Journal: Molecular Therapy. Nucleic Acids

Article Title: Overexpression of the mitochondrial anti-viral signaling protein, MAVS, in cancers is associated with cell survival and inflammation

doi: 10.1016/j.omtn.2023.07.008

Figure Lengend Snippet: MAVS overexpression in diverse types of cancer (A–C) Formalin-fixed and paraffin-embedded US-Biomax tissue microarray slides containing the indicated tissues from cancer patients (n = 20) and normal tissue (n = 5) were immunohistochemistry stained using anti-MAVS antibodies and hematoxylin stained, as described in . Representative images from sections of the different indicated tumors and corresponding healthy tissues are shown. The slides were incubated overnight at 4°C with anti-MAVS antibodies in PBS containing 1% BSA and then with secondary antibodies in PBS containing 1% BSA. The slides were subsequently treated with 3′3-diaminobenzidine tetra-hydrochloride (DAB) and counterstained with hematoxylin. Negative controls without primary antibody incubation were also performed. Sections of tissue were observed under an Leica microscope, and images were taken at 200× magnification with the same light intensity and exposure time. The percentage of the tumor sections stained at the intensity indicated on the scale above are presented. (D) Peripheral blood mononuclear cells (PBMCs) were obtained from chronic lymphocytic leukemia (CLL) patients (n = 16) and healthy donors (n = 13) using Ficoll-Paque PLUS (GE Healthcare, Israel) density gradient centrifugation, as described previously. Representative immunoblots of cell lysates of PBMCs derived from CLL patients and healthy donors subjected to SDS-PAGE and immunoblotting using anti-MAVS and anti-actin antibodies are shown. (E) Representative immunoblots using anti-MAVS antibodies of tissue lysates of lung cancer samples from tumor tissue (T, n = 22) and healthy tissue (H, n = 22), each derived from the same lung of a lung cancer patient. (F) Quantification of MAVS levels in CLL and lung cancer patients relative to healthy donors presented as fold change. The results are the mean ± SD. (G) HEK-293, Hela, SHSY-5Y, PC3, A549, MDA-MB-231, and UMUC3 cell lines were grown in the appropriate medium, and cell lysates (10 μg of protein) were subjected to SDS-PAGE and immunoblotting with anti-MAVS and citrate synthase (CS) antibodies (n = 3). (H) Quantitative analysis of MAVS and CS expression levels in the different cell lines were presented relative to the level in SHSY-5Y cells for MAVS and to A549 cells for CS. (I) T-REx-293 cells were transiently transfected with pcDNA3 or MAVS-pcDNA3 using metafectene as in the section. MAVS expression was analyzed by immunoblotting 48 h post transfection. (J) Schematic presentation of the bicistronic nature of the MAVS transcript depicting met1 (methionine1) and met142 (methionine142) as the two translation initiation sites.

Article Snippet: MAVS overexpression in diverse types of cancer (A–C) Formalin-fixed and paraffin-embedded US-Biomax tissue microarray slides containing the indicated tissues from cancer patients (n = 20) and normal tissue (n = 5) were immunohistochemistry stained using anti-MAVS antibodies and hematoxylin stained, as described in .

Techniques: Over Expression, Microarray, Immunohistochemistry, Staining, Incubation, Microscopy, Gradient Centrifugation, Western Blot, Derivative Assay, SDS Page, Expressing, Transfection