Journal: Plant Physiology

Article Title: Differential Effects of Prenylation and S -Acylation on Type I and II ROPS Membrane Interaction and Function ^{1 [W] [OA]}

doi: 10.1104/pp.110.166850

Figure Lengend Snippet: Subcellular localization of rop6CA and AGG1. A, Fluorescence images showing the subcellular distribution of GFP-rop6CA in wild-type Col-0 (WT) and pggt-Ib (gg-b) mutant leaf epidermis pavement cells. Note the nuclei and cytoplasmic strings in the pggt-Ib cells. Bars = 20 μm. B, Protein immunoblots decorated with anti-ROP polyclonal antibodies (6CA and EN-ROPs) or with anti-GFP monoclonal antibodies (AGG1). Soluble (Sup) and insoluble (Pellet) fractions were separated by floatation on Suc density gradients from wild-type Col-0 and pggt-Ib leaves. 6CA, GFP-His6-rop6CA; AGG1, YFP-AGG1; EN-ROPs, endogenous ROPs. Numbers on the side correspond to molecular mass in kD. Note the presence of rop6CA, endogenous ROPs, and AGG1 in the soluble fractions in pggt-Ib extracts.

Article Snippet: A cDNA of the improved, higher fluorescence Venus version of YFP ( Nagai et al., 2002 ) was inserted between Xho I and Hin dIII of the pLPCX multiple cloning site.

Techniques: Fluorescence, Mutagenesis, Western Blot, Bioprocessing

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: Generation and basic characterization of the SIK1 MT mice. (A) Genomic organization of SIK1 gene (top), domain structure of wild-type (middle), and mutant SIK1 (bottom) protein. The locations of the mutations identified in human patients are indicated on the domain structure of SIK1-WT. Nucleotide and amino acid sequences of target regions are shown. The target sequence of single guide RNA (sgRNA) is underlined. Converted amino acids after the mutated site are indicated in red. The SIK1 protein consists of four functional domains, including serine/threonine kinase, ubiquitin-associated domain (UBA), proline-glutamate-serine-threonine (PEST), and C-terminal nuclear localization regulatory domain (NLD). Fourteen exons exist in the mouse SIK1 gene. sgRNA was designed to target the coding region in exon 13 of SIK1. (B) Subcellular localization of Venus-tagged SIK1 mutants in HEK293T cells. Wild-type SIK1 was restricted to the nucleus in a punctate pattern, whereas SIK1-MT and pathological nonsense mutant of SIK1-Q614X were distributed both in the nucleus and in the cytoplasm. Scale bar indicates 15 μm. (C) The ratios between wild-type and mutant mRNA were analyzed in a wild-type, heterozygote SIK1-MT, and plasmid DNA mixture containing SIK1-MT at different ratios (WT:MT = 1:1 or 1:0.5), which are used as standards. Mutant SIK1 transcripts were detected at a similar level with plasmid DNA containing 50% of SIK1-MT. (D) Body weights of male wild-type (Male WT), male mutant (Male MT), female wild-type (Female WT), and female mutant (Female MT) mice are shown in the graph. Body weights are unchanged in the SIK1-MT mice. (E) The brain structure is unchanged in the SIK1-MT mice. Sagittal (left) and coronal (right) brain sections were stained with DAPI. Scale bars indicate 1 mm. (F) Representative traces of miniature excitatory postsynaptic currents (mEPSC) recorded from the layer 5 pyramidal neurons in the medial prefrontal cortex (mPFC) of wild-type and SIK1-MT mice. (G) Cumulative distribution of inter-event intervals of and the mean values of the frequency of mEPSCs were shown in the graph. The frequency of mEPSCs was increased in the SIK1-MT mice. (H) Cumulative distribution and the mean values of the amplitude of mEPSCs were shown in the graph. The amplitude of mEPSCs was unchanged in the SIK1-MT mice. (I) Representative traces of mIPSC recorded from the layer 5 pyramidal neurons in mPFC of SIK1-WT and SIK1-MT mice were shown. (J) Cumulative distribution of the inter-event intervals of mEPSC and the mean values of the frequency of mIPSCs were shown in the graph. The frequency of mIPSCs was unchanged in SIK1-MT mice. (K) Cumulative distributions and mean values of the amplitude of mIPSCs were shown in the graph. The amplitude of mIPSCs was unchanged in SIK1-MT mice. (L) The scatter plot shows the relationship between the frequencies of mIPSC (x-axis) and mEPSC (y-axis). Each dot represents a single neuron from WT (black) or MT (red). (M) Excitatory and inhibitory (E/I) synaptic balance index of WT and SIK1-MT mice is shown. E/I balance in SIK1-MT is shifted to excitatory dominance. The numbers of neurons and mice used in each analysis are shown on the bar (neurons/mice) in the graphs. Statistical analysis was made by a one-way ANOVA followed by Bonferroni's post-hoc test (for the EL of SIK1 gene expression) or Student's t -test (for the mean of the synaptic parameters). Statistical significance was indicated by asterisks (* p < 0.05 and *** p < 0.001).

