Journal: Physiological Reports
Article Title: Secreted mitochondrial aspartyl‐ tRNA synthetase ( DARS2 ) regulates TNFα signaling
doi: 10.14814/phy2.70627
Figure Lengend Snippet: Secreted DARS2 from airway epithelia binds to macrophages. (a) Immunoreactive levels of DARS2 in the cell lysate (top) and supernatant (bottom) and (b) quantitation of DARS2 in the supernatant from primary human small airway epithelial cells (HSAEC) treated with Pam, IL‐1β or increasing concentrations of TNFα (repeated for n = 3 representative blots shown). (c) Immunoreactive levels of DARS2 in the cell lysate (top) and supernatant (bottom) and (d) quantitation of DARS2 in the supernatant from human alveolar macrophages (HAM) treated with Pam, IL‐1β or increasing concentrations of TNFα (repeated for n = 3 representative blots shown). (e) Schematic of adoptive media transfer from BEAS‐2Bs to recipient PMA‐stimulated THP1‐macrophages. BEAS2Bs were transfected with FLAG‐tagged empty vector (EV) or DARS2‐Flag plasmid and media was transferred to THP‐1 cells. (f) Shown are levels of ectopically expressed DARS2 in THP1 cell lysates (each lane is a biologically independent replicate, repeated for n = 2). (g) Immunofluorescence of THP1‐macrophages receiving media from donor BEAS‐2B cells transfected with DARS2‐FLAG as in (e, f) showing co‐localization of DARS2‐Flag with the actin filament marker, phalloidin (arrows, merge panels) Scale bar = 20 μm (repeated for n = 2). (h) IL18 , (i) TNF , (j) CXCL1 mRNA levels by RT‐qPCR analysis in THP1‐macrophages 24 h after receiving media from donor BEAS‐2B cells transfected with EV‐FLAG or DARS2‐FLAG ( n = 8 biological replicates/group).
Article Snippet: BEAS‐2B (Human Bronchial Epithelial cells, ATCC, catalog no. CRL‐35588), THP‐1 (Tohoku Hospital Pediatrics‐1, an acute myeloid leukemia line used as a model of monocytes, ATCC, catalog no. TIB‐202), primary HSAEC (human small airway epithelial cells, ATCC, catalog no. PCS‐301‐010), and primary HBEC (human bronchial epithelial cells, ATCC, catalog no. PCS‐300‐010) were used for this study.
Techniques: Quantitation Assay, Transfection, Plasmid Preparation, Immunofluorescence, Marker, Quantitative RT-PCR