Journal: International Journal of Molecular Sciences

Article Title: Liraglutide Counteracts Endoplasmic Reticulum Stress in Palmitate-Treated Hypothalamic Neurons without Restoring Mitochondrial Homeostasis

doi: 10.3390/ijms24010629

Figure Lengend Snippet: Liraglutide prevents changes in ER proteostasis and ER morphology caused by palmitate in Neuro2A cells. Metformin is less effective in counteracting ER stress. ( A ) Neuro2A cells incubated for 6 h with 250 µM palmitate (Palm) in the presence and absence of 100 nM liraglutide (Lira) and 2 mM metformin (Met) were fixed, immunostained with anti-ATF6α antibody, and imaged by confocal microscopy with the 20× objective of the Olympus FluoviewFV1000 microscope. The graph shows the raw integrated fluorescence intensity (FI) of ATF6α immunostaining within a Region of Interest (ROI) drawn to include the entire cell (magenta line of a representative cell indicated by a magenta arrowhead and shown at higher magnification above the graph, each graph symbol represents one cell, n ≥ 45 cells per condition). ( B ) Neuro2A cells treated as in panel ( A ) were fixed, immunostained with anti-ATF6α antibody, counterstained with the nuclear stain Hoechst 33342, and imaged by confocal microscopy with the 60× objective. Graph shows FI of ATF6α within a ROI drawn to include the nucleus (red line of a representative cell indicated by a red arrowhead and shown at higher magnification above the graph, each graph symbol represents one cell, n ≥ 45 cells per condition). ( C ) Neuro2A cells treated as in panel ( A ) were fixed and immunostained with anti-GRP78/Bip antibody and imaged by confocal microscopy with the 20× objective. Graph shows the FI of GRP78/Bip per cell (n ≥ 25 cells per condition, each graph symbol represents one cell). ( D ) Neuro2A cells treated as in panel ( A ) were imaged with the 60× objective. Yellow arrowheads indicate cells that are shown at higher magnification in the lower panel. Using ImageJ software, a segment was drawn across the cytoplasm of cells treated under the indicated conditions (a representative red segment is drawn in the “No Palm” lower panel. Graphs show the FI values along ImageJ segments drawn in the cells indicated by arrows. ** p < 0.01 **** p < 0.0001, ns, not significant).

Article Snippet: Primary antibodies: chicken anti-MAP2 Antibody (Cat # NB300-213) and ATF6 (Cat # 70B1413.1) were from Novus Biologicals (Minneapolis, MN, USA); rabbit anti-PSD95 (Cat # ab18258), rabbit anti-COX IV antibody (Cat # ab16056), rabbit anti-GRP78/BiP (Cat # ab21685), rabbit anti-α-tubulin (Cat # ab18251) and goat anti-GFAP antibody (Cat # ab53554), from Abcam (Cambridge, MA, USA); mouse anti-KDEL antibody (Cat # ADI-SPA-827) from Enzo (Farmingdale, NY, USA); rabbit C29F4 anti-HA (Cat # 3724) antibody from Cell Signaling Technology (Danvers, MA, USA); and mouse SERCA2 Antibody (Cat # sc-376235) from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Confocal Microscopy, Microscopy, Fluorescence, Immunostaining, Staining, Software