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques: Mutagenesis, Sequencing, Functional Assay, Ubiquitin Proteomics, Plasmid Preparation, Staining, Gene Expression

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: Membrane and firing properties were changed in pyramidal neurons in layer 5 of the mPFC. (A–C) Resting membrane potential (A) , input resistance (B) , membrane capacitance (C) of pyramidal neurons in layer 5 of mPFC of SIK1-WT or -MT mice. (D–G) threshold (D) , half-peak width (E) , rise time (F) , and decay time (G) of the action potential of the pyramidal neurons in layer 5 of the mPFC. (H) Representative traces of induced action potentials responded to 240 pA injected currents. (I) Graph for the relationship between spike frequency and injected current. Input resistance and spike frequency of action potential were increased, and membrane capacitance and decay time of action potential were decreased in SIK1-MT mice. The numbers of neurons and mice used in each analysis are shown on the bar (neurons/mice) in the graphs. Statistical analysis was made by Student's t -test. Statistical significance was indicated by asterisks (* p < 0.05 and ** p < 0.01).

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques: Membrane, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: Repetitive and social behaviors were altered in SIK1-MT mice. (A) Trajectories of movement of SIK1-WT and -MT mice in the open field arena. (B) Total travel distance of SIK1-WT ( n = 9) and -MT ( n = 7) mice. (C) The percentage of the time spent in the center of the arena. (D) The total time of vertical activities in the open field. (E) The number of grooming during the observation period. The number of grooming was increased in SIK1-MT mice. (F) Moving trajectories of mice on the elevated plus-maze. (G) The number of entries into open arms of the elevated plus-maze. The number of the entry was unchanged in SIK1-MT mice. (H) The top views of the marble burying test field. Sixteen marbles were placed at the start of the test (top). Buried marbles were counted after 10–min of the test (bottom). (I) The numbers of marbles buried under the woodchip after the test. SIK1-MT mice buried more marbles in this test. (J) The sociability test evaluating the sociability by the time spent with the empty cage (E) vs. the cage with a stranger mouse (S1). Heatmaps represent the stay time of the test mouse. (K) Both WT and SIK1-MT mice interacted more with S1 than E and the preference to E vs. S1 was similar between these mice (graph). (L) Social novelty test evaluating social memory by the time spent with the stranger mouse (S1) versus novel stranger mouse (S2). Heatmaps represent the stay time of the test mouse. (M) WT mice interacted more with S2 than with S1, whereas the preference to S2 was significantly decreased in SIK1-MT mice (graph). Statistical analysis was made by Student's t -test. Statistical significance was indicated by asterisks (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: Ultrasonic vocalization was not altered between SIK1-WT and SIK1-MT mice. (A) Representative sonograms recorded from isolated mice (P14) were shown. (B) Both wild-type and mutant mice made a variety of vocalizations during the isolation from their mom. (C) The number of calls was not altered during the developmental stages we examined (no statistical significance was detected by t -test). Eight SIK1-WT and seven SIK1-MT mice were analyzed. Statistical analysis was made by Student's t -test.

