Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Functional Expression of the P2X7 ATP Receptor Requires Eros.

doi: 10.4049/jimmunol.1900448

Figure Lengend Snippet: FIGURE 2. Genome-wide CRISPR screen for genes that support P2X7-mediated PtdSer exposure. (A) Predominant expression of the P2X7k isoform in WR19L cells. The transcripts for the P2X7a and P2X7k isoforms were detected by RT-PCR in RNA from mouse resident peritoneal macrophages (rpMacs) and WR19L cells. In the lower panel, the structure of the mP2X7 gene is shown schematically with the positions of primers. The P2X7a and P2X7k isoforms were generated by alternative use of the first exon (exon 1 and exon 19). Forward primers on the first exon were specific for each isoform, whereas the reverse primer on exon 13 was common. (B) mP2X7k-mediated PtdSer exposure. In the upper panel, wild-type (WT), 16F2/2, DKO, and DKO-mP2X7k cells were stained with Alexa 488–anti-mP2X7 Ab and analyzed by flow cytometry. Staining profiles in the SYTOX Blue–negative population are shown. In the lower panel, WT, 16F2/2, DKO, and DKO-P2X7k cells were treated at 20˚C for 5 min with the indicated concentration of BzATP, stained with Cy5-labeled annexin V, and analyzed by flow cytometry. The experiments were performed three times, and the average percentage of annexin V–positive cells in the PI-negative population was plotted with SD (bars). (C) Identification of a gene that supports mP2X7-mediated PtdSer exposure. The upper panel shows the CRISPR-screening procedure. In the first step, DKO-P2X7k cells were transformed with Cas9 and the CRISPR library, and a population of cells with a reduced activity for BzATP-induced PtdSer exposure was sorted three times. In the second step, DNA from these sorted cells was subjected to deep sequencing, and the read sequences were processed to determine the abundance of each sgRNA. The middle panel shows the FACS profile of the first sorting, in which the population sorted for the next step is indicated. In the lower panel, the original cells and cells after the third sorting were left unstimulated or stimulated with BzATP and analyzed by flow cytometry. The annexin V–staining profiles in the PI-negative population are shown.

Article Snippet: The lentiCas9-Blast vector (28), mouse CRISPR Knockout Pooled Library (genome-scale CRISPR/Cas9 knockout [GeCKO] v2) (28), and pX330 and pX459v2 plasmids (29) were from Addgene. pCMV– vesicular stomatitis virus (VSV)-G–Rous sarcoma virus (RSV)-rev and pCMV-VSV-G (30) were provided by H. Miyoshi (RIKEN BioResource Center).

Techniques: Genome Wide, CRISPR, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Staining, Cytometry, Concentration Assay, Labeling, Transformation Assay, Activity Assay, Sequencing

Journal: Nature Communications

Article Title: PD-L1 deglycosylation promotes its nuclear translocation and accelerates DNA double-strand-break repair in cancer

doi: 10.1038/s41467-024-51242-8

Figure Lengend Snippet: a Schematic illustration of the CRISPR/Cas9 loss-of-function screen in human lung cancer H460 cells. b Volcano plot showing both negatively (left) and positively (right) selected genes at day 7 after radiation treatment (4 Gy). CD274 gene that encodes PD-L1 is highlighted in green. c Negative selection of CRISPR screen data ranked based on RRE method. d Differentially selected sgRNAs from ( b ) were subject to a functional enrichment analysis using over-representation test (ORT) as implemented in MAGeCKFlute.

Article Snippet: We used human CRISPR knockout pooled library (GeCKO v2) which is a pooled gRNA library in a 1 vector lentiCRISPRv2 containing Cas9 from Addgene.

Techniques: CRISPR, Selection, Functional Assay

Journal: Nature Communications

Article Title: PD-L1 deglycosylation promotes its nuclear translocation and accelerates DNA double-strand-break repair in cancer

doi: 10.1038/s41467-024-51242-8

Figure Lengend Snippet: a Endogenous PD-L1 was knocked out from H460 cells using the CRISPR/Cas9 system. PD-L1 was rescued by transfection with exogenous WT PD-L1. PD-L1 expression levels were analyzed by western blot. b , c H460 parental, H460 PD-L1 knockout cells and H460 PD-L1 knockout cells expressing exogenous WT PD-L1 were treated with increasing doses of IR, followed by colony formation analysis. Error bars represent ± s.d., n = 3 per group. *** P < 0.001, **** P < 0.0001, by two-tailed t test. d Endogenous PD-L1 was knocked out from H358 cells. PD-L1 was rescued by transfection with exogenous WT PD-L1. PD-L1 expression levels were analyzed by western blot. e , f H358 parental, H358 PD-L1 knockout cells and H358 PD-L1 knockout cells expressing exogenous WT PD-L1 were treated with increasing doses of IR, followed by colony formation analysis. Error bars represent ± s.d., n = 3 per group. *** P < 0.001, by two-tailed t test. g , h H460 parental and PD-L1 knockout H460 cells were treated with 4 Gy of IR at 2 Gy/min dose rate, followed by immunofluorescence analysis of γH2AX foci and DAPI at various time points. Foci ≥5/cell were counted as foci-positive cells. Error bars represent ± s.d., n = 3 per group. *** P < 0.001, by two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: We used human CRISPR knockout pooled library (GeCKO v2) which is a pooled gRNA library in a 1 vector lentiCRISPRv2 containing Cas9 from Addgene.

Techniques: CRISPR, Transfection, Expressing, Western Blot, Knock-Out, Two Tailed Test, Immunofluorescence