Fig. 5: Knockdown of SETD2 results in reduced microtubule number and polarization defects in cultured neurons.

a Representative images of hippocampal neurons cultured for 3 days from E14 mice. The neurons were electroporated with GFP reporter as well as shNC, mSETD2 shRNA2 or mSETD2 shRNA2 together with Flag-NES-SETD2(1469-1724) or α-tubulin^K40F. Fixed neurons were stained for GFP (green) and SMI-312 (red). Scale bar: 25 μm. b Quantitative analysis of a showed that the percentage of polarized GFP⁺ neurons was significantly decreased and the total number of neurite tips including all neurites and their protrusions was increased after SETD2 knockdown. These defects were rescued by expressing Flag-NES-SETD2(1469-1724) or α-tubulin^K40F. All data were shown as the mean ± s.e.m. (168, 174, 99 and 92 neurons respectively collected from 3 biological replicates with similar results). Data were analyzed using unpaired two-tailed Student’s t test. ***P < 0.001 versus shNC; ^###P < 0.001 versus shRNA2. c Immunoprecipitation by α-TubK40me3 antibody from soluble (S) and polymerized (P) tubulins and following immunoblotting with α-tubulin showed that α-TubK40me3 was preferably distributed on polymerized but not soluble tubulins in cultured E14 cortical neurons (n = 3 biological replicates). d PLA signals (red) representing α-TubK40me3 was present in soma and axon shaft but not grow cone of cultured hippocampal neurons at DIV 3. Neurons were stained for tubulin (green) and DAPI (blue) after PLA reaction (n = 3 biological replicates). Scale bar: 25 μm. e Representative images and kymographs of EB3-tdTomato in E14 hippocampal neurons at DIV 3. The neurons were electroporated with EB3-tdTomato plasmid as well as shNC, mSETD2 shRNA2 or mSETD2 shRNA2 together with Flag-NES-SETD2(1469-1724) or α-tubulin^K40F. Scale bar: 5 μm for the soma and 2.5 μm for the kymograph. f Quantitative analysis of e showed that the numbers of EB3 comets within both soma and neurites were significantly decreased after SETD2 knockdown. These defects were rescued by expressing Flag-NES-SETD2(1469-1724) or α-tubulin^K40F. All data were shown as the mean ± s.e.m. (41, 44, 39 and 35 neurons respectively collected from 3 biological replicates with similar results). Data were analyzed using unpaired two-tailed Student’s t test. ***P < 0.001 versus shNC; ^##P < 0.01, ^###P < 0.001 versus shRNA2. g Representative TEM images of microtubules in the neurites of E14 hippocampal neurons at DIV 2. shNC or mSETD2 shRNA2 was delivered into these neurons by lentivirus. Scale bar: 100 nm. h Quantitative analysis of g showed that the number of microtubules in neurites at different cross-sectional areas was significantly decreased after SETD2 knockdown. All data were shown as the violin plots (>500 neurites each from 2 trials). Red line indicated the median and black line indicated the quartiles. Data were analyzed using unpaired two-tailed Student’s t test. *P < 0.05, ***P < 0.001 versus shNC. i Representative images of E14 hippocampal neurons at DIV3. The neurons were electroporated with GFP reporter as well as shNC or mSETD2 shRNA2. 0.5 nM Taxol was applied to neurons 24 h after plating. Fixed neurons were stained for GFP (green) and SMI-312 (red). Scale bar: 25 μm. j Quantitative analysis of i showed that the defects in neuronal polarization and arborization were rescued by applying 0.5 nM Taxol. All data were shown as the mean ± s.e.m. (91, 99 and 88 neurons respectively collected from 3 biological replicates with similar results). Data were analyzed using unpaired Student’s t test. ***P < 0.001 versus shNC; ^###P < 0.001 versus shRNA2. Source data are provided as a Source Data file.