Journal: bioRxiv

Article Title: Identification of Injury-Response State Determinants in Glioblastoma Stem Cells

doi: 10.1101/2025.04.07.647626

Figure Lengend Snippet: A) Representative Flow cytometry experiments (3 independent experiments) measuring CD44 cell surface expression in IR GSC (G549) transduced with gRNA targeting AAVS1 (control) or EP300 (2 gRNAs). B) Representative Flow cytometry experiment (3 independent experiments) measuring CD44 surface expression following treatment of IR GSCs (G549) for 7 days with DMSO or A-485. C) Bar graph representing the quantification of CD44 cell surface expression (expressed as median fluorescent intensity relative to the control) in panel (A) and (B) (n=3). Error bars: mean ± SEM,* p<0.05 ** p<0.01. D) Quantification of IR GSC (G549) proliferation following treatment with control (DMSO) or with 400nM EP300 inhibitor (A-485) (n=3) using the Incucyte Live Cell Imaging system. Error bars: mean ± SEM. E) Quantification of IR GSC (G549) proliferation following control ( AAVS1 gRNA) or EP300 knockout ( EP300_KO) (n=3). Error bars: mean ± SEM. F) In vitro limiting dilution assay (LDA) of GSCs culture. Sphere forming Capacity (SFC) of the indicated GSC cultures expressing gRNA targeting AAVS1 (control) or EP300 . Error bars: estimated frequency +95% C, ** p<0.01. G) Schematic illustrating the GBM mouse model (Created with BioRender.com ). Mice carrying LSL-Cas9-GFP and triple floxed Tp53 , Pten and Rb1 alleles were intracranially injected with lentivirus carrying Cre-recombinase and gRNAs targeting either control or Ep300 . I) KaplanMeier curve for Tp53/Pten/Rb1 triple knockout GBM mouse model in tumors expressing the indicated gRNAs. Indicated p-value was calculated using the log-rank (Mantel-Cox) test.

Article Snippet: CRISPR-Cas9 KO screen was performed as previously described using the 70K TKOv3 library (Addgene Pooled Library #90294)( ; ).

Techniques: Flow Cytometry, Expressing, Transduction, Control, Live Cell Imaging, Knock-Out, In Vitro, Limiting Dilution Assay, Injection, Triple Knockout

Journal: European journal of immunology

Article Title: IRF8 regulates efficacy of therapeutic anti-CD20 monoclonal antibodies.

doi: 10.1002/eji.202250037

Figure Lengend Snippet: Figure 1. Top genes involved in complement-dependent cytotoxicity induced by anti-CD20 antibody rituximab (RTX) in a whole genome CRISPR- Cas9 screen. (A) Protocol used for identification of genes involved in CDC. Two conditions were performed: in condition A, a sort of remaining live cells was performed 24 hours post complement activation while in condition B the surviving cells were cultured for 8 days before DNA extraction. (B) Visualization of the enrichment of gRNA-coding sequences in conditions A (left panel) or B (right panel) represented with minus logarithm base 10 of the false discovery rate (FDR) on y-axis and logarithm base 2 of the fold change compared to control condition on x-axis. Genes with a FDR<1% are identified on the graph. (C) Percentage of gRNA-coding sequences detected in each condition: control, A, or B on the total number of gRNA in the library TKOv3. (D) Venn diagram representing the number of genes enriched in conditions A, B, or both. (E. Percentage of out-of-frame indels for each gRNA used to knock-out cells. IRF8 KO cells were electroporated with a mix of three gRNAs and the percentage depicted is the maximum of the 3. The mean of two to three experiments is represented. (F) Percentage of CDC in RAJI knocked-out for the top genes enriched in the screen with the indicated concentrations of normal human serum (NHS) added as a source of complement. The mean of three experiments is represented. The dotted line is the standard deviation and the area between the curve and the standard deviation is shaded. To take into account direct apoptosis triggered by RTX, the percentage of CDC was calculated as: (% dead cells in condition – % dead cells in no serum condition)/(100 – % dead cells in no serum condition).

Article Snippet: CRISPR-Cas9 genome-wide screen The genome-wide library Toronto KnockOut (TKO) CRISPR Library – Version 3 (TKOv3, #90294, Addgene) [42], was cloned in the lentiviral vector pLentiCRISPRv2 (#52961, Addgene).

Techniques: CRISPR, Activation Assay, Cell Culture, DNA Extraction, Control, Knock-Out, Standard Deviation

Journal: Acta neuropathologica communications

Article Title: Organelle resolved proteomics uncovers PLA2R1 as a novel cell surface marker required for chordoma growth.

doi: 10.1186/s40478-024-01751-w

Figure Lengend Snippet: Fig. 5 Effect of CRISPR/Cas9 knockdown on chordoma cell growth and survival. a Expression of PLA2R1 in UM-Chor1 following CRISPR-mediated knockdown of PLA2R1. b Digital image of colony formation assay after PLA2R1 knockdown. c Images of spheroid growth showing effect of PLA2R1 KD for a period of 28 days (Scale bar = 500 μM). Boxplots with jitters showing spheroid size (p-values calculated with t-test test between sg1 and sg2 compared against LacZ). e PLA2R1 KD (sg1 and sg2) and controls cells (NT and LacZ) were subcutaneously injected into NSG mice (N = 4). f Growth curve of xenograft tumor volume (average values with standard deviation) over a period of 120 days showing PLA2R1 KD negatively impacts in vivo tumor growth compared to controls. g Digital image of tumors after sacrifice of mice. h Tumor mass (mg) measurement after sacrifice of mice (p-values from comparing sg1 and sg2 against LacZ control)

Article Snippet: Two single guide RNA’s (sgRNA) targeting PLA2R1, sg1: CAC CGA TCA CAA CCT ACT TCT GCA G and sg2: CAC CGA GAC ATA ACC TCA TTA GCA G and control guide RNA LacZ: CAC CGC CCG AAT CTC TAT CGT GCG G were selected from Toronto KnockOut Library V3 (TKOv3) and obtained from Integrated DNA Technologies (IDT) and were cloned into a lentiCRISPRv2 construct (Addgene) with Cas9 and Puromycin cassette.

Techniques: CRISPR, Knockdown, Expressing, Colony Assay, Injection, Standard Deviation, In Vivo, Control