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques: Isolation, Mutagenesis

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: Risperidone reduces frequencies of mEPSCs and action potentials in the SIK1-MT mice. (A) Representative traces of mEPSC recorded from pyramidal neurons in layer 5 of the mPFC in saline (Sal)- or risperidone (Ris)-injected SIK1-MT mice. (B) Cumulative distribution of inter-event intervals and the mean values of the frequency of mEPSCs were shown in the graph. The frequency of mEPSCs was decreased in risperidone-injected SIK1-MT mice. (C) , Cumulative distribution and the mean values of the amplitude of mEPSCs were shown in the graph. The amplitude of mEPSCs was unchanged between saline- and risperidone-injected SIK1-MT mice. (D) Representative traces of mIPSC recorded from the layer 5 pyramidal neurons in mPFC of saline (Sal)- and risperidone (Ris)-injected SIK1-MT mice were shown. (E) Cumulative distribution of the inter-event intervals and the mean values of the frequency of mIPSCs were shown in the graph. (F) Cumulative distributions and mean values of the amplitude of mIPSCs were shown in the graph. (G) The scatter plot shows the relationship between the frequencies of mIPSC (x-axis) and mEPSC (y-axis). Each dot represents a single neuron from saline- or risperidone-injected SIK1-MT mice. (H) E/I balance index of saline (Sal)- and risperidone (Ris)-injected SIK1-MT mice is shown. E/I balance is unchanged between saline- and risperidone-injected SIK1-MT mice. (I–K) Resting membrane potential (I) , input resistance (J) , membrane capacitance (K) of pyramidal neurons in layer 5 of mPFC of saline (Sal)- and risperidone (Ris)-injected SIK1-MT mice. (L–N) half-peak width (L) , rise time (M) , and decay time (N) of the action potential of the pyramidal neurons in layer 5 of the mPFC of saline (Sal)- and risperidone (Ris)-injected SIK1-MT mice. (O) Representative traces of induced action potentials responded to 240 pA injected currents. (P) Graph for the relationship between spike frequency and injected current. Spike frequency of the action potential was decreased in risperidone-injected SIK1-MT mice. The numbers of neurons and mice used in each analysis are shown on the bar (neurons/mice) in the graphs. Statistical analysis was made by Student's t -test. Statistical significance was indicated by asterisks (* p < 0.05).

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques: Saline, Injection, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: Administration of risperidone ameliorated increased repetitive behaviors but not social deficits in SIK1-MT mice. (A) Total travel distance of saline (Sal)- and risperidone (Ris)-injected SIK1-MT mice. (B) The percentage of the time spent in the center of the open field arena. (C) Total time of the vertical activities of saline (Sal, n = 10) and risperidone (Ris, n = 9) -injected SIK1-MT mice. (D) The number of groomings during the observation period. (E) The number of marbles buried in the marble burying test. The number of buried marbles was less in risperidone-injected SIK1-MT mice compared to that of saline-injected. (F,G) Sociability and the social novelty test between saline- and risperidone-injected SIK1-MT mice. Risperidone did not affect social behavior in SIK1-MT mice. The numbers of neurons and mice used in each analysis are shown on the bar (neurons/mice) in the graphs. Statistical analysis was made by Student's t -test. Statistical significance was indicated by asterisks (* p < 0.05 and ** p < 0.01).

