Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A. Genes identified from the CRISPR screen. ACE2-expressing A549 (A549-ACE2) cells transduced with a CRISPR knockout library were infected with SARS-CoV-2 transcription-and replication-competent virus-like particles where the N gene is replaced by the reporter GFP (trVLP-GFP) (MOI 0.5, 24 h). trVLP-GFP was packaged in cells expressing the N gene and only replicated for single round in A549-ACE2 in the absence of N protein. GFP-positive cells were sorted for genomic extraction and sgRNA sequence analysis. The genes were analyzed by MAGeCK software and sorted based on the -log 10 (MAGeCK score). B. Experimental validation of top 20 genes from the screen in A549-ACE2 cells. Two independent sgRNAs per gene were used, and cells were infected with SARS-CoV-2 transcription- and replication-competent virus-like particles where the N gene is replaced by the NanoLuc luciferase (trVLP-Nluc) (MOI 0.5, 24 h). The infection efficiency was quantified by measuring the luciferase activity. C. High content imaging and quantification analysis of SARS-CoV-2 infection in DAZAP2 -edited A549-ACE2 (MOI 0.1, 24 h). D. Representative immunofluorescence images of SARS-CoV-2 infection in DAZAP2 -edited A549-ACE2 cells (MOI 0.1, 24 h). Scale bar, 100 μm. E. High content imaging and quantification analysis of SARS-CoV-2 infection in DAZAP2 -edited HeLa-ACE2 (MOI 0.1, 24 h). F. qRT-PCR was conducted to measure the N gene copies of SARS-CoV-2 infected Calu-3 cells (MOI 1, 24 h). GAPDH was used as internal control. G-H. High content imaging and quantification analysis ( G ) and immunofluorescence images ( H ) of SARS-CoV-2 infection in mouse Dazap2 -edited MEFs expressing human ACE2 (MEF-ACE2) (MOI 0.1, 24 h). Scale bar, 100 μm. I. Validation of DAZAP2 as a restriction factor during infection with other coronaviruses. Gene-edited cells were infected with alphacoronaviruses (HCoV-229E, MOI 1, 48 h; SADS-CoV, MOI 3, 24 h), betacoronaviruses (MHV, MOI 5, 24 h; HCoV-OC43, MOI 0.03, 12 h), gammacoronaviruses (IBV, MOI 0.5, 24 h), or deltacoronaviruses (PDCoV, MOI 0.3, 24 h). J-K. Overexpression of DAZAP2 inhibits SARS-CoV-2 infection (MOI 1, 24 h). Human DAZAP2 or mouse Dazap2 cDNA was expressed in A549-ACE2 or MEF-ACE2, respectively. L. Overexpression of human DAZAP2 in HeLa-ACE2 inhibits HCoV-OC43 (MOI 0.03, 24 h) and PEDV (MOI 1, 24 h) infection. The virus infection efficiency was determined by analyzing the percentage of viral N-positive cells using flow cytometry or Operetta High Content Imaging System. Data shown are from three independent experiments and each independent experiment was performed in duplicate or triplicate. B, two-way ANOVA with Dunnett’s test; the mean of two sgRNAs was compared with the control sgRNA; C, E, and G, one-way ANOVA with Dunnett’s test; F and I-L, unpaired t test; n=3; mean ± s.d.; *P < 0.05; **P < 0.01; ***, P < 0.001; ****P < 0.0001; ns, not significant.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: CRISPR, Expressing, Transduction, Knock-Out, Infection, Virus, Extraction, Sequencing, Software, Biomarker Discovery, Luciferase, Activity Assay, Imaging, Immunofluorescence, Quantitative RT-PCR, Control, Over Expression, Flow Cytometry

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A. Knockout efficiency of DAZAP2 in A549-ACE2, HeLa-ACE2, Calu-3, and MEF-ACE2 cells. Two sgRNAs or one representative sgRNA were used, and gene-edited bulk cells were subjected to western blotting. B. Overexpression of DAZAP2 in A549-ACE2 or HeLa-ACE2 cells. Cells were transduced with lentivirus bearing the human DAZAP2 cDNA, and subjected to western blotting. C. Western blotting to verify the DAZAP2 -knockout clones #2 and #3 of A549-ACE2. Clone #3 (11DAZAP2) was used in this study. D. The sequence traces of the DAZAP2 gene locus of WT and clonal cells. The sgRNA target site is indicated. E. The ICE analysis of the 11DAZAP2 clonal cell line.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Knock-Out, Western Blot, Over Expression, Transduction, Clone Assay, Sequencing

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A-C. Pseudovirus infection assay. The gene-edited A549-ACE2 cells were infected with murine leukemia retrovirus (MLV)-based pseudoviruses bearing the spike protein of SARS-CoV-2 ( A ), the glycoprotein of vesicular stomatitis virus (VSV-G) ( B ), or the spike protein of SARS-CoV-1 ( C ), and the luciferase activity was measured and normalized to the control. D-E. The inhibition of endosomal entry of SARS-CoV-2. Control and DAZAP2 -knockout clonal cell line of A549-ACE2 (11 DAZAP2 ) were infected with MLV-based pseudovirus bearing the spike protein of SARS-CoV-2 (D) or single-cycle trVLP-NLuc (E), in the presence of 100 μM E-64d (aloxistatin), an inhibitor that blocks the cysteine protease activity of cathepsins B and L, which are required for the endosomal membrane fusion, and/or 100 μM camostat mesylate, a TMPRSS2 inhibitor that blocks viral fusion at the plasma membrane. F. Virus binding and internalization assays. Cells were incubated with SARS-CoV-2 (MOI 5), and the bound or internalized virions were measured by qRT-PCR for genomic RNA. G-H. Trafficking of SARS-CoV-2 trVLP-Nluc particles in the presence of cysteine protease inhibitor E-64d (100 μM). The representative confocal images (G) were obtained, and the quantification of spike and N protein double-positive particles per field (H) was analyzed. The endolysosome marker LAMP1 was stained. Scale bar, 20 or 5 μm. I. Quantification of endosomal acidification. Control or DAZAP2 -deficient A549-ACE2 cells were pre-treated with or without 20 μM chloroquine (CQ) followed by staining of LysoSensor Green dye. The fluorescence intensity was quantified. J. The cleavage of the SARS-CoV-2 spike protein. Control or DAZAP2 -deficient A549-ACE2 cells were incubated with MLV-based pseudoviruses bearing the spike protein for 2 or 4 h, followed by western blotting analysis with anti-S2 antibody. The cysteine protease inhibitor E-64d (100 μM) was used as control. K. Split NanoLuc luciferase reporter-based virus-cell fusion assay. Cells expressing the LgBit were incubated with retrovirus particles encapsulated with CypA-HiBit to enable virion fusion in the endolysosomes. The re-complemented NanoLuc luciferase activity in the cytoplasm was determined and normalized to the control. L-M. Quantification of virions in the endolysosomes. Control or DAZAP2 -deficient A549-ACE2 cells were infected with SARS-CoV-2 for 4 h, and fixed to stain the spike, N, and endolysosome marker LAMP1. The colocalization of LAMP1 with N was visualized by confocal microscopy (L) and the number of colocalized foci per cell was counted (M). Three fields of view with a total of 27 to 42 cells were used for analysis. The representative confocal images (L) were shown. Scale bar, 20 or 5 μm. The cysteine protease inhibitor E-64d (100 μM) was used as control. N-O. Quantification of double-stranded RNA (dsRNA). Control or DAZAP2 -deficient A549-ACE2 cells were infected with SARS-CoV-2 for 4 h, then fixed to stain the dsRNA with J2 antibody. The dsRNA puncta were visualized by confocal microscopy (N) and the percentage of dsRNA-positive cells per field was counted (O). Six fields of view with a total of 109 control cells and 79 DAZAP2 -deficient cells were selected for analysis. The representative confocal images (N) were shown. Scale bar, 50 μm. Data shown are from three independent experiments and each independent experiment was performed in triplicate. A-C, F, H, K, and O, unpaired t test; D, E, I, and M, one-way ANOVA with Dunnett’s test; n=3; mean ± s.d.; *P < 0.05; ***, P < 0.001; ****P < 0.0001; ns, not significant.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Infection, Virus, Luciferase, Activity Assay, Control, Inhibition, Knock-Out, Membrane, Clinical Proteomics, Binding Assay, Incubation, Quantitative RT-PCR, Protease Inhibitor, Marker, Staining, Fluorescence, Western Blot, Cell Fusion Assay, Expressing, Confocal Microscopy

