Journal: bioRxiv
Article Title: Ceramide-Rich Microdomains Facilitate Nuclear Envelope Budding during the Biogenesis of LTB 4 -containing Exosomes
doi: 10.1101/2022.02.09.479761
Figure Lengend Snippet: A Representative Airyscan microscopy images showing the distribution of LBR (green) and 5-LO (red) in fixed PMNs uniformly stimulated with 20 nM fMLF (n=3). The yellow arrowheads point to nuclear buds and the blue arrowheads point to cytosolic vesicles. Scale bar is 5 µm. B Scatter dot plot showing the percentage of PMNs containing LBR-positive buds and cytosolic vesicles in a field of randomly selected images. Data collected from 50, 46, 74, and 66 cells in DMSO, 2min, 5 min, and 15 min fMLF treatment, respectively, pooled from three independent experiments are plotted as mean ± SEM. Each dot represents the value from one experiment. P=0.0076 as obtained from RM one-way ANOVA analysis. C Representative Airyscan microscopy images of PMNs chemotaxing towards 100 nM fMLF and stained for LBR (green) and 5-LO (red), obtained from six independent experiments. NE buds are shown in yellow boxes and cytosolic vesicles are shown in blue boxes. Scale bar is 5 µm. In magnified insets, the scale bar is 1 µm. D Histograms showing the normalized intensity of LBR and 5-LO across the diameter of LBR positive buds and cytosolic vesicles. The data points from 15 vesicles and 13 buds within 11 cells pooled from four independent experiments were plotted as mean +/- SEM, with the bold line showing the mean and bar representing the SEM. E Representative Airyscan microscopy images of fixed PMNs chemotaxing towards 100 nM fMLF and stained for LBR (green) and LTA 4 H (red), obtained from three independent experiments. See panel C for more details. Scale bar is 5 µm. In the inset, it is 1 µm. F Histograms showing the normalized intensity of LBR and LTA 4 H across the diameter of LBR positive buds and cytosolic vesicles. The data points from 5 vesicles and buds each within 5 different cells pooled from three independent experiments were plotted as mean +/- SEM, with the bold line showing the mean and bar representing the SEM.
Article Snippet: The agarose was removed and PMNs were blocked for 1 hour at room temperature followed by staining in blocking buffer (0.2% saponin, 2% goat serum in 1X mHBSS) at 4°C overnight with antibodies against FLAP (1µg/ml, Abcam 85227), 5-LO (1:500, BD biosciences 610694), nSMase1 (1:100, Abcam 131330), and Lamin B1 (1:100, Proteintech 66095-1), LBR (1:400, Abcam 32535), LTA 4 H (1:100, Santacruz biotechnology sc23070), Ceramide (1:50, Sigma C8104-50TST), CD63 (1:800, BD biosciences 556019), and ALIX (1:200, Abcam 117600).
Techniques: Microscopy, Staining
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