Journal: Nature Communications
Article Title: Nuclear lactate dehydrogenase A senses ROS to produce α-hydroxybutyrate for HPV-induced cervical tumor growth
Figure Lengend Snippet: HPV16/18 E7 induces LDHA nuclear translocation by ROS accumulation. a LDHA is significantly translocated into nucleus in HPV16 positive cervical cancer tissues. Representative IHC images for LDHA localization in HPV16-negative and positive cervical tumor samples. Scale bar, 100 μm. b Nuclear LDHA is dramatically increased in HPV16-positive cervical cancer tissues. Semi-quantitative cytoplasmic LDHA and nuclear LDHA scoring was performed in HPV16 negative ( n = 27) and positive ( n = 39) cervical tumor samples. c , d HPV16/18 E7 promotes LDHA nuclear translocation in a ROS-dependent manner. Primary human cervix keratinocytes (PHKs) stably expressing vector or Flag-tagged HPV16/18 E7 were treated with or without 1 mM NAC for 6 h, followed by staining with anti-LDHA (green), anti-Flag (red) antibodies, and DAPI (blue). Scale bars, 10 μm ( c ). The percentage of cells with nucleus-localized LDHA compared to total cell number was quantified ( d ). e HPV16/18 E7 enhances ROS production. Cellular ROS were measured in PHKs stably expressing vector or HPV16/18 E7 coupled with or without 1 mM NAC treatment for 6 h, followed by using the ROS-sensitive fluorescent dye (CM-H 2 DCFDA) with flow cytometry according to the manufacturer’s protocol. f , g LDHA nuclear translocation is profoundly increased in an H 2 O 2 -dose-dependent manner. Immunofluorescent images of LDHA (green) in HaCaT cells upon different dose of H 2 O 2 treatment as indicated. DAPI, blue. Scale bars, 10 μm ( f ). The percentage of cells with nucleus-localized LDHA compared with total cell number was quantified ( g ). h ROS promote LDHA nuclear translocation. HT-3 cells were treated with or without 10 μM H 2 O 2 for 6 h, and supplemented with or without 1 mM NAC for extended 6 h as indicated for nuclear isolating, followed by blotting with LDHA, Tubulin, and Lamin B1. Results are representative of three independent experiments. All data are shown as mean ± SEM. The p values were determined by two-tailed t -test. The values of p
Article Snippet: Hydrogen peroxide solution (H2 O2 ) (Sigma, 323381), N-acetyl cysteine (NAC) (Sigma, A7250), DAPI (Sigma, D9542), sodium pyruvate (Sigma, P5280), sodium (L) lactate solution (Sangon Biotech, A604046), sodium 2-ketobutyrate (Sigma, K0875), sodium 2-hydroxybutyrate (Santa Cruz, sc-258161), NADH (Sigma, N4505), EPZ004777 (Selleck, S7353), DPIC (Selleck, S8639), 50% glutaraldehyde solution (Sangon Biotech, A600875), and ML385 (MCE, HY-100523) were commercially obtained.
Techniques: Translocation Assay, Immunohistochemistry, Stable Transfection, Expressing, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Two Tailed Test