Journal: Cell Death & Disease

Article Title: RNAi-based ALOX15B silencing augments keratinocyte inflammation in vitro via EGFR/STAT1/JAK1 signalling

doi: 10.1038/s41419-025-07357-x

Figure Lengend Snippet: Analysis of membrane lipids via confocal microscopy in keratinocytes transfected with control or ALOX15B siRNA for 72 h. Ratiometric images of NR12A staining ( A ) and analysis ( B , N = 7) of the ratio of liquid order to liquid disorder. Immunocytochemistry staining ( C ) of endoplasmic reticulum marker Calnexin (magenta) in combination with plasma membrane marker MemGlow 488 (yellow) and cholesterol staining by filipin (Cyan). Mean pixel intensity of filipin staining at the plasma membrane and total per cell ( D ), Mander’s M1 and M2 co-localisation analysis of MemGlow 488 ( E ) or Calnexin ( F ) and filipin, data are mean per cell per biological replicate ( N = 6). Lipid raft staining ( G ) via Alexa Fluor 488 conjugated cholera toxin subunit B (CTX-AF488) and analysis (H) of mean pixel intensity per cell ( N = 6). Median fluorescence intensity of propidium iodide ( I ) following 1 h streptolysin O pore formation measured by flow cytometry ( N = 5). Individual biological replicates represented by different symbol sets, line is mean. t test ( B , H ) or two-way ANOVA performed ( D – F , I ); significance denoted by * P < 0.05, ** P < 0.01, *** P < 0.001. For all images, scale bar 20 µm, white boxes represent zoomed-in regions.

Article Snippet: For lipid order measurements, cells were stained with 20 nM MemGlow™ NR12A Membrane Polarity Probe (Cytoskeleton, Denver, Colorado, USA) for 10 min at room temperature.

Techniques: Membrane, Confocal Microscopy, Transfection, Control, Staining, Immunocytochemistry, Marker, Fluorescence, Flow Cytometry