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. 2021 May 26;19(5):e3001279. doi: 10.1371/journal.pbio.3001279

Fig 5. RHEBp.P37L overexpression induces an increase in axon length and branching both in vitro and in vivo.

Fig 5

(A) Representative images of primary hippocampal cultures transfected at DIV1 with either empty vector control or RHEBp.P37L constructs (tdTomato, in red) stained at DIV4 with a pan axonal marker SMI312 (in green); arrowheads indicate the axons; bar graphs represent mean ± SEM and single data points indicate the number of cells analyzed; numbers indicate number of neuronal cultures (N = 2) and total number of cells analyzed (n = 24, n = 20); axonal length: Mann–Whitney U = 32, p < 0.0001, Mann–Whitney test; axonal branches: Mann–Whitney U = 53, p < 0.0001, Mann–Whitney test. (B) Overview coronal sections in grey scale stained with anti-RFP antibody of an empty vector and a RHEBp.P37L mouse brain in utero electroporated on the left S1 and magnification of the axon terminals on the contralateral S1; scale bars: 500 μm. (C) Representative images of ipsilateral and contralateral S1 area of an empty vector and a RHEBp.P37L mouse coronal section (P50) with quantification of the axonal projections across the different layers in the contralateral cortex measured as normalized fluorescent intensity of the tdTomato signal; numbers in the legend indicate number of targeted mice (N = 3, N = 4) and number of contralateral pictures (n = 10, n = 23) analyzed; data are presented as mean (thick line) ± SEM (shading area); interaction group condition/cortical layers: F(9, 279) = 13.96, p < 0.0001, mixed-effects analysis; control vs RHEBp.P37L L2/3 (bin2–3 from the top): p < 0.0001; control vs RHEBp.P37L L5-L6: bin7, p = 0.0074, bin8, p < 0.0001, bin 9, p = 0.002; Bonferroni multiple comparisons test. The data underlying this figure can be found in S5 Data. **p < 0.01, ****p < 0.0001; scale bars: 50 μm (A), 500 μm (B), and 100 μm (C). DIV1, 1 day in vitro; DIV4, day in vitro 4.