Journal: eLife

Article Title: Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (p97-PROTAC)

doi: 10.7554/eLife.101496

Figure Lengend Snippet: ( A ) HeLa cells were co-transfected with a vector expressing αSynuclein mutant A53T fused to GFP (GFP-αSynuclein A53T) and empty or increasing concentrations of UBX-Nb (Syn87) . A53T-GFP degradation was determined by western blot. ( B ) Quantification of A, 2 μg ‘p-value’ 0.0651 (ns), 4 μg ‘p-value’ 0.0020 (** ). ( C ) Cells were co-transfected with a vector expressing untagged α-synuclein mutant A53T and an empty vector or increasing concentrations of UBX-Nb (Syn87) . Untagged αSynuclein A53T degradation was determined by western blot using an anti αSynuclein antibody. ( D ) Quantification of C, 2 μg ‘p-value’ 0.0240 (*), 4 μg ‘p-value’ 0.0210 (* ). ( E ) Quantification of α-synuclein aggregation via Thioflavin T (ThT) fluorescence. Non-transfected cells and cells transfected with GFP-tagged wild-type α-synuclein (pcDNA5 WT αSyn-GFP), either alone or co-transfected with the p97-based PROTACs containing nanobodies against GFP [UBX-Nb (GFP) ] or α-synuclein [UBX-Nb (Syn87) ], were analyzed for relative ThT fluorescence intensity normalized to total protein concentration (μg/μL). Bars represent mean ± SEM. Asterisks (*) indicate statistically significant differences compared to control (*p<0.05, Dunnett’s post hoc test); ‘ns’ indicates no significant difference. ( F ) Representative model of p97-PROTAC functioning in the degradation of proteins and protein aggregates. Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3. Figure 6—source data 1. Raw Western blots showing p97-PROTAC–mediated degradation of GFP-tagged and untagged α-synuclein A53T. Figure 6—source data 2. Annotated Western blots indicating protein bands for p97-PROTAC–mediated degradation of GFP-tagged and untagged α-synuclein A53T.

Article Snippet: Cells were transiently transfected with the following vectors: pcDNA5 FRT/TO GFP-ETV1, pcDNA5 FRT/TO GFP-Emerin, pEYFP-Coilin, pcDNA FRT/TO GFP, VCP(wt)-EGFP (Addgene, #23971), pEGFP-C1-tagged plasmids containing the exon 1 of HTT with 23 CAG repeats (pEGFP-Q23: wild-type HTT, Addgene, #40261) or 74 CAG repeats (pEGFP-Q74: mutant HTT, Addgene, #40262) and pEGFP-Q24 (generously provided by Dr. Maite Castro, Universidad Austral de Chile).

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Fluorescence, Protein Concentration, Control

Journal: European Biophysics Journal

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

doi: 10.1007/s00249-025-01774-8

Figure Lengend Snippet: Localization and recruitment kinetic of p53 at the DNA damage sites in MCF7 cells. a Fluorescence intensities measured in the control condition (DMSO) and 30 min after 1 µM camptothecin (CPT) or 30 min after 3 Gy γ-rays treatments from different channels for each protein specified along the distance (µm) measured and represented with the white line . The graphs represent the mean fluorescence intensity (y-axis), which is expressed as a function of the distance measured inside the nuclei (distance measured in µm on the x-axis). Nucleus delimited by dotted lines , scale bar 7 µm. b On the left , the fluorescence intensity in MCF-7 nuclei measured for 53BP1 and phospho-Ser 15-p53 in control (DMSO), 40 min after CPT, and 40 min after 3 Gy treatment. Fluorescence intensity (y-axis in the graphs) was measured along the distance (µm) (x-axis in the graphs) represented by the white line . Nuclei delimited by dotted lines , scale bar 7 µm. On the right , higher magnification of a 3 Gy-irradiated MCF7 cell nucleus containing co-localizing 53BP1 and phosphor-ser15-p53 spots. The scale bar shows 3 µm. c Recruitment kinetics for p53-GFP and 53BP1-GFP transiently transfected in MCF7 cells. Cells were microirradiated in the regions of interest ( red dotted circle , diameter 2 µm) with a 355 nm UV laser, and representative pictures were obtained by scanning with WLL 488 nm at the specified time points after irradiation. The scale bar indicates 8 µm

Article Snippet: MCF-7 cells were transiently transfected with GFP-tagged p53 (a gift from Tyler Jacks, Addgene 12091) (Boyd et al. ) and GFP-tagged 53BP1 (a gift from Daniel Durocher, Addgene 60813) (Fradet-Turcotte et al. ) plasmids using Metafectene pro Kit (Biotex Laboratories, GimbH, Munchen, Germany) and following the manufacturer’s instructions.

Techniques: Fluorescence, Control, Irradiation, Transfection

Journal: European Biophysics Journal

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

doi: 10.1007/s00249-025-01774-8

Figure Lengend Snippet: γH2AX and 53BP1 detection in 3 Gy-irradiated p53 wild-type and mutant cell lines. a Representative images of γH2AX foci for the cell line considered; cell nuclei are delimited by dotted lines. Scale bars 15 µm. b Bar chart shows the quantification of the number of foci for γH2AX per cell nucleus. c Representative images of 53BP1 foci, each cell nuclei are delimited by dotted lines. The scale bar shows 10 µm. d 53BP1 number of foci measured in each cell nucleus. The mean ± S.D. was calculated from three biological replicates ( N > 500 cells). Statistical significance is shown in Supplementary Table 1

Article Snippet: MCF-7 cells were transiently transfected with GFP-tagged p53 (a gift from Tyler Jacks, Addgene 12091) (Boyd et al. ) and GFP-tagged 53BP1 (a gift from Daniel Durocher, Addgene 60813) (Fradet-Turcotte et al. ) plasmids using Metafectene pro Kit (Biotex Laboratories, GimbH, Munchen, Germany) and following the manufacturer’s instructions.

Techniques: Irradiation, Mutagenesis

Journal: European Biophysics Journal

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

doi: 10.1007/s00249-025-01774-8

Figure Lengend Snippet: Fluorescence recovery after photobleaching for 53BP1-GFP and recruitment of RIF to DSBs. a Representative images of the cell lines assayed for fluorescence recovery at 1 h after 3 Gy exposure. Scale bar 5 µm. Cells were subjected to a single bleach pulse ( red circle ) followed by a real-time recording of 53BP1-GFP fluorescence recovery kinetics. The graphs represent the measurements obtained from b untreated cells and c within irradiated spots where 53BP1 accumulated. The mean ± S.E.M. is shown, n = 50 cells per the condition of two biologically independent experiments were analyzed, one-way ANOVA test was used; *** means p ≤ 0.001. d A representative panel of RIF1 foci. Scale bars show 15 µm. e Number of foci for RIF1 per cell nucleus. Bars represent the mean ± S.D. Data are from >500 cells across three biological replicates. Statistical significance is shown in Supplementary Table 1

Article Snippet: MCF-7 cells were transiently transfected with GFP-tagged p53 (a gift from Tyler Jacks, Addgene 12091) (Boyd et al. ) and GFP-tagged 53BP1 (a gift from Daniel Durocher, Addgene 60813) (Fradet-Turcotte et al. ) plasmids using Metafectene pro Kit (Biotex Laboratories, GimbH, Munchen, Germany) and following the manufacturer’s instructions.

Techniques: Fluorescence, Irradiation