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. 2019 May 14;10(3):e00266-19. doi: 10.1128/mBio.00266-19

FIG 4.

FIG 4

Patterns of NF-κB activation and TNF-α expression differ. Dissected swimbladders from C. albicans-infected fish were imaged at 24 hpi. (A) z-projection of 3 slices of a dissected Tg(NF-κB:EGFP) swimbladder with moderate EGFP expression. (B) Single z-slice from the blue square in the z-stack in panel A, with outlines of fungi, EGFP+ cells, and epithelial layers based on the DIC image. (C) z-projection of 7 slices of a TgBAC(tnfa:GFP) swimbladder with high GFP expression levels. (D) Single z-slice from the blue square in the z-stack in panel A, with outlines of fungi, GFP+ cells, and epithelial layers based on the DIC image. (E and F) Still images from time-lapse images taken at 24 hpi. (E) Tg(NF-κB:EGFP) × mpeg1:dTomato (red macrophage) zebrafish at time 0:00 of the time-lapse image in Movie S1 in the supplemental material. The leftmost image is a maximum-projection overlay of all colors using a middle plane from the DIC image. (i) Zoomed-in images of the areas outlined in the blue square. Dotted lines outline example cells that either moved (white outlines [cells 1 and 3]) or remained stationary (yellow outlines [cell 2]) over the 16-min-long time-lapse experiment. (ii) The GFP channel was eliminated to demonstrate red fluorescence of macrophages. Cells 1 and 3 are dTomato+ (macrophages), while cell 2 is not. (iii) Schematics showing the positions of each cell at the times indicated in the grayscale legend. Only cells 1 and 3 change shape or position. (F) TgBAC(tnfa:GFP) × mpeg1:dTomato zebrafish at time 0:00 of the time-lapse imaging in Movie S2. (i) Outlines of example cells (white, moved [cells 5 and 6]; yellow, stationary [cell 4]). (ii) Cells 4, 5, and 6 are dTomato+ (macrophages). (iii) Schematics showing movement over time. Cells 5 and 6 change shape and position over the course of the time-lapse experiment, but cell 4 does not. Color channels show z-projections of 13 slices (E) or 11 slices (F). DIC was performed for a single z-slice. Bars, 150 μm (A, C, E, and F) and 50 μm (B, D, Ei to Eiii, and Fi to Fiii).