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques: Saline, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1

doi: 10.3389/fnmol.2021.706494

Figure Lengend Snippet: A schematic illustration of neuronal abnormality in SIK1-MT mice. In SIK1-MT mice, excitatory synaptic input onto pyramidal neurons in layer 5 of mPFC is increased, resulting in the disruption of E/I balance (middle). Risperidone restores the increased excitatory synaptic input. Risperidone also decreases the inhibitory input onto the pyramidal neurons. As a result, disrupted E/I balance is maintained in the risperidone-treated SIK1-MT mice (right).

Article Snippet: For the overexpression of SIK1 variants, we made cDNAs of SIK1 fused with Venus YFP (Nagai et al., ) at N-terminal of SIK1.

Techniques: Disruption

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: Interaction of DCC with TUBB3 mutants. HeLa cells were transfected with human full-length DCC-Myc plus wild type human TUBB3 (A–D) or FLAG-tagged TUBB3 mutants (G82R, T178M, E205K and A302V in A, A302T, M388V, R262C and R62Q in B, M323V, E410K, D417H and D417N in C, and truncation mutants TUBB3-1-232 and TUBB3-233-449 in D, respectively). The anti-DCC antibody was used to immunoprecipitate DCC and the blot analyzed with anti-FLAG and anti-DCC antibodies. Relative binding of DCC to TUBB3 in A, B and C was quantified in the corresponding lower panel. The Y axis is the normalized ratio of mean intensity (arbitrary units) of FLAG to DCC. Data are mean ± s.e.m from three separate experiments. *p<0.05, **p<0.01, ***p<0.0001, ns, no significant difference (one-way Anova and Tukey’s test for post-hoc comparsions).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Transfection, Binding Assay

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: TUBB3 mutations reduce the subcellular overlap of DCC with TUBB3 in the GC of primary neurons. A. Knockdown of endogenous TUBB3 in primary neurons. E15 mouse cortical neurons were nucleofected with several shRNAs targeting 3'UTR of human and mouse TUBB3. shRNA #1 and #4 dramatically reduced endogenous TUBB3 protein levels. ** indicates p<0.01 (one-way Anova and Tukey’s test for post-hoc comparsions). B. Expression of wild type human TUBB3, R262C and A302V could restore TUBB3 protein levels after knockdown of endogenous TUBB3. E15 cortical neurons were transfected with control shRNA, TUBB3 shRNAs (#1 and #4), TUBB3 shRNAs (#1 and #4) plus wild type human TUBB3, TUBB3 shRNAs (#1 and #4) plus R262C, or TUBB3 shRNAs (#1 and #4) plus A302V, respectively. TUBB3 and DCC were examined by Western blotting. C–K. TUBB3 shRNAs were co-transfected with the wild type human TUBB3 (C–E), R262C (F–H) or A302V (I–K) into mouse E15 cortical neurons. Primary neurons were cultured for 2 days and immunostained with anti-DCC and anti-FLAG antibodies. E, H and K are the merged images of C and D, F and G, and I and J, respectively. Scale bar: 10µm. L. Quantitative analysis of PCC in the ROI of the GC. The value of PCC in C–E, F–H, and I–K is 0.53 ± 0.08, 0.28 ± 0.08, and 0.3 ± 0.07, respectively. 30 GCs in each group were analyzed. *** indicates p<0.0001 (one-way Anova and Tukey’s test for post-hoc comparisons). M. Analysis of PCC in the ROI of the GC after 90 degree counterclockwise rotation of the red fluorescence channel. ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparisons).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Knockdown, shRNA, Expressing, Transfection, Control, Western Blot, Cell Culture, Fluorescence

