Journal: Nature medicine

Article Title: Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

doi: 10.1038/nm.3610

Figure Lengend Snippet: Differential regulation of HCV strains by SPHK2-mediated lipid peroxidation. ( a ) Dose-dependent effects of PUFAs on H77S.3/GLuc and HJ3-5/GLuc RNAs in Huh-7.5 cells. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. ( b ) Growth kinetics of H77S.3/GLuc and HJ3-5/GLuc RNAs in the presence of 50 μM PUFAs. Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. ( c ) Cells transfected with HCV RNAs encoding GLuc were treated with DMSO, 100 μM LA or 100 μM LA plus 1 μM SKI. Data shown represent percent GLuc activity secreted between 48–72 h relative to DMSO control. L.O.D. = limit of detection. ( d ) Effect of 1 μM SKI, 1 μM VE, 100 μM CoQ10 or 50 μM ARA or DHA, on intracellular malondialdehyde (MDA) abundance in cells transfected with the indicated HCV/GLuc RNAs at 72 h. MDA was significantly increased by PUFAs and reduced by SKI or lipophilic antioxidants ( P < 0.01). ( e ) Analysis of 8-isoprostane abundance in cells electroporated with the indicated HCV RNAs and grown in the presence of 1 μM SKI or VE, or 50 μM LA with or without 1 μM SKI and VE for 48 h. ( f ) Effect of siRNA targeting SPHK isoforms (see ) on MDA accumulation after treatment with increasing concentrations of LA (6.25, 12.5, 25, 50, 100 μM) for 24 h (left panel). MDA levels in Huh-7.5 cells treated with increasing concentrations of LA in the presence of DMSO or 1 μM SKI (right panel). ( g ) Effects of increasing concentrations of VE (left), 1 μM VE alone, or 1 μM VE plus 1 μM SKI (right) on replication of H77S.3/GLuc and HJ3-5/GLuc RNAs. Data shown represent GLuc secreted between 48–72 h relative to DMSO control. ( h ) GLuc secretion from Huh-7.5 cells transfected as in a and treated with 10 μM CuOH with or without 10 μM VE. ( i ) Influence of SKI or VE (each 1 μM) on replication of H77S.3 and HJ3-5 viruses expressing GLuc in cells cultured in the presence or absence of 10% FBS. Medium containing 10% FBS was replaced with FBS-free or 10% FBS media containing SKI, VE or DMSO 6 h after RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from two ( a–f,i ) or three ( g,h ) independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Vitamin E (α-, rac-β-, and γ-tocopherols), 4-deoxypyridoxine hydrochloride (DOP), coenzyme Q10, butylated hydroxytoluene, N-acetyl-L-cysteine, diphenyleneiodonium chloride, oleic acid, and cyclosporine A were from Sigma-Aldrich. nSMase spiroepoxide and cumene hydroperoxide were from Santa Cruz Biotechnology, D609 was from Enzo Life Sciences, and sofosbuvir (PSI-7977) was from Chemscene.

Techniques: Activity Assay, Control, Transfection, Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: Silencing of circRERE(4-5) inhibits ONECUT2-mediated tumorigenesis and metastasis in gastric cancer

doi: 10.3389/fimmu.2026.1686702

Figure Lengend Snippet: CircRERE(4-5) upregulates ONECUT2 by sponging miR-571. (A) qRT–PCR demonstrating the enrichment of circRNAs in a representative anti-AGO2 RIP assay conducted in GC cells. (B) Venn diagram illustrating the overlap of candidate miRNAs predicted to interact with circRERE(4-5) by two open-access databases. (C) qRT–PCR showing the enrichment of miRNAs following circRERE(4-5) pull-down in lysates of GC cells. (D) Venn diagram depicting the intersection of candidate mRNAs targeted by miR-571 as predicted by three open-access databases. (E) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)). (F) qRT–PCR analysis of ONECUT2 mRNA expression in GC cells transfected with control inhibitors or miR-571 inhibitors. (G) Western blotting revealing ONECUT2 protein levels in GC cells under control conditions (sh-NC) or with circRERE(4-5) knockdown (sh-circRERE(4-5)), with GAPDH serving as a loading control. (H) Western blotting showing ONECUT2 protein levels in GC cells transfected with control inhibitors or miR-571 inhibitors. (I) qRT–PCR analysis of ONECUT2 mRNA expression in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (J) Representative western blot of ONECUT2 in AGS cells under control conditions (sh-NC), after circRERE(4-5) knockdown (sh-circRERE(4-5)), or following cotransfection with sh-circRERE(4-5) and miR-571 inhibitors. (K) Sequence alignment analysis highlighting the binding sites of circRERE(4-5) and ONECUT2 3’ UTR on miR-571. Data are presented as the mean ± SD. P -values were calculated using a two-tailed unpaired Student’s t -test (E, F, I) ; ** P < 0.01, *** P < 0.001. See also Supplementary Figure 4 .

Article Snippet: The antibodies used were anti-ONECUT2 (Proteintech, #21916-1-AP; 1:1,000), anti-AGO2 (Proteintech, # 67934-1-Ig; 1:1,000), anti-RERE (abcam, ab217756; 1:1,000), and anti-GAPDH (Proteintech, #10494–1-AP; 1:6,000).

Techniques: Quantitative RT-PCR, Expressing, Control, Knockdown, Transfection, Western Blot, Cotransfection, Sequencing, Binding Assay, Two Tailed Test