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A-B. Cell-cell fusion assay. Control and 11 DAZAP2 A549-ACE2 acceptor cells were co-cultured with 293T donor cells that express SARS-CoV-2 spike protein. Spike protein-induced syncytia was visualized under brightfield microscope (A), and syncytial nuclei were counted after Giemsa staining (B). Scale bar, 100 μm. C-D. Schematic (C) and the results (D) of split NanoLuc luciferase reporter-based cell-cell fusion assay. A549-ACE2 acceptor cells expressing the LgBit were incubated with 293T donor cells expressing both HiBit and SARS-CoV-2 spike protein. The functional NanoLuc luciferase was re-complemented after cell-cell fusion and the activity was measured and normalized to the control. E-F. The inhibition of plasma membrane entry of SARS-CoV-2. Control and 11 DAZAP2 A549-ACE2 cells ectopically expressing the TMPRSS2 (A549-ACE2-TMPRSS2) were infected with MLV-based pseudovirus bearing the spike protein of SARS-CoV-2 (E) or single-cycle trVLP-NLuc (F), in the presence of cysteine protease inhibitor E-64d (100 μM), and/or TMPRSS2 inhibitor camostat mesylate (100 μM). Data shown are from three independent experiments and each independent experiment was performed in triplicate. B and C, unpaired t test; E and F, one-way ANOVA with Dunnett’s test; n=3; mean ± s.d.; ***, P < 0.001; ****P < 0.0001; ns, not significant.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Cell-Cell Fusion Assay, Control, Cell Culture, Microscopy, Staining, Luciferase, Expressing, Incubation, Functional Assay, Activity Assay, Inhibition, Clinical Proteomics, Membrane, Infection, Protease Inhibitor

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A. Replicon RNA assay in HeLa cells edited with control or DAZAP2 sgRNA. The SARS-CoV-2 replicon system was constructed by replacing the portion of the genome encoding the spike protein all the way through ORF8 with NanoLuc luciferase. The in vitro transcribed replicon RNA was electroporated into cells. The RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir (10 μM) was added as a control to verify the utility of the replicon system. One representative sgRNA was used to edit the DAZAP2 . The luciferase activity was determined and normalized to the control. B. Replicon RNA assay in empty vector-and DAZAP2 -overexpressing HeLa cells, and the results were normalized to the control. C. Quantification of genomic RNA replication. The in vitro transcribed replicon RNA was electroporated into cells, and the levels of genomic RNA replication was determined by qRT-PCR targeting the NSP10 gene at the indicated timepoints. The results were normalized to the control. D. Schematic of the construction of inactivated replicon system to assess the primary translation of viral replicases. The NanoLuc luciferase reporter gene, flanked by a P2A cleavage site, was inserted between NSP1 and NSP2 of the SARS-CoV-2 replicon. The D760N and D761N double mutations were introduced into the NSP12 to inactivate the RdRp activity, ensuring that only translation could be assessed. E. Detection of the primary translation of viral replicases as indicated by the luciferase activity. Control and DAZAP2-edited HeLa cells were electroporated with the modified replicon RNA, and the luciferase activity was monitored. F. Confocal analysis of the localization of DAZAP2 and SARS-CoV-2 N protein. SARS-CoV-2-infected A549-ACE2 cells were fixed and stained with anti-DAZAP2 or anti-N antibody. The representative confocal images were shown. Scale bar, 50 or 20 μm. G-H. Validation of SERPINE1 gene. A549-ACE2 cells were edited with two independent sgRNAs targeting the SERPINE1 , followed by infection with authentic SARS-CoV-2 (G) or trVLP-NLuc particles (H). Data shown are from three independent experiments and each independent experiment was performed in triplicate. As for the results shown as relative change, data are normalized to the control of individual experiment. A-C, G and H, unpaired t test; E, two-way ANOVA; n=3; mean ± s.d.; ****P < 0.0001; ns, not significant.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Control, Construct, Luciferase, In Vitro, Activity Assay, Plasmid Preparation, Quantitative RT-PCR, Modification, Infection, Staining, Biomarker Discovery

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A-C. A549-ACE2 cells were treated with 0 or 1,000 U/ml of IFNα-2b for various time points, and cellular RNA was extracted for detection of DAZAP2 ( A ), IFITM3 ( C ), or MX1 ( C ) by qRT-PCR. The experiment was performed in triplicate and data were normalized to the treatment with 0 U/ml of IFNα-2b at each time point.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A-C. The antiviral effect of DAZAP2 on SARS-CoV-2 infection in STAT1 -, MAVS -, or IRF3 -knockout A549-ACE2 cells. Cells were infected with different MOIs and the percentage of N positive cells were analyzed (left panels of A to C). The representative fluorescence images from high-content analysis in cells were shown on right panels of A to C. D-E. Verification of STAT1- or IRF3- knockout efficiency in clonal cell lines by Western blotting. The clone #3 for STAT1- or IRF3- knockout cells was selected for use.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Infection, Knock-Out, Fluorescence, High Content Screening, Western Blot

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A-B. The A549-ACE2 cells were infected with SARS-CoV-2, then fixed and stained with anti-DAZAP2 (A and B), anti-EEA1 (A), or anti-LAMP1 (B) antibody. The representative confocal images were shown. Scale bar, 50 or 20 μm.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: DAZAP2 functions as a pan-coronavirus restriction factor by inhibiting viral entry and genomic replication

doi: 10.1101/2025.02.04.636569

Figure Lengend Snippet: A. Flow cytometry analysis of surface expression of ACE2 in WT or ACE2-overexpressing HeLa cells stained with anti-ACE2 antibody. B. Flow cytometry analysis of surface expression of ACE2 in WT or ACE2-overexpressing HeLa cells stained with isotype or anti-ACE2 antibody. The percentage of ACE2 positive cells and geometric mean fluorescence intensity (MFI) were analyzed. C-E. Flow cytometry analysis of surface expression of ACE2, AXL, or heparan sulfate in A549-ACE2 (C) or HeLa-ACE2 (D and E) cells edited with control or DAZAP2 sgRNA. The percentage of positive cells and geometric mean fluorescence intensity (MFI) were analyzed. F. Flow cytometry analysis of surface expression of TIM-1, SIGLEC1, or DC-SIGN in A549-ACE2 cells edited with control or DAZAP2 sgRNA. G. Western blotting analysis of CTSL, Furin, or TMPRSS2 in A549-ACE2 cells edited with control or DAZAP2 sgRNA.