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: TUBB3 mutations inhibit netrin-1-induced interaction of DCC with TUBB3. A–D. TUBB3 shRNAs were co-transfected with wild type human TUBB3 (A–D), R262C (A and B) or A302V (C and D) into mouse E15 cortical neurons. E–F. Mouse E15 cortical neurons were transfected with either wild type human TUBB3 or A302V. Primary neurons were cultured overnight and stimulated with purified netrin-1 or sham-purified control for 20 min. Cell lysates were immunoprecipitated with anti-DCC and followed by probing with anti-DCC, anti-FLAG antibodies. B, D and F. Quantification of relative binding of DCC to TUBB3-FLAG (B, D and F), R262C-FLAG (B) and A302V-FLAG (D and F). The Y axis is the normalized ratio of mean intensity (arbitrary units) of FLAG to DCC. Data are mean ± s.e.m from three separate experiments. *p<0.05, **p<0.01, ***p<0.0001, ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparsions).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Transfection, Cell Culture, Purification, Control, Immunoprecipitation, Binding Assay

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: TUBB3 mutations decrease the interaction of DCC with polymerized TUBB3 mutants. A. DCC was cotransfected with wild type human TUBB3, R262C or A302V into HeLa cells and a cosedimentation assay was performed. DCC and TUBB3 proteins in the pellet (P) and supernatant (S) fractions were analyzed by immunoblotting using anti-DCC and anti-FLAG antibodies, respectively. B. Quantification of P/S ratio of DCC and TUBB3 in A (three independent experiments). *** indicates p<0.0001, ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparisons). C-F. TUBB3 shRNAs were co-transfected with wild type TUBB3 (C–F), R262C (C and D) or A302V (E and F) into mouse E15 cortical neurons. Primary neurons were stimulated with purified netrin-1 or sham-purified control. The co-sedimentation assay of cell lysates was conducted with taxol to stabilize MTs in vitro. DCC and TUBB3-FLAG in the P and S fractions were examined by Western blotting. D and F are quantification of P/S ratio of DCC and TUBB3 in C and E, respectively. Data are mean ± s.e.m from three separate experiments. *p<0.05, **p<0.01, ***p<0.001, ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparisons). WT, wild type human TUBB3-FLAG.

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Western Blot, Transfection, Purification, Control, Sedimentation, In Vitro

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: TUBB3 mutations inhibit netrin-1-induced neurite outgrowth of cortical neurons. A–J. Venus YFP was co-transfected with TUBB3 control shRNA (A and B), TUBB3 shRNAs (C and D), TUBB3 shRNAs plus the wild type human TUBB3 (E and F), TUBB3 shRNAs plus R262C (G and H) or TUBB3 shRNAs plus A302V (I AND J), into E15 cortical neurons. Primary neurons were incubated with purified netrin-1 (B, D, F, H and J) or sham-purified control (A, C, E, G and I) for 20 h and stained with Alexa Fluor® 555 phalloidin (red) and DAPI (blue). Scale bar, 10 µm. (K) Quantification of netrin-1-induced neurite outgrowth. The Y axis is the longest neurite length per neuron. Data are mean ± s.e.m. 100 neurons/group from three independent experiments were analyzed. *p<0.05, ***p<0.0001, ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparisons).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Transfection, Control, shRNA, Incubation, Purification, Staining

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: Expression of either R262C or A302V in cortical neurons without knockdown of endogenous TUBB3 inhibits netrin-1-induced neurite outgrowth. Venus YFP was co-transfected with the wild type human TUBB3 (A and B), R262C (C and D) or A302V (E and F) into E15 cortical neurons. Primary neurons after transfection were incubated with purified netrin-1 (B, D and F) or sham-purified control (A, C and E) for 20 h and stained with Alexa Fluor® 555 phalloidin (red) and DAPI (blue). Scale bar, 10 µm. (G) Quantification of netrin-1-induced neurite outgrowth. Data are mean ± s.e.m. 79 neurons/group from three independent experiments were analyzed. ***p<0.0001 (one-way Anova and Tukey’s test for post-hoc comparisons).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Expressing, Knockdown, Transfection, Incubation, Purification, Control, Staining

