Journal: eLife

Article Title: Genome editing in the unicellular holozoan Capsaspora owczarzaki suggests a premetazoan role for the Hippo pathway in multicellular morphogenesis

doi: 10.7554/eLife.77598

Figure Lengend Snippet:

Article Snippet: To generate a plasmid expressing Venus and Geneticin resistance (pJP114), the Venus reading frame was amplified from p EF1α -CoH2B:Venus (Addgene #111877, ) using primers oJP103 and oJP104 and cloned into a KpnI and AflII digest of pJP72 by Gibson assembly.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Amplification

Journal: Cells

Article Title: Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

doi: 10.3390/cells12030427

Figure Lengend Snippet: Figure 1. The representative illustrations of the cloned Spastin constructs used in the study. The full-length wild-type or mutant Spastin constructs expressing both M1 and M87-Spastin isoforms are named SpastinM1&M87 (Thr292Pro&Thr303Pro), SpastinM1&M87_mutA (Ala292Pro&Ala303Pro), or SpastinM1&M87_mutD (Asp292Pro & Asp303Pro), and wild-type or mutant Spastin constructs express- ing only the M87 isoform of Spastin are named SpastinM87, SpastinM87_mutA, and SpastinM87_mutD, respectively. In addition, the wild-type or mutant constructs expressing only the microtubule- binding domain (MBD) of Spastin consisting of amino acids from 198 to 338 are named SpastinMBD, SpastinMBD_mutA, and SpastinMBD_mutD.

Article Snippet: SpastinMBD vec- Cells 2023, 12, 427 3 of 21 tors (SpastinMBD, SpastinMBD_mutA, and SpastinMBD_mutD) containing only the MBD domain (198-338 aa) of Spastin and M87-Spastin vectors (SpastinM87, SpastinM87_mutA, and SpastinM87_mutD) expressing only the M87 isoform of Spastin (87-617 aa) were constructed by cloning the DNA fragments amplified from wild-type and mutant full-length Spastin vectors into pcDNA3.1 (+)/myc-His A vector (Addgene, Watertown, MA, USA) at HindIII/XhoI restriction sites.

Techniques: Clone Assay, Construct, Mutagenesis, Expressing, Binding Assay

Journal: Cells

Article Title: Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

doi: 10.3390/cells12030427

Figure Lengend Snippet: Figure 5. Pin1 interacts with Spastin through Pin1 recognition motifs in its microtubule-binding domain (MBD). (A) The expression levels of the proteins expressed from either the full-length or MBD-only Spastin constructs were analyzed through Western blot (WB). Green bands indicate the expressions of either the full-length Spastin (left panel) or MBD-only proteins (right panel) in the wild-type or mutant forms. β-Actin observed as red bands was used as the loading control. (B,C) The pull-down assay was used to examine the interaction of the full-length Spastin with Pin1 (B) and actin (C), respectively. SpastinM1&M87 proteins were precipitated from cell lysates (HEK293T in B, and T98G in C) overexpressing the wild-type or mutant SpastinM1&M87 proteins dually with Pin1- Flag. Then, co-precipitation of Pin1 or actin with each wild-type and mutant Spastin proteins was investigated by WB. (D) The bar graphs represent the amount of co-precipitated actin with the full- length wild-type or mutant SpastinM1&M87 protein. The quantification was performed by dividing the amount of precipitated actin to the amount of precipitated wild-type or mutant Spastin proteins. (E) The interaction between Spastin’s MBD region with Pin1 was investigated by pull-down assay. Each SpastinMBD protein was precipitated from HEK293T cells overexpressing either the wild-type or mutant SpastinMBD constructs singly. Then, co-precipitation of the endogenous Pin1 or actin with each wild-type or mutant Spastin protein was investigated by WB. (F) To analyze the impact of EGF treatment in the interaction of Spastin with Pin1, pull-down experiment was performed. Exogenous wild-type Spastin protein was precipitated from lysates isolated from SpastinM1&M87-overexpressed T98G cells either treated with EGF or not. The co-precipitation of Flag-Pin1 or actin with Spastin was investigated by WB.

Article Snippet: SpastinMBD vec- Cells 2023, 12, 427 3 of 21 tors (SpastinMBD, SpastinMBD_mutA, and SpastinMBD_mutD) containing only the MBD domain (198-338 aa) of Spastin and M87-Spastin vectors (SpastinM87, SpastinM87_mutA, and SpastinM87_mutD) expressing only the M87 isoform of Spastin (87-617 aa) were constructed by cloning the DNA fragments amplified from wild-type and mutant full-length Spastin vectors into pcDNA3.1 (+)/myc-His A vector (Addgene, Watertown, MA, USA) at HindIII/XhoI restriction sites.