Article Snippet: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), A549-ACE2 , HeLa (ATCC #CCL-2), HeLa-ACE2 , Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MEF expressing the human ACE2 (MEF-ACE2), BHK-21, Huh7, swine testicular (ST), LLC-MK2, HRT-18, and 11 MAVS A549 , all were cultured at 37°C in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.

Techniques: Flow Cytometry, Expressing, Staining, Fluorescence, Control, Western Blot

Journal: Frontiers in Immunology

Article Title: Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2023.1270843

Figure Lengend Snippet: Phenotypic characterization of CD33-CAR-T cells from AML patients. (A) Population doublings of CAR-T cells generated from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors during CAR-T cell production. (B) Percentage of transduced cells (CAR + ) at the end of each CAR-T cell production. (C) Analysis of the phenotype of CAR-T cells at resting state for each group. CAR-T cell subpopulations within CD4 + and CD8 + cells are depicted. T N , naïve; T SCM , stem central memory; T CM , central memory; T EM , effector memory; T E , effector. (D) Analysis of the expression of CD69, HLA-DR, PD1 and LAG3 in CD8 + T cells from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors. Mean ± SEM for each group is depicted. 2-way ANOVA with Tukey’s multiple comparisons test (A) , Kruskal-Wallis test with Dunn’s multiple comparisons test (B, D) . ns, not significant; *p<0.05; **p<0.01.

Article Snippet: Isolated T cells were activated with 10 μl/ml T cell TransAct (Miltenyi Biotec) for 48h and infected with the CAR lentiviral vector at MOI 2 with 10 μl/ml of LentiBoost (Sirion Biotech).

Techniques: Generated, Expressing

Journal: Frontiers in Immunology

Article Title: Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2023.1270843

Figure Lengend Snippet: Functional characterization of CD33-CAR-T cells from AML patients. (A) Quantification of the cytotoxic activity of CAR-T and UTD cells generated from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors, against CD33 + (left) and CD33 knock-out (right) MOLM-13 AML cell line at different E:T ratio. The percentage of specific lysis for each CAR-T cell production is depicted. (B) Quantification of IFN-γ levels in supernatants from cytotoxic assays (ratio 1:3) measured by ELISA. The cytokine concentration (ng/ml) for each CAR-T cell production is depicted. Analysis of the expression of PD1 and LAG3 in CD4 + (C) and CD8 + (D) CAR-T cells from AML patients, adult, and senior healthy donors, before (basal) and after continuous repeated in vitro stimulation (reest) for 21 days with MOLM-13 tumoral cells. (E) Cytotoxic activity of CAR-T cells from AML patients (n=7), adult (n=5) and senior (n=5) healthy donors after continuous repeated in vitro stimulation for 21 days with MOLM-13 tumoral cells. (F) Survival of mice treated with CAR-T cells from AML patients, adult, and senior healthy donors. Untreated animals or treated with UTD cell form same groups were use as control. All groups included 12 animals (6 male and 6 female). Mean ± SEM of the average of three technical replicates for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test (C, D) , 2-way ANOVA with Tukey’s multiple comparisons test (E) , Logrank test (F) . ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Isolated T cells were activated with 10 μl/ml T cell TransAct (Miltenyi Biotec) for 48h and infected with the CAR lentiviral vector at MOI 2 with 10 μl/ml of LentiBoost (Sirion Biotech).

Techniques: Functional Assay, Activity Assay, Generated, Knock-Out, Lysis, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, In Vitro, Control

Journal: Frontiers in Immunology

Article Title: Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2023.1270843

Figure Lengend Snippet: Transcriptomic characterization of CD33-CAR-T cells from AML patients. The transcriptomic landscape of CAR-T cells generated from AML patients (n=4), adult (n=3) and senior (n=3) healthy donors was profiled using high-throughput RNA sequencing (RNA-seq) (A) RNA-seq principal components (PC) analysis, corrected by patient heterogeneity, of sorted CD4 + and CD8 + CAR-T cell subsets. Percentage of variance explained by PC1 and PC2 are depicted. (B) Left: Heatmap of differentially expressed genes associated to stem cell memory and T cell activation shared between CD8 + CAR-T cells from AML patients and senior healthy donors (age-related) compared to adult CAR-T cells. Right: Quantification of CD28 and CIITA gene expression. (C) Left: Heatmap of differentially expressed genes specific for CD8 + CAR-T cells from AML patients (AML-specific) compared to adult and senior CAR-T cells. Right: Quantification of LAG3, NR4A1, CCR7 and OAS1 gene expression. (D) Quantification of CD81, CCL5, IRF1 and KLF2 gene expression as example of genes with disrupted expression pattern in AML CAR-T cells after stimulation with tumoral cells. Mean ± SEM for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test (B, C) , 2-way ANOVA with Tukey’s multiple comparisons test (D) . ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Isolated T cells were activated with 10 μl/ml T cell TransAct (Miltenyi Biotec) for 48h and infected with the CAR lentiviral vector at MOI 2 with 10 μl/ml of LentiBoost (Sirion Biotech).

