deposited by dyche mullins  (Addgene inc)

Journal: bioRxiv

Article Title: Dual Regulatory Role of Nuclear FNBP4 in Actin Binding and Formin FMN1 Inhibition

doi: 10.64898/2026.01.05.697755

Figure Lengend Snippet: (A) Cells were transfected with the pmCherry-C1 actin-3×NLS P2A mCherry construct. After transfection, the cells were fixed with 3.7% paraformaldehyde and subjected to immunofluorescence staining for FNBP4. FNBP4 was detected using an anti-FNBP4 antibody, and nuclei were counterstained with DAPI. The representative image shows a single optical section from the middle of the Z-stack. Scale bar: 20 µm. (B) Quantification of FNBP4 and actin colocalization using Manders’ Colocalization Coefficient. The scatter dot plot shows the distribution of data points along with standard deviations.

Article Snippet: For transfection studies, the pmCherry-C1 actin-3×NLS P2A mCherry plasmid was obtained from Addgene (RRID:Addgene_58475; deposited by Dyche Mullins).

Techniques: Transfection, Construct, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Nuclear F-actin and Lamin A antagonistically modulate nuclear shape.

doi: 10.1242/jcs.259692

Figure Lengend Snippet: Fig. 5. Dynamic nuclear F-actin alters nuclear shape in HeLa cells. HeLa cells were transfected with the indicated plasmids: mCh (pEmCherry-C2), actinWT–NLS (actin-3×NLS P2A mCherry), actinR62D–NLS (pmCherry-C1 R62D actin-3×NLS P2A) and mCh–Exp6 (pcDNA3.1-mCherry-Exp6). For knockdowns, cells were transfected with a scrambled control (siSCR) or siRNA targeted against Lamin A/C (siLMNA). Fixed cells were stained for Lamin B1 (green) and DNA (Hoechst 33342, blue). Only transfected cells expressing mCherry were quantified. (A) Representative images are shown. (B) Nuclear circularity measurements for A. Based on three independent experiments, the nucleus numbers quantified were: siSCR+mCh (n=283), siSCR+actinWT–NLS (n=357), siLMNA+mCh (n=244) and siLMNA+actinWT–NLS (n=332). (C) Representative images are shown. (D) Nuclear circularity measurements for C. Based on four independent experiments, the nucleus numbers quantified were: siLMNA+mCh (n=382), siLMNA+actinWT–NLS (n=412) and siLMNA+actinR62D–NLS (n=443). (E) Representative images are shown. (F) Nuclear circularity measurements for E. Based on two independent experiments, the nucleus numbers quantified were: siSCR+mCh (n=223), siSCR+mCh–Exp6 (n=195), siLMNA+mCh (n=255) and siLMNA+mCh–Exp6 (n=165). Images were obtained by confocal microscopy. Mean values and 95% c.i. error bars are shown. One-way ANOVA with multiple comparisons and post-hoc Tukey tests were performed, showing statistical significance relative to controls. ns, not significant; **P≤0.01; ***P≤0.001; ****P≤0.0001.

Article Snippet: F-actin was stained using 4 U/ml of Alexa Fluor 488 phalloidin (Invitrogen, A12379). siRNAs and plasmids The following plasmids were used: control plasmid pEmCherry-C2 (a gift from Anne Schlaitz, University of Heidelberg, Heidelberg, Germany); nuclear-targeted actin plasmids pmCherry-C1 actin-3×NLS P2A mCherry (Addgene #58475, deposited by Dyche Mullins) and pmCherry-C1 R62D actin-3×NLS P2AmCherry (Addgene #58477, deposited by DycheMullins) (Belin et al., 2015); nuclear actin-chromobody-GFP plasmid (pnAC-TagGFP; Chromotek, acg-n); and pcDNA3.1-mCherry-Exp6 (gifted by Kei Miyamoto, Kindai University, Osaka, Japan; Okuno et al., 2020).

Techniques: Transfection, Control, Staining, Expressing, Confocal Microscopy