Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 1. Overexpression of mutant α-synuclein (α-syn) E46K attenuated neurite outgrowth. We transfected SK-N-SH cells with human wild-type (WT) α-syn, A53T or E46K mutant α-syn, or an empty

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Over Expression, Mutagenesis, Transfection

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 2. Proliferation of mutant α-syn E46K transfectants. We performed cell proliferation assays using the alamarBlue reagent for 72 h after transfection with pcDNA3.1+-α-syn (WT, A53T, or E46K). The fluorescence of the alamarBlue indicator (excitation, 530 nm; emission, 590 nm) was analyzed using a microplate fluorometer. All samples were prepared in triplicate. Data are presented as means ± SD.

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Mutagenesis, Transfection, Fluorescence

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 3. Cell division control 42 effector protein 2 (Cdc42EP2) negatively regulated neurite outgrowth by downregulating βIII-tubulin in E46K α-syn transfectants. (A) Quantitative analysis of Cdc42EP2 mRNA expression. SK-N-SH cells were seeded into six-well plates (1 × 105 cells/well) and cultured for 16 h to achieve attachment and confluency. After transfection with each pcDNA3.1+-α-syn (WT, A53T, and E46K), the cells were maintained at 37 ◦C in an atmosphere of 5% CO2 for 24 h. Then, total RNA was isolated for cDNA synthesis, followed by real-time quantitative PCR using SYBR Green TOPrealTM qPCR PreMIX (Enzynomics) on an Eco Real-Time PCR System (Illumina). Data are presented as means ± SD from four independent experiments. (B) Quantitative analysis of βIII- tubulin gene expression using the same techniques and statistical analyses as described in Figure 3A. (C) Western blot analysis of Cdc42EP2 protein levels in α-syn transfectants, including the empty vector group (control). (D) Western blot analysis of βIII-tubulin levels in α-syn transfectants, including the empty vector group (control). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of prepared cell lysate samples and transferring the proteins onto membranes, western blotting was performed using specific antibodies for human α-syn, Cdc42EP2, or βIII-tubulin. Figure S1 presents representative blots from three independent experiments. Data are presented as means ± SD for three independent experiments performed in triplicate. Analysis was performed using one-way ANOVA (* p < 0.05, ** p < 0.01) and Student t-test (# p < 0.05, ## p < 0.01).

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Control, Expressing, Cell Culture, Transfection, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Gene Expression, Western Blot, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Transferring

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 4. α-Syn-induced Cdc42EP2 expression regulated neurite outgrowth in SK-N-SH cells. Confocal microscopy analysis of SK-N-SH cells transfected with WT, A53T, and E46K α-syn, including empty

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Expressing, Confocal Microscopy, Transfection

Journal: Brain sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells.

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Figure 5. Cdc42EP2 knockdown abrogated α-syn-induced neurite outgrowth. Small interfering RNA (siRNA) targeting Cdc42EP2 was transfected into SK-N-SH cells. Unrelated siRNA was used as a

Article Snippet: For permeabilization, 0.1% Triton X-100 in phosphate-buffered saline (PBS) was applied for 1 min and then blocked with the blocking buffer (10% FBS in PBS) for 1 h. Immunofluorescence was processed with an anti-α-syn antibody (1:1000 dilution) and anti-Cdc42EP2 mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 594 donkey anti-mouse IgG (H+L) conjugated (sc-362278) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (sc-516592) (all from Santa Cruz Biotechnology, 1:1500 dilution) for 1 h at room temperature.

Techniques: Knockdown, Small Interfering RNA, Transfection