Techniques: Generated, High Throughput Screening Assay, RNA Sequencing, Activation Assay, Gene Expression, Expressing

Journal: Frontiers in Immunology

Article Title: Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2023.1270843

Figure Lengend Snippet: Characterization of HLA-I KO /TCR KO CD33-CAR-T cells. Selected CRISPR RNPs were combined with the Sleeping Beauty transposon system to generate HLA-I KO /TCR KO CD33-CAR-T (CAR-T KO ) cells from healthy donors (n=8 independent samples) (A) Distribution of the different edited populations observed after simultaneous HLA-I and TCR targeting of CAR-T and UTD cells with CRISPR systems. (B) Percentage of transduced cells (CAR + ) at the end of CAR-T and CAR-T KO cell production. (C) Percentage of HLA-I KO /TCR KO double negative cells in CAR-T and UTD cells before and after selection. (D) Percentage of transduced cells (CAR + ) at the CAR-T KO cell production before and after selection of HLA-I KO /TCR KO double negative cells. (E) Population doublings of CAR-T and CAR-T KO cells during CAR-T cell production. UTD and UTD KO cells were use as control. (F) Analysis of CD4/CD8 ratio in CAR-T and CAR-T KO cells. (G) Analysis of the phenotype of CAR-T and CAR-T KO cells at resting state for each group. CAR-T cell subpopulations within CD4 + and CD8 + cells are depicted. T N , naïve; T SCM , stem central memory; T CM , central memory; T EM , effector memory; T E , effector. (H) Quantification of the cytotoxic activity of CAR-T and CAR-T KO cells against CD33 + MOLM-13 AML cell line at different E:T ratio. The percentage of specific lysis (average of three technical replicates) for each CAR-T cell production is depicted. UTD and UTD KO cells were used as control. (I) Survival of mice treated with CAR-T and CAR-T KO . Untreated animals or treated with UTD and UTD KO cells were used as control. Mean ± SEM for each group is depicted. Kruskal-Wallis test with Dunn’s multiple comparisons test (B, D) , 2-way ANOVA with Tukey’s multiple comparisons test (E, H) , Logrank test (I) . ns, not significant; *p<0.05; ***p<0.001.

Article Snippet: Isolated T cells were activated with 10 μl/ml T cell TransAct (Miltenyi Biotec) for 48h and infected with the CAR lentiviral vector at MOI 2 with 10 μl/ml of LentiBoost (Sirion Biotech).

Techniques: CRISPR, Selection, Control, Activity Assay, Lysis

Journal: Frontiers in Immunology

Article Title: Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2023.1270843

Figure Lengend Snippet: Safety analysis of HLA-I KO /TCR KO CD33-CAR-T cells. (A) Analysis of the SB copy number integrations in CAR-T and CAR-T KO cells (n=8 independent productions). (B) Histogram plot showing the genomic annotation of SB integration sites in CAR-T and CAR-T KO cells (n=3 independent productions. (C) Sequences of cleavage sites identified by iGUIDE for B2M (left) and TRAC (right) sgRNAs annotated by on target or off target, with the total number of unique alignments associated with the site. Wilcoxon matched-pairs signed rank test (A) . ns, not significant.

Article Snippet: Isolated T cells were activated with 10 μl/ml T cell TransAct (Miltenyi Biotec) for 48h and infected with the CAR lentiviral vector at MOI 2 with 10 μl/ml of LentiBoost (Sirion Biotech).

Techniques:

Journal: Frontiers in Immunology

Article Title: Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia

doi: 10.3389/fimmu.2023.1270843

Figure Lengend Snippet: Preclinical production of HLA-I KO /TCR KO CD33-CAR-T cells. (A) Percentage of HLA-I KO /TCR KO double negative cells before and after selection (n=3 independent productions). (B) Percentage of transduced cells (CAR + ) at the CAR-T KO cell production after selection of HLA-I KO /TCR KO double negative cells (n=3 independent productions). (C) Population doublings of CAR-T KO cells during CAR-T cell production (n=3 independent productions). (D) Quantification of total number of CAR-T KO cells obtained at the end of the production after HLA-I KO /TCR KO double negative selection (n=3 independent productions). (E) Quantification of the cytotoxic activity of CAR-T KO cells against CD33 + MOLM-13 AML cell line at different E:T ratio. The percentage of specific lysis (average of three technical replicates) for each CAR-T cell productions (n=3) is depicted. (F) Quantification of IFN-γ levels in supernatants from cytotoxic assays (ratio 1:3) measured by ELISA. The cytokine concentration (ng/ml; average of three technical replicates) for each CAR-T cell production (n=3) is depicted. Mann Whitney test (B, C, F) , 2-way ANOVA with Tukey’s multiple comparisons test (E) . ns, not significant.

Article Snippet: Isolated T cells were activated with 10 μl/ml T cell TransAct (Miltenyi Biotec) for 48h and infected with the CAR lentiviral vector at MOI 2 with 10 μl/ml of LentiBoost (Sirion Biotech).

Techniques: Selection, Activity Assay, Lysis, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 3. Enhancer activity compacts SOX9 promoter-enhancer hub in individual TNBC cells. (A and B) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (A) and SOX9.EC2, SOX9. EC3, or both (B) in MB157 (n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9.EC3 (magenta), and SOX9.EC1 (A, red) or SOX9.EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). (C and F) SOX9 enhancers inactiva- tion expands SOX9-EC1-EC3 and SOX9-EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9-EC1-EC3 (C) and SOX9-EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/SOX9.EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/SOX9.EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/SOX9.EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/SOX9.EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/SOX9.EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/SOX9.EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). (D and G) Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (D) and SOX9.EC1, SOX9. EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA (n = alleles). (E and H) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. (I) SOX9 promoter inactivation decreases SOX9-EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percent- ages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA (SOX9.P sgRNA) (n = alleles). Bottom-left: CDFs of SOX9-EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/SOX9.P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Activity Assay, Expressing, Control

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 6. SOX9 regulates oncogene MYC by positioning its enhancers. (A) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter- enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC.EC1 (magenta), MYC.EC2 (red), MYC.EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. (B) A total of 80% of differ- entially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. (C to E) SOX9 loss significantly in- creases 3D distances between the MYC promoter and SOX9-bound MYC.EC2 (C) or MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC.EC2 (C) and MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC.EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC.EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC.EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Represen- tative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 7. SOX9 loss decompacts MYC promoter-enhancer hubs. (A) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER+ MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC.EC1, SOX9-bound MYC.EC3, or both MYC.EC1 and MYC.EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in Fig. 6A top genome track (n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. (B and C) SOX9 loss expands MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC-EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC-EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm (n = cells). Middle: Allele per- centages with MYC promoter interacting (<350 nm) with MYC.EC1, MYC.EC2, or both MYC.EC1 and MYC.EC2 (B) and MYC.EC1, MYC.EC3, or both MYC.EC1 and MYC.EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Expressing

Journal: Cell Reports

Article Title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.109479

Figure Lengend Snippet: Standardization of an electrical resistance-based assay as a measure of SARS CoV-2-induced CPE and anti-SARS-CoV-2 activity Vero E6 cells were seeded into a CytoView-Z 96-well plate and allowed to stabilize overnight, as measured by electrical resistance. (A) Resistance was measured every minute over the course of 72 hours in wells that were mock infected or infected with SARS-CoV-2 in 10-fold dilutions ranging from an MOI of 10-0.0001. Solid lines indicate the mean, dotted lines indicate the standard error of three replicates. (B) Median time-to-death calculations based on raw resistance data for each MOI. (C and D) Remdesivir was titrated in 6-fold dilutions ranging from 50–0.006 μM. (C) After infection at an MOI of 0.01, resistance was monitored for 48 hpi and (D) percent inhibition for remdesivir based on the data from the 48-h time point is presented (solid circles). Resistance measurements were monitored between 1–20 hpi for potential compound-induced cytopathic effects prior to SARS-CoV-2 induced cytopathic effects, as indicated in (C). The data represent the mean and SE of three replicates.