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: TUBB3 mutations block netrin-1-induced axon branching of cortical neurons. A-J. Venus YFP was co-transfected with control shRNA (A, B), TUBB3 shRNAs (C, D), TUBB3 shRNAs plus wild type TUBB3 (E, F), TUBB3 shRNAs plus A302V (G, H) or TUBB3 shRNAs plus R262C (I, J) into E15 mouse cortical neurons. Primary neurons after nucleofection were cultured with purified netrin-1 (B, D, F, H and J) or sham-purified control (A, C, E, G, I) for 72 h. Scale bar: 10 µm. (K) Quantification of total branching numbers. 100 neurons/group from three independent experiments were analyzed. Data are mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.0001, ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparisons).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Blocking Assay, Transfection, Control, shRNA, Cell Culture, Purification

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: Expression of either R262C or A302V in primary cortical neurons without knockdown of endogenous TUBB3 inhibits netrin-1-induced axon branching. A–F. Venus YFP was co-transfected with wild type TUBB3 (A, B), R262C (C, D), or A302V (E, F), into E15 mouse cortical neurons. Transfected neurons were incubated with purified netrin-1 (B, D, and F) or sham-purified control (A, C, and E) for 72 h. Scale bar: 10 µm. (G) Quantification of total branching numbers. 78 neurons/group from three independent experiments were analyzed. Data are mean ± s.e.m. ***p<0.0001 (one-way Anova and Tukey’s test for post-hoc comparisons).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Expressing, Knockdown, Transfection, Incubation, Purification, Control

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: TUBB3 mutations inhibit netrin-1-induced CA attraction. A. Schematic diagram showing in ovo electroporation and an open-book CA turning assay (Huang et al., 2015; Li et al., 2008; Liu et al., 2004; Liu et al., 2007; Liu et al., 2009; Qu et al., 2013a; Qu et al., 2013b). B–G. Venus YFP together with wild type human TUBB3 (B–C), R262C (D–E) or A302V (F–G) were electroporated into chick neural tubes and explants cultured with either netrin-1 cells (C, E and G) or control HEK293 cells (B, D and F). The scale bar is 100 µm. L. Quantification of axon turning. Data are mean ± s.e.m ***p<0.001 (One-way ANOVA with Tukey’s test for post-hoc comparisons). The axons from commissural neurons transfected with wild type TUBB3, but not R262C or A302V, were attracted to the netrin-1 source.

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: In Ovo, Electroporation, Cell Culture, Control, Transfection

Journal: Neuroscience

Article Title: Human TUBB3 Mutations Disrupt Netrin Attractive Signaling

doi: 10.1016/j.neuroscience.2018.01.046

Figure Lengend Snippet: A. Schematic diagram showing chick spinal cord CA projection after electroporation. B. The chick neural tube was electroporated with Venus YFP only. C. Chick commissural neurons were electroporated with Venus YFP plus wild type human TUBB3. D. Venus YFP plus A302V were electroporated into the chick neural tube. E. Commissural neurons with Venus YFP plus R262C. The red arrows in D and E point to misguided axons. Scale bar, 100 µm. F. Quantification of the percentage of axons reaching the midline. G. Quantification of the average distance of chick CAs away from the midline. H. The percentage of embryos with misguided axons. Data are presented as the mean ± s.e.m in F–G. ***p<0.001 (One-way ANOVA with Tukey’s test for post-hoc comparisons); ns, not significant. The numbers of embryos tested were: 12 (the Venus YFP group); 15 (the wild type human TUBB3 group; 14 (the A302V group); 14 (the R262C group).

Article Snippet: E15 cortical neurons were dissociated and nucleofected with Venus YFP plus missense human TUBB3 mutants, TUBB3 control shRNA, TUBB3 shRNAs (combination of TUBB3 shRNA #1 and #4), TUBB3 shRNAs plus wild type human TUBB3 or TUBB3 shRNAs plus TUBB3 mutants (Amaxa Biosystems).

Techniques: Electroporation