Techniques: Binding Assay, Expressing, Construct, Western Blot, Mutagenesis, Control, Pull Down Assay, Isolation

Journal: Cells

Article Title: Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

doi: 10.3390/cells12030427

Figure Lengend Snippet: Figure 6. M87-Spastin is directed to actin filaments through phosphorylation of the microtubule- binding domain (MBD). (A) The expression levels and molecular weights of the proteins expressed from SpastinM87, or SpastinM1&M87 vectors were analyzed through Western blot (WB). T98G cells were transfected with either mock, SpastinM87, or SpastinM1&M87 vectors, and then endogenous or overexpressed Spastin expressions were detected by WB using a Spastin-specific antibody. β-actin was used as the loading control. (B) Immunocytochemistry (ICC) analysis was used to examine the impact of EGF treatment on the localization of wild-type M87-Spastin. T98G cells were transfected with SpastinM87 vector for 24 h with or without EGF treatment. Then, cells were fixed with PFA and stained with Myc-tag antibody for Spastin (red), phallacidin for actin filaments (green), and tubulin antibody for microtubule (gray). (C,D) ICC was used to detect the localization of mutant M87 proteins relative to both microtubules and actins (C) or relative to actin filaments with only higher magnification (D). T98G cells were transfected with either SpastinM87_mutA or SpastinM87_mutD vectors for 24 h. Then, cells were fixed with PFA and stained with Myc-tag antibody for Spastin (red), phallacidin for actin filaments (green), and tubulin antibody for microtubule (gray). Arrows indicate color transformation caused by the co-localization of Spastin with actin filaments (in B,C; lane of Spastin actin overlay), while arrowheads indicate microtubule structure that enriched with Spastin and actin filaments (in C; lane of microtubule).

Article Snippet: SpastinMBD vec- Cells 2023, 12, 427 3 of 21 tors (SpastinMBD, SpastinMBD_mutA, and SpastinMBD_mutD) containing only the MBD domain (198-338 aa) of Spastin and M87-Spastin vectors (SpastinM87, SpastinM87_mutA, and SpastinM87_mutD) expressing only the M87 isoform of Spastin (87-617 aa) were constructed by cloning the DNA fragments amplified from wild-type and mutant full-length Spastin vectors into pcDNA3.1 (+)/myc-His A vector (Addgene, Watertown, MA, USA) at HindIII/XhoI restriction sites.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Western Blot, Transfection, Control, Immunocytochemistry, Plasmid Preparation, Staining, Mutagenesis, Transformation Assay

Journal: Cells

Article Title: Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

doi: 10.3390/cells12030427

Figure Lengend Snippet: Figure 7. M87-Spastin is directed to actin filaments due to its interaction with Pin1. T98G cells were dually transfected with each mutant M87 vector (SpastinM87_mutA or SpastinM87_mutD) and Pin1-Flag vector. After 24 h of transfection, cells were fixed with PFA and stained with Myc-tag antibody for Spastin (red), Flag-tag antibody for Pin1 (yellow), phallacidin for actin filaments (green), and tubulin antibody for microtubule (gray). Arrows indicate color transformation caused by the co-localization of Spastin with actin filaments.

Article Snippet: SpastinMBD vec- Cells 2023, 12, 427 3 of 21 tors (SpastinMBD, SpastinMBD_mutA, and SpastinMBD_mutD) containing only the MBD domain (198-338 aa) of Spastin and M87-Spastin vectors (SpastinM87, SpastinM87_mutA, and SpastinM87_mutD) expressing only the M87 isoform of Spastin (87-617 aa) were constructed by cloning the DNA fragments amplified from wild-type and mutant full-length Spastin vectors into pcDNA3.1 (+)/myc-His A vector (Addgene, Watertown, MA, USA) at HindIII/XhoI restriction sites.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Staining, FLAG-tag, Transformation Assay

Journal: Cells

Article Title: Spastin Promotes the Migration and Invasion Capability of T98G Glioblastoma Cells by Interacting with Pin1 through Its Microtubule-Binding Domain.

doi: 10.3390/cells12030427

Figure Lengend Snippet: Figure 9. Real-time monitoring and measurement of T98G cell motility upon M87 overexpression. (A) Expression levels of exogenous mutant M87-Spastin proteins were analyzed by Western blot (WB). T98G cells were transfected with either mock, SpastinM87_mutA, or SpastinM87_mutD vectors and analyzed 24 h post-transfection with immunoblotting using Myc-tag antibody for M87 proteins. β-actin was used as a loading control. Representative WB is shown, and relative densitometric values of M87-Spastin/β-actin are reported in the right panel. (B,C) xCelligence impedance-based system was used for the migration (B) and invasion (C) analysis. Results were statistically analyzed by one-way ANOVA from two independent biological replicates containing at least three independent technical replicates. Data are represented as mean ± SD; n.s. p > 0.05, * p <0.05, *** p <0.001, and **** p < 0.0001 (right panel).

Article Snippet: SpastinMBD vec- Cells 2023, 12, 427 3 of 21 tors (SpastinMBD, SpastinMBD_mutA, and SpastinMBD_mutD) containing only the MBD domain (198-338 aa) of Spastin and M87-Spastin vectors (SpastinM87, SpastinM87_mutA, and SpastinM87_mutD) expressing only the M87 isoform of Spastin (87-617 aa) were constructed by cloning the DNA fragments amplified from wild-type and mutant full-length Spastin vectors into pcDNA3.1 (+)/myc-His A vector (Addgene, Watertown, MA, USA) at HindIII/XhoI restriction sites.

Techniques: Over Expression, Expressing, Mutagenesis, Western Blot, Transfection, Control, Migration