection, immunoblot analysis using the indicated antibodies was performed. (C) Effect of TRIM38ΔRING/B-box mutant on TRIF-induced IFN-β promoter activation. 293T cells were transfected with IFN-β-Luc plasmid, Flag-TRIF plasmid, together with increasing amounts plasmid expressing of Flag-tagged full length TRIM38 or TRIM38ΔRING/B-box mutant (0, 50, and 100 ng). Luciferase assays were performed 24 h after transfection.</p

Interaction of TRIM38 and TRIF.

<p>(A) TRIM38 specifically interacts with TRIF. 293T cells transfected with plasmids expressing TRIM38-Myc and Flag-TRIF, Flag-TBK1, or Flag-IKKi. After 24 h, cell lysates were immunoprecipitated using anti-Flag agarose beads. The lysates and immunoprecipitates were analyzed by immunoblot with anti-Myc and anti-Flag antibodies. (B) Endogenous interaction of TRIF with TRIM38. Hela cells were left untreated or treated with poly(I:C) for 4 h. Cell lysates were immunoprecipitated with goat anti-TRIF antibody or control goat IgG and analyzed with rabbit anti-TRIM38 or rabbit anti-TRIF antibodies. (C, D) Schematic presentations of truncation mutants of TRIF (C) and TRIM38 (D). (E) TRIF interacts with TRIM38 through its N-terminus. 293T cells were transfected with Myc-TRIM38 together with full length Flag-tagged TRIF or TRIF truncation mutants. The immunoprecipitations were performed using anti-Flag agrose beads. Immunoblot analysis was carried out similarly as in (A). (F) TRIM38 is associated with TRIF through its PRYSPRY domain. Experiments were performed as described in panel (E). The co-immunopricipitated proteins were marked by asterisks.</p