Journal: Nature Communications

Article Title: Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages

doi: 10.1038/s41467-021-24225-2

Figure Lengend Snippet: a Amino acid sequence showing the ADPRylation (blue) and phosphorylation (red) sites in the TA domain of STAT1α. b ADPRylation at D721 in the TA domain of STAT1α is required for IFNγ-dependent phosphorylation. Immunoblots showing total STAT1α, STAT1α S727p and STAT1α Y701p from iBMDMs expressing Wt, DBDmut, or TAmut STAT1α. Uncropped immunoblots are provided as a Source Data file. c ADPRylation of STAT1α stimulates p300 autoacetylation. Immunoblots showing the acetylation of p300 from in vitro reactions performed in the presence of ADPRylated STAT1α under the conditions indicated. The immunoblots are representative of 3 independent experiments. Uncropped immunoblots are provided as a Source Data file. d ADPRylation of STAT1α on its TA domain is required for p300 autoacetylation. Immunoblots show autoacetylation of p300 in the presence of STAT1α Wt, DBDmut or TAmut from ADPRylation reactions with PARP-1 as indicated. The immunoblots are representative of 3 independent experiments. Uncropped immunoblots are provided as a Source Data file. e Loss of ADPRylation on the STAT1α TA domain results in reduced H3K27ac levels at maintained STAT1α binding sites. Box plots of ChIP-seq data showing STAT1α and H3K27ac enrichment in Wt- or TAmut-expressing iBMDMs ( n = 492 peaks; Wilcoxon Signed-Rank test; p < 2.2 × 10 -16 ). Boxes represent 25 th –75 th percentile (line at median) with whiskers at 1.5*IQR. The cells were treated with IFNγ for 1 h. f Line plots representing fold change in IFNγ-stimulated gene expression in iBMDMs expressing Wt or TAmut STAT1α relative to an untreated control. The iBMDMs ectopically expressing Wt or TAmut STAT1α had concurrent shRNA-mediated knockdown of endogenous STAT1α. g Model showing the regulation of pro-inflammatory responses in macrophages by PARP-1-mediated site-specific ADPRylation of STAT1α. See the text for details.

Article Snippet: The following antibodies were used for immunoblotting and immunofluorescent staining: STAT1 rabbit polyclonal antibody (Cell Signaling, 9172 L); Phospho-STAT1α (Ser727) rabbit monoclonal antibody (Cell Signaling, 8826 S); Phospho-STAT1α (Ser727) rabbit polyclonal antibody (Cell Signaling Technologies, 9177); Phospho-STAT1α (Tyr701) rabbit polyclonal antibody (Cell Signaling Technologies, 9167); Flag mouse monoclonal antibody (Sigma-Aldrich, F3165); β-tubulin rabbit polyclonal antibody (Abcam, ab6046); p300 mouse monoclonal antibody (Active motif, 61401); Acetyl-CBP (Lys1535)/p300 (Lys1499) rabbit polyclonal antibody (Cell Signaling, 4771); rabbit IgG (ThermoFisher Scientific, 10500 C); goat anti-rabbit HRP-conjugated IgG (Pierce, 31460); and goat anti-mouse HRP-conjugated IgG (Pierce, 31430).

Techniques: Sequencing, Phospho-proteomics, Western Blot, Expressing, In Vitro, Binding Assay, ChIP-sequencing, Gene Expression, Control, shRNA, Knockdown

Journal: bioRxiv

Article Title: Induction of PARP7 Creates a Vulnerability for Growth Inhibition by RBN2397 in Prostate Cancer Cells

doi: 10.1101/2022.09.02.506406

Figure Lengend Snippet: (A) Treating PC3-AR cells with si PARP7 partially reduces the growth inhibitory effect of RBN2397. PARP7 knockdown in the cell growth experiments (A-C) was confirmed by immunoblotting using TUBULIN as a loading control (upper panels). For the cell growth in A-C, each RBN2397 concentration reflects eight biological replicates, error bars show the standard deviation, and the statistical differences between cell lines at each RBN concentration are ****, p<0.0001; ***, p<0.001; **, <0.01; *, <0.05 (lower panels). (B, C) Stable knockdown of PARP7 in PC3-AR and CWR22Rv1 cells partially protects from the growth inhibitory effects of RBN2397. CWR22Rv1( shGFP ) cell growth is copied from . (D) Immunoblot detection of CDK inhibitor p21 and PARP7 in prostate cancer lines grown +R1881 and +RBN2397 for 24 hrs. (E) Treating PC3-AR cells with si-p21 (20 nM) partially reverses the growth inhibitory effect of RBN2397. The expression levels of p21 and PARP7 +R1881 and +RBN2397 were determined by immunoblotting (upper panel). (F) CRISPR knockout of CDKN1A (p21) in PC3-AR cells reduces the growth inhibitory effect of RBN2397. The knockout was confirmed by DNA sequencing (not shown) and by immunoblotting for p21 (upper panel).

Article Snippet: Commercial antibodies used were TUBULIN (1:10,000–20,000, mouse mAb TUB-1A2, Sigma T9028), HA tag (1:1,000, mouse mAb 16b12, Covance A488-101L, also used for IP), p21 (1 μg/ml, mouse mAb SX118, BD Pharmingen 550827), AHR (1 μg/ml, Rabbit mAb JM34-10, Thermo Fisher Scientific MA5-32576), CYCLIN B1 (1:10,000, rabbit mAb Y106, Epitomics 1495-1), CYCLIN A (1:500, mouse mAb 6E6, Leica Microsystems NCL-CYCLIN A), STAT1α (1:1,000, rabbit mAb EPR4407, abcam ab109320), p-STAT1(Tyr701)(1:1,000, rabbit, Cell Signaling 9171), LAMIN A/C (1:40, mouse mAb JOL2, Millipore MAB3211), Alexa Fluor 680 donkey anti-Rabbit IgG(H + L) (1:20,000, Invitrogen A10043), IRDye800-conjugated anti-mouse IgG(H&L) (1:20,000, Rockland 610-132-121), and AR (mouse mAb G122-434, BD Pharmingen 554225, used for IP).

Techniques: Western Blot, Concentration Assay, Standard Deviation, Expressing, CRISPR, Knock-Out, DNA Sequencing