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. 2021 May 10;6(9):e138484. doi: 10.1172/jci.insight.138484

Figure 2. FL2 depletion results in a more dynamic MT array near the growth cone in regenerating neurites.

Figure 2

(A and B) Immuno-micrographs of DRG neurites of GFP AV– and Cre AV–treated neurons 2 days after replating, dual-stained for tubulin isoform β3 (TUBB3) (green) and tyrosinated tubulin (Tyr tub) (red) (A) or for TUBB3 (green) and acetylated tubulin (Act tub) (red). Brightness/contrast adjusted linearly to more clearly see acetylated tubulin stain (B). Scale bars: 50 μm. (C) Tyr/TUBB3 ratios in GFP AV– and Cre AV–treated neurons from 1 representative experiment, starting at the tip of the growth cone and moving toward the soma (dark line is the mean intensity ratio; shaded area represents the SEM). (D) Tyr/TUBB3 fluorescence intensity ratios in the 50 distalmost micrometers of neurites, normalized to the GFP AV mean (GFP AV: 1.00 ± 0.03, Cre AV: 1.26 ± 0.05, n = 86 GFP, n = 101 Cre). (E) Distribution of Act/TUBB3 ratios in the 50 distalmost micrometers of neurites, normalized to the GFP AV mean (GFP AV: 1 ± 0.06, Cre AV: 0.74 ± 0.04. n = 87 GFP; n = 116 Cre). (F) Act/TUBB3 ratios in the proximal to mid region of the axonal shaft (GFP AV: 1.00 ± 0.13; Cre AV: 0.94 ± 0.07, P = 0.68, n = 43 GFP, n = 51 Cre). Experiment performed in duplicate. (G) Inverted images of MTs in the growth cones (GCs) of GFP AV– and CRE AV–treated neurons, immunostained for TUBB3. Brightness/contrast adjusted to better visualize individual MTs. Scale bar: 10 μm. (H) Mean TUBB3 fluorescence intensities in the distalmost 50 μm of neurites (GFP: 1.00 ± 0.06, Cre: 1.18 ± 0.09, Welch’s t test, P = 0.12, n = 91 GFP, n = 101 Cre). Experiments performed in triplicate unless otherwise indicated. Data were analyzed using unpaired 2-tailed Welch’s t test. Bars represent mean ± SEM. ****P < 0.0001.