pc  (ATCC)

Journal: Journal of Translational Medicine

Article Title: Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancer

doi: 10.1186/s12967-023-04156-w

Figure Lengend Snippet: NF-κB-triggered α-Actinin-2 positive auto-regulatory loop enhances ACTN2 gene transcription. A and B α-Actinin-2, RelA, α-Actinin-2 siRNAs and/or RelA siRNAs were cotransfacted with ( A ) ACTN2 Luc-R or ( B ) 3xRelA/NF-κB Luc-R into SNU-16 cells for 12 h, then dual-luciferase reporter assays were performed. The statistical analysis information in A, B: The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. * P < 0.05 using Ordinary one-way ANOVA. C NAGC and HAGC tissue sections obtained after infusion were immunostained for α-Actinin-2 (green) and nucleus (blue). D AGS cells were transfected with α-Actinin-2 for 24 h, then the PM, Cyto and Nu of cells were separated by the Subcellular Protein Fractionation Kit. IP assays were performed in PM, Cyto and Nu by using α-Actinin-2 antibodies. E and F α-Actinin-2, RelA and/or α-Actinin-2 siRNAs were cotransfacted into SUN-16 cells for 24 h, then cell nuclear extracts were subjected to ChIP analysis using the p-RelA antibody. E Western blotting analysis with the p-RelA antibody demonstrates the IP specificity and efficiency. F DNA isolated and purified from immunoprecipitated material was amplified with primers spanning the RelA binding site (Fs/Rs) or primers far away from the RelA binding site (Ff/Rf) of the ACTN2 gene promoter

Article Snippet: si α-actinin-1 a (siRNA ID: 147017), si α-actinin-2 a (siRNA ID: 147020), si α-actinin-4 a (siRNA ID: 147360) or si RelA a (siRNA ID: 109424) specific siRNAs were from Thermo Fisher Scientific and si α-actinin-1 b (Cat No. sc-43095), si α-actinin-2 b (Cat No. sc-43097), si α-actinin-4 b (Cat No. sc-43101) or si RelA b (Cat No. sc-72100) specific siRNAs were from Santa Cruz Biotechnology.

Techniques: Luciferase, Transfection, Fractionation, Western Blot, Isolation, Purification, Immunoprecipitation, Amplification, Binding Assay

Journal: PLoS ONE

Article Title: Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

doi: 10.1371/journal.pone.0008427

Figure Lengend Snippet: (A) Localization of α-actinin-1 and α-actinin-4 in U-373 MG cells cultured on collagen-coated glass. Solid and open arrowheads mark the localization of individual α-actinin isoforms at ruffles and along actin stress fibers, respectively. Scale Bar = 10 µm. (B) Measurement of siRNA efficacy by Western Blot of U-373 MG cells transfected with either scrambled control siRNA (siCTL) or siRNA directed against α-actinin-1 (siACTN1) or α-actinin-4 (siACTN4) (*p<0.05). (C) Subcellular localization of α-actinin-1 and α-actinin-4 following siRNA treatment. Arrowheads depict the redistribution of α-actinin-4 to FAs when α-actinin-1 is suppressed. Scale Bar = 20 µm.

Article Snippet: To suppress α-actinin expression, cells were transfected with isoform-specific α-actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer's specifications.

Techniques: Cell Culture, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

doi: 10.1371/journal.pone.0008427

Figure Lengend Snippet: Effect of α-actinin depletion on mean speeds (mean±SEM, *p<0.001) of cells tracked over 6 hours.

Article Snippet: To suppress α-actinin expression, cells were transfected with isoform-specific α-actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer's specifications.

Techniques:

Journal: PLoS ONE

Article Title: Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

doi: 10.1371/journal.pone.0008427

Figure Lengend Snippet: (A) Projected cell-ECM adhesion area of siCTL (squares), siACTN1 (circles), and siACTN4 (triangles) cells on collagen I-coated polyacrylamide ECMs of varying elasticity. Cell spreading differences between control and α-actinin depleted cells are statistically significant (p<0.001) for both isoforms on 2 kPa and 8 kPa ECMs. (B) Cortical cell elasticity of siCTL, siACTN1, and siACTN4 cells on variable-rigidity ECMs by AFM. Differences in cortical elasticity between control and α-actinin-depleted cells are statistically significant (p<0.001) for all ECM stiffness.

Article Snippet: To suppress α-actinin expression, cells were transfected with isoform-specific α-actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer's specifications.

Techniques:

Journal: PLoS ONE

Article Title: Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

doi: 10.1371/journal.pone.0008427

Figure Lengend Snippet: (A) Effect of α-actinin isoform suppression on vinculin expression by Western Blot. Vinculin expression is significantly lower (*p<0.05) in both α-actinin-1 and α-actinin-4-depleted cells than in control cells. (B) Effect of α-actinin depletion on vinculin localization. Both α-actinin-1 and α-actinin-4 depleted cells contain more vinculin-positive FAs (arrows) than controls. Scale bar = 20 µm. (C) Quantitative analysis of size and number of vinculin-positive adhesions. The right-shift of the siACTN1 and siACTN4 data relative to control demonstrates that both α-actinin-1 and α-actinin-4 depleted cells possess larger and more numerous adhesions than controls. (D) Circularity of vinculin-positive FAs of siCTL, siACTN1 and siACTN4 cells (*p<0.001). In all cases, data are mean±SEM.

Article Snippet: To suppress α-actinin expression, cells were transfected with isoform-specific α-actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer's specifications.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

doi: 10.1371/journal.pone.0008427

Figure Lengend Snippet: Mean speeds of control, α-actinin-1-depleted, and α-actinin-4-depleted cells for 6 hr following 1 hr of incubation with 10 µM ML7 or 10 µM Y27632. Compared to motility of ML7 treated control cells, knockdown of either isoform significantly reduces cell motility (**p<0.001). Depletion of α-actinin-4 (*p<0.05), but not of α-actinin-1, reduces the motility of Y27632 treated cells to levels observed in untreated cells.

Article Snippet: To suppress α-actinin expression, cells were transfected with isoform-specific α-actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer's specifications.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

doi: 10.1371/journal.pone.0008427

Figure Lengend Snippet: (A) Effect of α-actinin suppression on NMMII expression by Western Blot. NMMII expression is significantly lower (*p<0.05) in both α-actinin-1 and α-actinin-4-depleted cells than in controls. (B) Effect of α-actinin depletion on traction force generation measured by traction force microscopy. Box-whisker plots of root-mean squared (RMS) traction of siCTL, siACTN1 and siACTN4 cells on 2 kPa and 18 kPa ECM substrates. The error bars mark the maximum and minimum of the data sets, the three horizontal lines mark the 25th, 50th, and 75th percentile of the data, and the point marks the mean. N>10 cells in all cases.

Article Snippet: To suppress α-actinin expression, cells were transfected with isoform-specific α-actinin siRNA sequences (siACTN1 or siACTN4) or non-targeting sequences (siCTL) (Santa Cruz, CA) per manufacturer's specifications.

Techniques: Expressing, Western Blot, Microscopy, Whisker Assay