Article Snippet: To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 therapeutics, we first tested remdesivir, a well-described inhibitor of SARS-CoV-2 that has been granted emergency use authorization (EUA) for the treatment of COVID-19 ( ; ).

Techniques: Activity Assay, Infection, Inhibition

Journal: Cell Reports

Article Title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.109479

Figure Lengend Snippet: VPS34 inhibitors exhibit anti-SARS-CoV-2 activity Vero E6 cells were seeded into a CytoView-Z 96-well plate and allowed to stabilize overnight. Cells were pre-treated with serial half-log 10 dilutions of VPS34-IN1 (A and B) or PIK-III (C and D) and infected with SARS-CoV-2 at an MOI of 0.01. Resistance (A and C) was measured every minute over the course of 48 h and percent inhibition (B and D) was determined at the 48-h time point (solid circles) as compared to the infected DMSO-treated control (red). Uninfected cells are indicated in blue. Resistance measurements were monitored between 1–20 hpi for potential compound-induced cytopathic effects prior to SARS-CoV-2 induced cytopathic effects, as indicated in (A) and (C). The data represent the mean and SE of three replicates.

Article Snippet: To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 therapeutics, we first tested remdesivir, a well-described inhibitor of SARS-CoV-2 that has been granted emergency use authorization (EUA) for the treatment of COVID-19 ( ; ).

Techniques: Activity Assay, Infection, Inhibition

Journal: Cell Reports

Article Title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.109479

Figure Lengend Snippet: Screening of fatty acid inhibitors for anti-SARS-CoV-2 activity Vero E6 cells were seeded into a CytoView-Z 96-well plate and allowed to stabilize overnight. Cells were pre-treated with serial half-log 10 dilutions of orlistat (A and B) or triacsin C (C and D) and infected with SARS CoV-2 at an MOI of 0.01. Resistance (A and C) was measured every minute over the course of 48 h and percent inhibition (B and D) was determined at the 48-h time point (solid circles) as compared to the infected DMSO-treated control (red). Uninfected cells are indicated in blue. Resistance measurements were monitored between 1–20 hpi for potential compound-induced cytopathic effects prior to SARS-CoV-2-induced cytopathic effects, as indicated in (A) and (C). The data represent the mean and SE of three replicates.

Article Snippet: To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 therapeutics, we first tested remdesivir, a well-described inhibitor of SARS-CoV-2 that has been granted emergency use authorization (EUA) for the treatment of COVID-19 ( ; ).

Techniques: Activity Assay, Infection, Inhibition

Journal: Cell Reports

Article Title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.109479

Figure Lengend Snippet: Attenuation of VPS34 kinase activity and fatty acid metabolism inhibit SARS-CoV-2 replication in human airway epithelial cell line Calu-3 cells were plated onto a 96-well plate and allowed to reach confluency. Cells were then pre-treated with a series of 3-fold dilutions of VPS34-IN1 (A), PIK-III (B), orlistat (C), triacsin C (D), DMSO, or mock-treated with media alone for 1 h, then infected with SARS-CoV-2 at an MOI of 0.01. Supernatants were collected at 48 hpi, and virus was quantified by plaque assay on VeroE6 cells. The data are reported as plaque forming units per milliliter (pfu/mL) (left panels) and percent inhibition (right panels). Cell viability over 48 h was determined in parallel and plotted with the percent inhibition data. IC 50 and IC 90 were calculated from the plaque assay data and are indicated on the curves. For the plaque assay data, the dotted lines labeled DMSO and LOD indicate the level of virus growth in the DMSO control and the limit of detection, respectively. The data represent the mean and SE of three replicates. See also Figure S1 .

Article Snippet: To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 therapeutics, we first tested remdesivir, a well-described inhibitor of SARS-CoV-2 that has been granted emergency use authorization (EUA) for the treatment of COVID-19 ( ; ).

Techniques: Activity Assay, Infection, Plaque Assay, Inhibition, Labeling

Journal: Cell Reports

Article Title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.109479

Figure Lengend Snippet: Mechanistic characterization of anti-SARS-CoV-2 activity To determine which steps involved in fatty acid metabolism contribute to the observed anti-SARS-CoV-2 activity, Calu-3 cells were pre-treated with DMSO or a series of 3-fold dilutions of the indicated compounds, or mock-treated with media alone for 1 h, then infected with SARS-CoV-2 at an MOI of 0.01. (A–D) Supernatants were collected at 48 hpi and virus was quantified by focus forming assay on VeroE6 cells. Cytotoxicity assays were performed in parallel. IC 50 and CC50 values were calculated for each compound (A). To discern whether or not genomic and subgenomic RNA levels are affected by compound treatment, Calu-3 cells were pre-seeded in 24-well format, allowed to grow to confluency, and infected at an MOI of 1. Two hours post-infection, cells were treated with VPS34-IN1 (5 μM), orlistat (500 μM), triacsin C (5 μM), TOFA (50 μM), 2-bromopalmitate (50 μM), A922500 (30 μM), remdesivir (1 μM), or DMSO. At 4, 10, and 24 hpi, total RNA was extracted from the cell monolayers, and 24 hpi supernatants were harvested for viral titers. Virus titers were determined by plaque assay (B). (C–E) Levels of genomic RNA, subgenomic N RNA, and NSP14 RNA were quantified via qPCR. Data are represented as fold change of RNA levels in infected compound treated samples versus infected DMSO-treated samples. The virus titer at 24 hpi for compound treated cells (orange bars) are plotted alongside the qPCR data and represented as fold-change compared to titers from DMSO-treated cells. The data represent the mean and SE of three replicates. See also , , and .

Article Snippet: To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 therapeutics, we first tested remdesivir, a well-described inhibitor of SARS-CoV-2 that has been granted emergency use authorization (EUA) for the treatment of COVID-19 ( ; ).

Techniques: Activity Assay, Infection, Focus Forming Assay, Plaque Assay

Journal: Cell Reports

Article Title: Inhibitors of VPS34 and fatty-acid metabolism suppress SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.109479

Figure Lengend Snippet: Fatty acid metabolism is essential for efficient SARS-CoV-2 replication FASN CRISPR KO Caco2 cells and corresponding NT Caco2 cells were pre-seeded in 96-well plates, grown to confluency, and then infected at an MOI 0.01 in minimal media. Post-adsorption, cells were maintained in either 2% FBS DMEM (A) or 1% fatty-acid free (FAF)-BSA DMEM (B). Supernatants were collected at 1, 24, 48, 72, and 96 hpi and viral titers were determined by plaque assay. Protein samples were obtained from cell monolayers and analyzed by western blot to confirm FASN knockout and look for changes in SARS-CoV-2 N levels. FASN KO and WT cells were pre-seeded and infected as previously described. Post adsorption, inoculum was removed and replaced with 2% FAF-BSA DMEM or 2% FAF-BSA DMEM containing 250 μM palmitic acid + 250 μM oleic acid (C). Supernatants were collected at 1, 24, 48, and 72 hpi and viral titers were determined via plaque assay. The data represent the mean and SE of three replicates.

