plvx ef1alpha ires mcherry  (TaKaRa)

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARP inhibitors increase MMEJ at ISceI-mediated deDSBs. ( A, B ) Schematic ( A ) and experimental timeline ( B ) of MMEJ reporter used in panels (C)–(F), Figs and , , , and E, and , and and . Cutting the reporter with ISceI and repair by microhomology annealing to an in-frame, functional mCherry gene. Flow cytometry was used to identify the mCherry + cells (MMEJ+) within the BFP+ (ISceI+) population. ( C–E ) MMEJ quantification using reporter and timeline from panel (A) and (B) in HT1080 cells. Values are normalized to DMSO. Drugs used were olaparib (5 µM or as indicated), DNA-PKcsi (NU7441, 1 µM), Polθi (ART558 10 µM), niraparib (2.5 µM), rucaparib (2.5 µM), and talazoparib (2.5 µM). ( F ) MMEJ quantification in HT1080 parental cells compared to isogenic POLQ -KO cells following olaparib treatment. Values are normalized to wild-type DMSO. Statistical analyses for panels (C)–(F): Data represent three independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, *** P <.001, **** P <.0001.

Article Snippet: The PARP2 plasmid used to complement PARP2 -KO clones was a generous gift from the Karolin Luger lab and was derived by cloning the human PARP2 coding region into plasmid mCherry C1 (obtained from Dyche Mullins [ ], Addgene #58476).

Techniques: Functional Assay, Flow Cytometry, Comparison

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARPi-dependent MMEJ increase does not occur at Cas9-induced DSBs. ( A, B ) Schematic ( A ) and experimental timeline ( B ) of MMEJ reporter used in panels (C)–(E), Figs and . Repair of the Cas9-induced cut with microhomology annealing leads to an in-frame, functional mCherry gene. Flow cytometry was used to identify the mCherry + cells (MMEJ+) within the GFP+ (Cas9+) population. ( C–E ) MMEJ quantification using the reporter and timeline from panels (A) and (B) in HT1080 cells. Values are normalized to DMSO. Drugs used were olaparib (5 µM or indicated dose), DNA-PKcsi (NU7441, 1 µM), Polθi (ART558, 10 µM). ( F ) HR quantification using the DR-GFP reporter in HT1080 cells after an ISceI- or Cas9-induced DSB, ± olaparib treatment (5 µM). Statistical analyses ( C–F ): Data represent three (D–F) or four ( C ) independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, *** P <.001, **** P <.0001.

Article Snippet: The PARP2 plasmid used to complement PARP2 -KO clones was a generous gift from the Karolin Luger lab and was derived by cloning the human PARP2 coding region into plasmid mCherry C1 (obtained from Dyche Mullins [ ], Addgene #58476).

Techniques: Functional Assay, Flow Cytometry, Comparison

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: The Journal of Biological Chemistry

Article Title: Three mammalian VDAC isoforms distinctly regulate mitochondrial function and proteome to maintain cell metabolism

doi: 10.1016/j.jbc.2025.111009

Figure Lengend Snippet: HK1 mitochondrial localization is dependent on VDAC1 isoform . A , representative images of HK1-GFP ( green ) colocalization with Omp25-mCherry ( red ) shown in the merged image ( yellow ) for HeLa cells with WT, VDAC1 KO, VDAC2 KO, and VDAC3 KO. (Scale bar represents 50 μm). B , colocalization analysis of HK1 with Omp25 measured by Pearson’s correlation coefficient (PCC) shows a significant decrease in HK1 mitochondrial localization in VDAC1 KO. Data from three independent experiments are represented. The symbols represent PCC for each cell, and error bars indicate the SD from the mean. Significance was tested using one-way ANOVA followed by Dunnett's post hoc test (∗∗∗∗ p < 0.0001). KO, knockout; VDAC, voltage-dependent anion channel.

Article Snippet: Cells were transfected with HK1-GFP (Addgene, 21917) or HK2-GFP (Addgene, 21920) along with Omp25-mCherry (Addgene, 157758) using Lipofectamine Stem transfection reagent (Thermo Fisher Scientific, STEM00008) according to the manufacturer's protocol.

Techniques: Knock-Out