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

Figure 3. FL2 depletion attenuates the effects of inhibitory substrates on adult DRG GC advancement during regeneration.

Figure 3

(A) Micrographs of GFP AV– and Cre AV–treated neurons immunostained for TUBB3 (green) 72 hours after plating on aggrecan stripes (red). Scale bar: 200 μm. (B) High-magnification image of a GFP AV–treated neurite turning in response to an aggrecan border and Cre AV–treated neurites crossing through the stripe border. Scale bar: 50 μm. (C) Fraction of neurites of GFP AV– and Cre AV–treated neurons that crossed aggrecan stripe borders (GFP AV mean = 0.26 ± 0.026; Cre AV mean = 0.41 ± 0.06. P = 0.0169, unpaired 2-tailed Welch’s t test, GFP n = 65 neurons, 257 neurite/border encounters; Cre AV n = 79 neurons, 200 neurite/border encounters. Experiment performed 4 times). (D) Immuno-micrographs showing an active, viable GC (left) and a collapsed GC (right), stained for MTs (TUBB3) in red and actin in green. Scale bar: 10 μm. (E) Fraction of collapsed GCs in untreated, GFP AV–treated, and Cre AV–treated neurons, in the presence and absence of Sema3A. GFP and Cre AV experiments performed in quadruplicate, untreated +/– Sema3A performed in triplicate (untreated, no Sema3A: 0.38 ± 0.04, n = 425 GCs; untreated, +Sema3A: 0.56 ± 0.03, n = 376; GFP AV, no Sema3A: 0.35 ± 0.01, n = 379; GFP AV, +Sema3A: 0.55 ± 0.04, n = 410; Cre AV, no Sema3A: 0.35 ± 0.05, n = 440; Cre AV, +Sema3A: 0.40 ± 0.02, n = 422. Mean ± SEM; *P < 0.05, **P < 0.01; unpaired 2-tailed Welch’s t test). Imaging and analyses performed in a blinded manner.