Article Snippet: To establish the Maestro Z as a potential instrument for screening of anti-SARS-CoV-2 therapeutics, we first tested remdesivir, a well-described inhibitor of SARS-CoV-2 that has been granted emergency use authorization (EUA) for the treatment of COVID-19 ( ; ).

Techniques: CRISPR, Infection, Adsorption, Plaque Assay, Western Blot, Knock-Out

Journal: Diabetes

Article Title: Applying CRISPR Screen in Diabetes Research

doi: 10.2337/dbi20-0047

Figure Lengend Snippet: Strategies for modifying gene expression with CRISPR/Cas9 construct. A: CRISPR/Cas9 knockout induces a double-stranded DNA break in gDNA. Error-prone nonhomologous end joining (NHEJ) repair causes an indel mutation, causing a reading frameshift in the exon. B: dCas9 fused with VP64 and additional activator domains binds to promoter region and enhances transcription. C: dCas9 fused with KRAB binds to and blocks promoter region, interfering with transcription. Created with BioRender (BioRender.com).

Article Snippet: A large collection of knockout, activation, or inhibition CRISPR libraries, either genome-wide libraries or smaller, more focused, subpool libraries, for both the human and mouse genome, is readily available on Addgene, a nonprofit global repository of plasmids dedicated to academic research.

Techniques: Gene Expression, CRISPR, Construct, Knock-Out, Mutagenesis

Journal: Diabetes

Article Title: Applying CRISPR Screen in Diabetes Research

doi: 10.2337/dbi20-0047

Figure Lengend Snippet: Illustration of a general workflow of a high-throughput CRISPR screen. A: A plasmid library containing CRISPR gRNAs is amplified and used to generate a lentiviral library. B: A cell line or purification of primary cells is infected with the lentiviral library. C: Mutated cells containing the gRNA and selection marker are isolated. D: Cells are selected or sorted based on the phenotype of interest. E: gDNA of the selected cells is isolated. F: The CRISPR gRNA region of the purified gRNA is amplified via PCR and sequenced. G: Enriched and/or depleted gRNAs are analyzed to identify hits related to the phenotype of interest. Created with BioRender (BioRender.com).

Article Snippet: A large collection of knockout, activation, or inhibition CRISPR libraries, either genome-wide libraries or smaller, more focused, subpool libraries, for both the human and mouse genome, is readily available on Addgene, a nonprofit global repository of plasmids dedicated to academic research.

Techniques: High Throughput Screening Assay, CRISPR, Plasmid Preparation, Amplification, Purification, Infection, Selection, Marker, Isolation

Journal: Diabetes

Article Title: Applying CRISPR Screen in Diabetes Research

doi: 10.2337/dbi20-0047

Figure Lengend Snippet: Overview of the design process of a CRISPR screen.

Article Snippet: A large collection of knockout, activation, or inhibition CRISPR libraries, either genome-wide libraries or smaller, more focused, subpool libraries, for both the human and mouse genome, is readily available on Addgene, a nonprofit global repository of plasmids dedicated to academic research.

Techniques: CRISPR

Journal: Molecular Cell

Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

doi: 10.1016/j.molcel.2018.06.032

Figure Lengend Snippet: HuD Increases Global and Target-Specific Translation (A) Top enriched Gene Ontology terms among HuD mRNA targets are related to RNA processes, including splicing, transport, stability, and translation (highlighted in bold). (B) Metaprofile of HuD binding sites along protein coding transcripts, showing binding enrichment in 3′UTRs. (C) Right panel: representative sucrose gradient profiles in control and HuD overexpressing NSC-34 cells. Left panel: calculation of the global translation efficiency upon HuD silencing and overexpression. (D) Right: schematic representation of Click-iT AHA assay to quantify de novo protein synthesis in NSC-34 cells. Left: detection of de novo protein synthesis upon HuD silencing and overexpression. Puromycin, a translation inhibitor, was used as negative control. (E) Transcriptome-wide translation efficiency changes upon HuD overexpression in NSC-34 cells. Scatterplot displaying for each gene the average expression signal (CPM) against the log2 change in translation efficiency (delta TE) upon HuD overexpression. Genes with increased or decreased TE are highlighted. (F) Enrichment analysis of HuD RNA targets among genes with increased or decreased TE upon HuD overexpression, compared to enrichments associated with genes changing at either the polysomal or the total RNA level. Fisher’s test ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (G) Enrichment of mTOR responsive mRNAs among HuD targets, as listed in multiple literature sources. (H) Western blot analysis of HuD targets (Eef1a1, Eif4a1, Eif4a2, Pabpc1) and negative control (Eif4a3) in HEK293 cells transiently transfected with HuD. Tubulin was used as reference. Experiments were performed at least in triplicate. In (C), (D), and (H), data are represented as mean ± SEM; t test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S2 .

Article Snippet: The following antibodies were used: mouse anti HuD (sc-28299, Santa Cruz), rabbit anti HA (A190-1081, Bethyl Laboratories), rabbit anti eIF4A1 (ab312-17, Abcam), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-eIF4A3 (homemade, generously provided from Prof. Macchi’s lab), rabbit anti eEF1A1 (SAB2108050, Sigma), mouse anti Tubulin (sc-53140, Santa Cruz) and rabbit Anti-PABPC1 (SAB2101708, Sigma), rabbit anti Rpl26 (Ab59567, Abcam), rabbit anti S6 (2217, Cell Signaling Technology).

Techniques: Binding Assay, Control, Over Expression, Negative Control, Expressing, Western Blot, Transfection

Journal: Molecular Cell

Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

doi: 10.1016/j.molcel.2018.06.032

Figure Lengend Snippet: Y3 Counteracts HuD-Induced Neurogenesis (A) Differentiating ESCs cultures assayed for Y3 and HuD expression levels by Northern blot and western blot, respectively. Cultures were immunostained for stage-specific markers: Oct4 (ESCs; red), Nestin (NPCs; red), and beta3-tubulin (early neurons; red); the scale bar corresponds to 75 μm. Relative quantification of Y3 and HuD levels are shown (right). (B) Differentiated NSC-34 cells (control or silenced for Y3) immunostained with anti-tubulin antibody (yellow) to detect neurites (left panel); GFP (green) identified transfected cells subjected to high content analysis; the scale bar corresponds to 100 μm. Multiple parameters were analyzed using Operetta HCS device (right panel). (C) Differentiation assay in control Y3 silenced cells, Y3 silenced cells transfected with wild-type HuD or with mutant HuD. A schematic representation of HuD constructs used in the experiment is provided. (D) PC12 cells were co-transfected with HA-tagged HuD and mock or Y3 WT or Y3 “deleted” vectors. Co-transfected cells were immunostained with anti-HA antibody, and the neurites were stained for tubulin. In (A)–(D), data are represented as mean ± SEM t test ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: The following antibodies were used: mouse anti HuD (sc-28299, Santa Cruz), rabbit anti HA (A190-1081, Bethyl Laboratories), rabbit anti eIF4A1 (ab312-17, Abcam), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-eIF4A3 (homemade, generously provided from Prof. Macchi’s lab), rabbit anti eEF1A1 (SAB2108050, Sigma), mouse anti Tubulin (sc-53140, Santa Cruz) and rabbit Anti-PABPC1 (SAB2101708, Sigma), rabbit anti Rpl26 (Ab59567, Abcam), rabbit anti S6 (2217, Cell Signaling Technology).

