Journal: Nature

Article Title: Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes

doi: 10.1038/s41586-023-06857-0

Figure Lengend Snippet: a , Representative fluorescence microscopy images depicting IEC-6 cells treated for 48 h with actinonin (100 μM), Atpenin A5 (AA5, 1 μ M) or 1% dimethyl sulfoxide; DMSO (control). Short treatment (6 h) with Brefeldin A (BFA, 5 μg/ml) was used as a positive control for Golgi dispersal. Scale bars, 50 μm b , Representative fluorescence microscopy images of IEC-6 cells grown under the same conditions as described in a were incubated with oleic acid (OA, 600 μM) for the last 24 h prior imaging. In this case, BFA was applied in the last 6 h of OA treatment to avoid cytotoxicity. Anti-TGN38 (red) antibody was used to visualize Golgi, Anti-COX1(green) to stain mitochondrial networks (a) , BODIPY (green) to stain lipid droplets (b) , and DAPI (blue) for nuclei (top). TGN38 staining is additionally depicted in white (bottom). Scale bars, 50 μm. Quantification of the observed Golgi morphology of the IEC-6 cells based on five distinct categories, as illustrated at the right ( n = 100–300 inspected IEC-6 cells from three independent biological experiments). c , Representative confocal images and graphs depicting quantification of GFP signal in C. elegans expressing α-mannosidase II fused to GFP under the control of the gut-specific vha-6 promoter grown either on a control empty vector (EV) or RNAi against dars-2 at the first (D1) and fourth day of adulthood (D4) (EV, n = 19 ( D1 ), n = 13 ( D4 ), dars-2 , n = 20 ( D1 ), n = 19 ( D4 )). Scale bars, 1 μm. d , Representative confocal images of GFP signal in C. elegans expressing SPCS-1 fused to GFP under the control of the gut-specific vha-6 promoter grown either on a control empty vector (EV) or RNAi against dars-2 at the first (D1) and fourth day of adulthood (D4) EV, n = 19 ( D1 ), n = 19 ( D4 ), dars-2 , n = 20 ( D1 ), n = 19 ( D4 )). Scale bars, 1 μm. e , Immunofluorescence micrographs of C. elegans carrying vit-2::GFP reporter on a control empty vector (EV) ( n = 10) and RNAi against sar-1 , sec-13 , fum-1 and dars-2 at D1 ( n = 10). Insets show magnification of a selected area of the worm. Scale bars, 100 μm. In bar graphs data are represented as mean ± s.e.m. and P values were calculated by two-sided Chi-squared test ( a , b ) and two-sided Student’s t -test with assumption of equal variance ( c ).

Article Snippet: Immunofluorescence staining was performed on IEC-6 cells cultured on coverslips and fixed in 4% paraformaldehyde for 15 min. Reactive aldehydes were quenched with 50 mM NH 4 Cl for 10 min and the cells were permeabilized with 0.1% Triton-X-100 in PBS for 5 min. After 20 min in blocking solution (0.2% fish-skin gelatin diluted in PBS), IEC-6 cells were incubated with primary antibodies against TGN38 (bio-techne, AF8059-SP, 1:200) and MTCO1/COX1 (Molecular Probes, 459600, 1D6E1A8, 1:100) for 30 min at room temperature, followed by incubation with anti-sheep IgG NorthernLights NL557 (bio-techne, NL010, 1:300) or anti-mouse Alexa 488 (Molecular Probes, A1101, 1:300) fluorescence-conjugated secondary antibodies for 30 min at room temperature.

Techniques: Fluorescence, Microscopy, Positive Control, Incubation, Imaging, Staining, Expressing, Plasmid Preparation, Immunofluorescence