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. 2016 Jul 13;7:12187. doi: 10.1038/ncomms12187

Figure 6. Augmin disruption phenocopies the defects observed in γ-tubulin-depleted neurons.

Figure 6

(ac) Hippocampal neurons were infected at 1 DIV with the indicated lentivirus, transfected with the reporter EB3-Tomato at 3 DIV (a,b) or 4 DIV (c), and imaged by time-lapse microscopy 24 h later. (a) Projection of frames from time-lapse recordings with tracings of EB3-comets. (b) Quantification EB3-comets density in control, HAUS7- and HAUS1-depleted neurons. n=25, 25 and 24 somas, respectively, three independent experiments. *P<0.05, ***P<0.001 by two-tailed t-test. Error bars: s.e.m. (c) Quantification EB3-comets density in control and NEDD1-depleted neurons. n=32 (control), 39 (NEDD1#1), 34 (NEDD1#2) somas, three independent experiments. ns: not significant by two-tailed t-test. Error bars: s.e.m. (dj) Hippocampal cultures were infected with lentivirus as indicated at 1 DIV, simultaneously permeabilized and fixed at 4 DIV, and stained with the indicated antibodies. (d) Representative images showing microtubules stained with α-tubulin antibody in control and HAUS1-depleted neurons. Red arrowheads mark microtubule bundles in the soma. The areas in dashed squares are shown magnified for a better visualization of microtubule configurations. Scale bar, 5 μm. (e) Quantification of the mean intensity of α-tubulin staining normalized to the average intensity in control neurons. Red bars show average. (f) Scoring of neurons with bundled or tangled microtubules. (e,f) n=72 (control), 70 (HAUS7), 69 (HAUS1) somas, three independent experiments. *P<0.05, ***P<0.001, ****P<0.0001 by two-tailed t-test. Error bars: s.e.m. (g) Examples of α-tubulin and acetylated-α-tubulin co-stainings in axons of control, HAUS7 and HAUS1-depleted neurons. Scale bar, 5 μm. (h) Quantification of the mean intensity of the axonal α-tubulin signal normalized as in e. (i) Quantification of the mean intensity of the acetylated-α-tubulin signal in the same axon segments as in h. Values were normalized as in e. (j) Ratios of the intensities of acetylated-α-tubulin and total α-tubulin staining in axons. (hj) n=72 (control), 69 (HAUS7), 69 (HAUS1) axons, three independent experiments. ****P<0.0001, ns: not significant by two-tailed t-test. Red bars in dot plots show average. Boxes show 25–75 percentiles, whiskers show 5–95 percentiles, black bars show median.

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