Techniques: Expressing, Northern Blot, Western Blot, Quantitative Proteomics, Control, Transfection, High Content Screening, Differentiation Assay, Mutagenesis, Construct, Staining

Journal: Molecular Cell

Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

doi: 10.1016/j.molcel.2018.06.032

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: mouse anti HuD (sc-28299, Santa Cruz), rabbit anti HA (A190-1081, Bethyl Laboratories), rabbit anti eIF4A1 (ab312-17, Abcam), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-eIF4A3 (homemade, generously provided from Prof. Macchi’s lab), rabbit anti eEF1A1 (SAB2108050, Sigma), mouse anti Tubulin (sc-53140, Santa Cruz) and rabbit Anti-PABPC1 (SAB2101708, Sigma), rabbit anti Rpl26 (Ab59567, Abcam), rabbit anti S6 (2217, Cell Signaling Technology).

Techniques: Virus, Recombinant, Magnetic Beads, Western Blot, Luciferase, Amplified Luminescent Proximity Homogenous Assay, Imaging, Sequencing, CRISPR, Knock-Out, Control, Northern Blot, Mutagenesis, Binding Assay, Activity Assay, Plasmid Preparation, Software, Microscopy, High Content Screening

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: Selection of gRNAs targeting EWSR1–FLI1 and analysis of gene editing evolution. ( a ) Experimental design: Ewing sarcoma cell line A673 expressing Cas9 protein (A673/Cas9) were generated by lentiviral infection. After clonal selection, A673/Cas9 cells were infected with a multiplex lentiviral CRISPR library of 18,479 different sgRNAs targeting 1983 transcription factors including ten gRNAs targeting FLI1. After this screening phase, two gRNAs were selected for functional and molecular characterization. A673/Cas9 were infected with lentiviral sgRNAs to generate A673/Cas9/sgRNA cells and then maintained in continuous growth to assess gene editing, cell proliferation, senescence, and studies of mRNA and protein expression at different time points. A673/TR/shEF, which expresses a specific EWSR1–FLI1 shRNA upon doxycycline stimulation, were cultured and analyzed in a similar way. The results obtained upon gene editing and gene silencing were then compared. ( b ) Schematic representation of native FLI1 and EWSR1–FLI1 fusion genes, location of FLI1 gRNAs, and enrichment scores obtained for each gRNA in the CRISPR screening assay (mean ± SD of two independent experiments).

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Selection, Expressing, Generated, Infection, Multiplex Assay, CRISPR, Functional Assay, shRNA, Cell Culture, Screening Assay

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: Effect of EWSR1–FLI1 gene editing on EWSR1–FLI1 expression and EWSR1–FLI1 target genes. ( a ) EWSR1–FLI1, NR0B1, and CD44 mRNA levels were quantified by RT-qPCR in A673/Cas9 cell lines infected with sgRNA vectors targeting exon 2 and exon 9. The graphs represent the results of two independent experiments performed with MOI = 1 (1) and MOI = 5 (2). A673/sgRNA was used as the control (Ctrl). A673/TR/shEF cells were stimulated with doxycycline (1 µg/mL) for 72 h to induce the expression of the EWSR1–FLI1-specific shRNA. Data shown are the mean ± SD of experiments conducted in triplicate (ns, not significant; ** p < 0.01; *** p < 0.001; Student’s t -test). ( b ) EWSR1–FLI1, NR0B1, and CD44 protein levels were detected by Western blot. β-Tubulin was used as a control for loading and transferring. ( c ) Immunostaining of cells with anti-FLI1 antibody. Intense nuclear staining for EWSR1–FLI1 (red fluorescence colocalized with DAPI staining) was observed in both the A673 control cell line and A673/Cas9/FLI1-EX2, while it was undetectable in the majority of the A673/Cas9/FLI1-EX9 cells.

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Infection, shRNA, Western Blot, Transferring, Immunostaining, Staining, Fluorescence

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: Effect of EWSR1–FLI1 gene editing on cell proliferation. ( a ) A673/Cas9/sgRNA cells were continuously maintained in culture and the cumulative population doubling was recorded. The A673/TR/shEF cells were also continuously maintained in culture in the absence or presence of doxycycline (1 µg/mL). ( b ) Quantification of the population doubling time in each cycle of cell seeding–trypsinization. Mean ± SD of three independent experiments is shown (ns, not significant; ** p < 0.01, *** p < 0.001; two-way ANOVA Tukey’s post hoc vs. control). ( c ) The cell cycle was analyzed by flow cytometry in non-synchronized cells. The percentages of cells in each cell cycle phase are indicated. One representative experiment out of two independent experiments performed with similar results is shown.

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Flow Cytometry

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: EWSR1–FLI1 gene inactivation induced generalized senescence. ( a ) β-Galactosidase activity was measured in control (Ctrl) cells (A673/sgRNA) and A673/Cas9/sgRNA cells using a β-galactosidase activity assay. β-Galactosidase-positive cells were counted, and the percentage of positive cells were determined. The graphs represent the results of two independent experiments performed with MOI = 1 (1) and MOI = 5 (2). The A673/TR/shEF cells were stimulated with doxycycline (1 µg/mL) for 7 days to induce the expression of the EWSR1–FLI1-specific shRNA (mean ± Scheme 0. Student’s t -test). Representative micrographs of each cell line are also shown. ( b ) Representative micrographs of A673 and A673/Cas9/FLI1-EX9 cells, showing the characteristic appearance of senescent cells in A673/Cas9/FLI1–EX9 cells. ( c ) The A673/Cas9/sgRNAs cells were seeded at a low density and then isolated clones were reseeded independently in 96-well plates. Afterward, Sanger sequencing/ICE CRISPR analysis was performed for each clone to determine the gene edition percentage (% indels). The tables show the phenotype of each picked-up clone, if it expand after reseeding and the percentage of gene edition.

