Journal: Structure (London, England : 1993)
Article Title: PARG inhibition induces nuclear aggregation of PARylated PARP1.
doi: 10.1016/j.str.2024.09.006
Figure Lengend Snippet: Figure 4. PARP1 catalytic activity is required for the formation of PARP1 aggre- gates (A) Representative images of the validation of the U2OS PAR sensor cell line with 5 mM olaparib treatment. Upon olaparib treatment, no PAR sensor signal is detected 1 min after laser-induced damage. (B) Bright spot intensity was quantified and normalized to maximum PAR intensity across 4 different experiments, ***p < 0.001 (Welch’s two- sample t test). (C) Representative images of PARP1-eGFP, APLF- PBZ-mCherry, and the merged images taken pre- bleach, and 15 min and 60 min after laser-induced damage. Cells were pre-treated with 10 mM PARGi for 1 h. The co-localization of PARP1 with PAR and PAR with PARP1 aggregates were represented as percentages calculated using ImageJ with a co- localization tolerance of 1 pixel. Data are pre- sented as mean ± SD, n = 4 nuclei. Scale bar: 5 mm. (D) Schematic of full-length PARP1 protein showing the catalytic domain and the site of the point mu- tation. Western blot showing overexpression of PARP1 catalytic variants, E988D, producing me- dium PAR chains, and E988A, producing no PAR chains in the PARP1/ cell line. The PARP1 cat- alytic variants show reduced PAR chain expression compared to PARP1 wildtype (WT). (E) Representative images of PARP1-WT, -E988A, and -E988D-eGFP cells pre-bleach and 7.5 min post-bleach after pre-treatment with 10 mM PARGi for 1 h. (F) Quantification of normalized PARP1 intensity at the site of damage with and without PARGi treat- ment after laser-induced damage over 15 min. n = 3 nuclei, n.s. = not significant (Welch’s two-sample t test). (G) Quantification of PARP1 aggregates in PARP1- WT, -E988A, and -E988D-eGFP cell lines with and without PARGi treatment at 7.5 min after laser- induced damage. PARP1 aggregates form only in the PARP1 catalytically proficient cell line. Data are presented as mean ± SD, n = 3 nuclei, *p < 0.05 (two-tailed unpaired t test). Scale bar: 10 mm. See also Figure S5.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-PARP1 Cell Signaling Technology Cat#9542; RRID: AB_2160739 Rabbit anti-PAR Millipore Cat#MABE1031; RRID: AB_2665467 Rabbit anti-SIRT6 Cell Signaling Technology Cat#12486; RRID: AB_2636969 Rabbit anti-Histone H3 GeneTex Cat#GTX122148; RRID: AB_10633308 Mouse anti-GAPDH Proteintech Cat#60004; RRID: AB_2737588 Mouse anti-PARG Millipore Cat#MABS61; RRID:AB_10806473 Mouse anti-PARP1 R&D Biosystems, discontinued Cat#4338-MC; RRID: AB_354246 Rabbit anti-XRCC1 Cell Signaling Technology Cat#2375; RRID: AB_2218471 Mouse anti-PAR Abcam Cat#ab14459; RRID: AB_301239 Rabbit anti-cleaved PARP (Asp214) Cell Signaling Technology Cat#5625; RRID: AB_10699459 Rabbit Anti-b-tubulin Cell Signaling Technology Cat#2146; RRID: AB_2210545 Anti-rabbit IgG HRP-linked Cell Signaling Technology Cat#7074; RRID: AB_2099233 Anti-mouse IgG HRP-linked Thermo Fisher Scientific Cat#31432; RRID: AB_228302 Mouse anti-phospho-Histone H2A.X (Ser139) Millipore Cat#05–636; RRID: AB_309864 Rabbit anti-53BP1 Novus Biologicals Cat#NB100-904; RRID: AB_10002714 Alexa Fluor 488 goat anti-mouse Thermo Fisher Scientific Cat#A-11001; RRID: AB_2534069 Alexa Fluor 555 goat anti-mouse Thermo Fisher Scientific Cat#A-21422; RRID: AB_2535844 Alexa Fluor 555 goat anti-rabbit Thermo Fisher Scientific Cat#A-21428; RRID: AB_2535849 Alexa Fluor 647 goat anti-rabbit Thermo Fisher Scientific Cat#A-21245; RRID: AB_2535813 Anti-phospho-Histone H2A.X (Ser139), Alexa Fluor 488 Conjugate Millipore Cat#05-636-AF488; RRID:N/A Bacterial and virus strains MAX Efficiency DH5a Competent Cells Invitrogen Cat#18258012 PARP1 exon 7-targeting lentivirus Dr. Gregory Breuer, Yale University N/A Chemicals, peptides, and recombinant proteins Dimethyl sulfoxide Sigma-Aldrich Cat#D4540 Methyl methanesulfonate Sigma-Aldrich Cat#129925 PDD 00017273 (PARGi) Tocris Cat#5952 Olaparib MedChem Express Cat#HY-10162 5-Bromo-20-Deoxyuridine Sigma-Aldrich Cat#B5002 Talazoparib Selleckchem Cat#S7048 Hydrogen peroxide Sigma-Aldrich Cat#H1009 Temozolomide Selleckchem Cat#S1237 Z-VAD-FMK Selleckchem Cat#S7023 Critical commercial assays Subcellular Fractionation Kit for Cultured Cells Thermo Fisher Scientific Cat#78840 Deposited data Original western blot images This study Mendeley Data https://doi.org/10.17632/ ktpzmk93yg.1 Experimental models: Cell lines U2OS (female) ATCC RRID:CVCL_0042 OVCAR-8 (female) DCTD Tumor Repository RRID:CVCL_1629 U2OS PARP1 / (female) This study N/A (Continued on next page) e1 Structure 32, 2083–2093.e1–e5, November 7, 2024
Techniques: Activity Assay, Biomarker Discovery, Western Blot, Over Expression, Expressing, Two Tailed Test
