Journal: Redox Biology

Article Title: SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione

doi: 10.1016/j.redox.2021.102041

Figure Lengend Snippet: Nrf2, Membrane transporters, and GCLC in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Immunoblot of Nrf2 protein expression ( A , left panels) was assessed 6 hpi and 24 hpi, and by semi-quantitative fluorescence analysis 48 hpi ( A , right panels). Fluorophores were FITC (green) for Nrf2 protein labelling, DAPI (blue) for nuclei and Phalloidin-Alexa Fluor595 (orange) for the cytosolic space. Immunoblot of xCT and MRP1 membrane transport proteins ( B ), and GCLC protein ( C ) were carried out as described in the section Methods 24 hpi. Infection conditions and treatments with antivirals were the same of . Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **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.)

Article Snippet: The primary antibodies used were: Nrf2 Polyclonal Antibody, FITC Conjugated (bs-1074R-FITC, Bioss Antibodies, 1:50); NQO1 (A180, Mouse mAb #3187, CST, 1:50); GST-pi (610,718, mouse mAb, BD Biosciences, 1:500); PE anti-human IL-6 Antibody (BioLegend, 1:1000); PE anti-human IL-10 Antibody (BioLegend, 1:1000).

Techniques: Infection, Western Blot, Expressing, Fluorescence

Journal: Molecular Medicine Reports

Article Title: Effective elimination of chronic lymphocytic leukemia cells in the stromal microenvironment by a novel drug combination strategy using redox-mediated mechanisms

doi: 10.3892/mmr.2015.4364

Figure Lengend Snippet: Effect of SAHA on levels of GSH-associated enzyme and GSH in CLL cells co-cultured with stromal cells. (A) SAHA increased the expression of Nrf2 in CLL cells. CLL cells were treated with 2 µ M SAHA for 20 h, and cell lysates were assayed for Nrf2 using western blot analysis. Representative western blot results from three samples from patients with CLL are shown. The right panel shows the quantification of Nrf2 band density of eight CLL samples, with β-actin expression as an internal control (mean ± standard deviation; * P<0.05; Ctrl, control cells without treatment; S, SAHA treatment). (B) SAHA induced the translocation of Nrf2 between the cytosol and nucleus. SAHA (2 µ M) was added to the CLL cells for 20 h, and the cells were cytospun and immunostained with Nrf2 antibodies, and observed using a confocal laser scanning microscope. The nuclei were stained with 4,6-diamidino-2-phenylindole. (C) Upregulation of mRNA expression of GCLC following SAHA treatment. CLL cells were treated with 2 µ M SAHA for 22 h and the GCLC mRNA expression was examined using reverse transcription-quantitative polymerase chain reaction. (D) SAHA increases the expression of GCLC in CLL cells. CLL cells were treated with 2 µ M SAHA for 24 h, and the cell lysates were then assayed for the expression levels of GCLC by western blot analysis. The upper panel shows the representative western blot results from samples of four patients with CLL. The lower panel shows the quantification of GCLC band density of eight CLL samples, with β-actin as the internal control (mean ± standard deviation; ** P<0.01. (E) Treatment with SAHA enhanced stromal-mediated GSH upregulation in CLL cells. The CLL cells were treated with 2 µ M SAHA for 48 h in the presence or absence of HS5 cells. In another treatment group, CLL and HS5 cells in co-culture were incubated with cystine transporter inhibitor S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Values are presented as the mean ± standard deviation of three independent experiments using three CLL samples. (F) Sensitization of CLL cells to SAHA by inhibiting the cystine transporter with S-4-CPG. CLL and HS5 cells in co-culture were incubated with S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Cell viability was analyzed using an Annexin V/PI assay. Representative dot plots are shown. CLL, chronic lymphocytic leukemia; SAHA, suberoylanilide hydroxamic acid; GSH, glutathione; PI, propidium iodide; Nrf2, nuclear factor-E2-related factor 2; CPG, carboxyphenylglycine Ctrl, untreated control; S, SAHA treatment.

Article Snippet: The fixed CLL cells were incubated with rabbit anti-human polyclonal Nrf2 antibody (1:50; cat no. sc-13032; Santa Cruz Biotechnology, Inc.), at 4°C overnight, followed by incubation with Alexa-Fluor-594 goat anti-rabbit polyclonal antibody (1:400; cat. no. A11594; Molecular Probes at room temperature for 1 h. Finally, the slides were washed with phosphate-buffered saline, mounted and counterstained with mounting medium supplemented with DAPI prior to examination with a Nikon Eclipse TE2000 confocal microscope and analysis with Nikon EZ-C1 3.80 software (Nikon Corporation, Tokyo, Japan).

Techniques: Cell Culture, Expressing, Western Blot, Control, Standard Deviation, Translocation Assay, Laser-Scanning Microscopy, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Co-Culture Assay, Incubation