sgrnas targeting exon 2  (Synthego Inc)

Journal: Cancer Research Communications

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

doi: 10.1158/2767-9764.CRC-23-0086

Figure Lengend Snippet: Growth inhibition by RBN2397 is PARP7 dependent and associated with elevated CDKN1A (p21) expression. 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 for PARP7, with TUBULIN as a loading control (top). For the cell growth in A – C and E and F , each condition reflects eight biological replicates, error bars show the SD, and the statistical differences between cell lines at each RBN concentration are ****, P < 0.0001; ***, P < 0.001; **, <0.01; *, <0.05 (bottom). B, Stable knockdown of PARP7 in PC3-AR treated with R1881 partially protects cells from the growth-inhibitory effects of RBN2397. C, Stable knockdown of PARP7 in CWR22Rv1 treated with R1881 partially protects cells from the growth-inhibitory effects of RBN2397. D, Immunoblot detection of CDK inhibitor CDKN1A and PARP7 in prostate cancer lines grown ±R1881 and ±RBN2397 for 24 hours. E, Treating PC3-AR cells with si-CDKN1A (20 nmol/L) partially reverses the growth-inhibitory effect of RBN2397. The expression levels of CDKN1A and PARP7 ±R1881 and ±RBN2397 were determined by immunoblotting (top). F, CRISPR knockout of CDKN1A in PC3-AR cells reduces the growth-inhibitory effect of RBN2397. The knockout was validated by immunoblotting for CDKN1A (top).

Article Snippet: A pair of single-guide RNAs (sgRNA) targeting nonoverlapping sequences within Exon 2 of the CDKN1A gene, encoding p21, were designed using Benchling, and cloned into the CAS9-expresssing pX330 expression vector (RRID:Addgene_42230).

Techniques: Inhibition, Expressing, Knockdown, Western Blot, Control, Concentration Assay, CRISPR, Knock-Out

Journal: Nucleic acids research

Article Title: PARP1 associates with R-loops to promote their resolution and genome stability.

doi: 10.1093/nar/gkad066

Figure Lengend Snippet: Figure 5. PARP1 depletion or inhibition triggers R-loop-mediated genomic instability. (A) Representative images of anti-S9.6 immuno-fluorescence in RNH1-D210N-GFP-expressing U2OS cells treated with DOX and/or PARPiTal. RNases T1 and III were used to degrade unspecific RNAs. S9.6 signal is shown in red, RNH1-D210N-GFP in green, and DNA in blue after DAPI staining. White scale bars represent 10 m. (B) Quantification of relative S9.6 signal intensity from S9.6 immuno-fluorescence shown in (A). Data in black are from cells treated with RNases T1 and III and data in grey are from cells treated with RNases T1, III and H1. Orange bars represent mean S9.6 intensity. Data were collected from three independent experiments in which 100– 200 nuclei per condition were counted. Statistical analyses were performed using ordinary one-way ANOVA. (C) Representative images of anti-H2AX immunofluorescence in RNH1-D210N-GFP-expressing U2OS cells treated with DOX and/or PARPi for 24 h and pulsed with EdU for 15 min prior to fixation. H2AX foci are shown in red, DNA is shown in blue after DAPI staining, and EdU signal is shown in grey. EdU-negative nuclei are outlined in white. White scale bars represent 10 m. (D) Quantification of the number of H2AX foci per nucleus for each experimental condition shown in (C). The pink box represents EdU-positive nuclei. Orange bars represent the mean. Data are from three individual experiments in which 80–150 nuclei were counted per condition. Statistical analyses were performed using ordinary one-way ANOVA. (E) Representative images of anti-H2AX immunofluorescence in RNH1-D210N-GFP-expressing U2OS cells, RNH1-D210N-GFP-expressing PARP1ko U2OS cells and RNH1-D210N-GFP-expressing PARP1ko U2OS cells complemented with Cas9-insensitive Flag-PARP1. H2AX foci are shown in red, DNA is shown in blue after DAPI staining. White scale bars represent 10 m. (F) Quantification of the number of H2AX foci per nucleus for each experimental condition shown in (E). Data were collected from three independent experiments in which 300–500 nuclei per condition were counted. Statistical analyses were performed using ordinary one-way ANOVA.

Article Snippet: A Cas9 sgRNA targeting exon 2 of the PARP1 gene (sgPARP1-1, 5’- CAGCAGAATTCCCCGATCCG-3’) was cloned into lentiCRISPRv2 (Addgene #52961) and transformed into Stbl3 competent cells.

Techniques: Inhibition, Fluorescence, Expressing, Staining, Immunofluorescence