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Activity Assay, Expressing, shRNA, Isolation, Clone Assay, Sequencing, CRISPR

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, b, Centrosome localization of Piezo2 (a) and Piezo1 (b) in C2C12 myoblast cells visualized by IF for Piezo1/2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue) in mitotic and interphase cells. c, d, Centrosome localization of Piezo2 (d) and Piezo1 (c) in IMCD3 and Neuro-2A cells visualized by IF in interphase cells as in (a-b). e-f, STORM imaging performed for fixed unsynchronized C2C12 cells, stained with Piezo2 (green) and γ-Tubulin (magenta). The STORM image (e) showed co-localization of Piezo2 and γ-Tubulin, suggesting Piezo2 localized to the pericentrosomal region, and the insets (f) of the centrosomes are shown with x3 zoom-in. g, Rosa26 (off target control), Piezo1 and 2 CRISPR-Cas9 polyclonal KO (pKO) of C2C12 cells at day 1 post selection imaged by IF as in (a-b), showing supernumerary centrosomes in mitotic cells. h, Quantitative analysis of supernumerary centrosomes in interphase and mitotic C2C12 pKO cells from the IF experiments in (g). i, Quantitative analysis of mitotic cell populations from the IF experiments in . In 3 independent experiments, 190-280 cells were scored for each category based on the mitotic stage. Pro: prometaphase, Meta: metaphase, Ana: anaphase, Telo: telophase, Cyto: cytokinesis, scored as per Methods. j, Piezo2 -/- myoblasts derived from newborn mice imaged by IF as in (a-b), showing supernumerary centrosomes in Piezo2 -/- myoblasts. k, Quantitative analysis of supernumerary centrosomes in interphase and mitotic cells in WT and Piezo2 -/- myoblasts from IF experiments in (j). m, Cell cycle analysis by flow cytometry for WT and Piezo2 -/- myoblasts. All images are maximum intensity Z projections. Centrosomes and centrosome-localized Piezo proteins are marked with white arrowheads. Scale bars are 5 μm for (a-d), 1 μm (e), and 10 μm (g, j). For (h, k), data are represented by mean ± SEM from three independently quantified experiments counting 50-200 cells each. Statistical significance between an experimental group and a control group was assessed by 2-tailed t-test with ***, ** and * denote p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Imaging, Staining, Control, CRISPR, Selection, Derivative Assay, Cell Cycle Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, b, A field view of interphase C2C12 cells, showing Piezo2 (a) and Piezo 1 (b) localization at the centrosomes. Cells were visualized by IF for Piezo2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue). c, Controls for the fixation method, and primary and secondary antibodies in IF. Top: C2C12 cells were fixed in PFA (instead of methanol) and stained using primary antibodies rabbit anti-Piezo2 and mouse anti-γ-Tubulin, and secondary antibodies goat anti-rabbit Alexa Fluor 488 (green) and goat anti-mouse Alexa Fluor 647 (magenta), as well as Hoechst dye for DNA (blue). Middle: C2C12 cells were stained similarly but with a rabbit IgG replacing rabbit anti-Piezo2. Bottom: C2C12 cells were stained only with secondary antibodies. d, Quantitative analysis of the percentage of C2C12 cells with co-localized centrosome and Piezo1 or 2 from IF images as in (a) and (b). e, f, Centrosome localization of Piezo1 (e) and Piezo2 (f) in IMCD3 cells visualized by IF at different cell cycle stages, imaged by IF as in (a) and (b). g, Snapshots from live imaging of C2C12 cells stably expressing Piezo1-GFP during the cell cycle (Supplementary Video 1). Centrosomes are marked with green arrowheads. h , Co-localization of Piezo1-GFP fluorescence (green) with anti-Piezo1 IF (magenta). Cells were also stained for DNA (Hoechst, blue). i, Localization of Piezo1-GFP fluorescence (green) at the γ-Tubulin (magenta)-marked centrosome by IF. All IF mages are maximum intensity Z projections. Centrosomes and centrosome-localized Piezo1 are marked with white arrowheads. All scale bars are 10 μm, except that the scale bar for (g) is 20 μm.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining, Imaging, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Centrosomal localization of Piezo1 ( left) and Piezo2 ( right ) in E14.5 WT mouse forelimb sections, imaged by IF for Piezo1/2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue). b, A thoracic dorsal root ganglion (DRG) section of a WT mouse at E17.5, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). Right panel shows a 3x zoom-in of the dotted region in the left panel. Piezo2 puncta consistent with a centrosomal localization are labeled with white arrowheads. Of note, Piezo2 positive puncta are often present in DRG neurons whether or not the cell bodies are also Piezo2 positive. c, A thoracic DRG section of a Piezo2 -/- KO mouse at E17.5, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). Piezo2 expression at DRG neuronal cell body or centrosome is largely undetectable . d, A lung bronchial section of a WT mouse at P0, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). The cells labeled in green represent scattered pulmonary neuroepithelial cell bodies (NEBs). e, A comparable lung bronchial section of a Piezo2 -/- KO mouse at P0, imaged by IF with anti-Piezo2 antibody and Hoechst dye (blue), showing absence of Piezo2 staining. f, A comparable lung bronchial section of a WT mouse at P0, stained with rabbit IgG (green) as primary antibody and Hoechst dye (blue). g, h, Thoracic DRG sections of WT mice (g) and Piezo2 -/- KO mice, shown at 2 different contrast levels (h) at E17.5, stained with anti-Piezo2 antibody (green), γ-Tubulin (magenta) and Hoechst dye (blue). White arrowheads identify γ-Tubulin-marked centrosomes (magenta) and/or Piezo2. All scale bars are 10 μm.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining, Labeling, Expressing

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: iSIM was performed for fixed unsynchronized C2C12 cells, stained with Piezo2 (green) and γ-Tubulin (magenta). The iSIM image (left) shows co-localization of Piezo2 and γ-Tubulin, and the insets of the centrosomes (right) are shown with x4 zoom-in.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Piezo1 and Piezo2 protein levels in Piezo1 pKO, Piezo2 pKO, or Rosa26 pKO (control) of C2C12 cells analyzed by Western blots with GAPDH as the loading control. Densitometric analysis of Western blot bands of Piezo1 or 2 are shown. b, Piezo1 pKO ( left ) and Piezo2 pKO ( right ) interphase C2C12 cells at day 1 post-pKO selection, imaged by IF for Piezo1 or Piezo2 (green), γ-Tubulin (magenta) and DNA (Hoechst, blue). Centrosomes are marked with white arrows, and supernumerary centrosomes are seen in Piezo pKO cells, in comparison with Rosa26 pKO control. c, Piezo1 pKO ( middle row ), Piezo2 pKO ( bottom row ) and Rosa26 pKO ( top row ) of mitotic C2C12 cells at day 1 post-pKO selection, imaged by IF for α-Tubulin (green), γ-Tubulin (magenta) and DNA (Hoechst, blue). Centrosomes and lagging chromatin are marked with white arrows, and misaligned microtubules are seen in Piezo pKO cells, in comparison with the Rosa26 pKO control d, Quantitative real-time RT-PCR (qRT-PCR) of Piezo 1 or 2 transcripts upon small hairpin RNA (shRNA) KD of Piezo1 or Piezo2 in C2C12 cells using two and three different shRNAs for Piezo1 and Piezo2, respectively, in comparison with an off target shRNA against firefly luciferase mRNA as the control. e, Control KD, Piezo1 shRNA KD (one shRNA) or Piezo2 shRNA KD (two shRNAs) C2C12 cells at day 4 post-KD selection. The cells were imaged by IF for γ-Tubulin (red) and DNA (Hoechst, blue), showing supernumerary centrosomes upon Piezo1 or Piezo2 KD. f, Quantitative analysis of C2C12 cells with supernumerary centrosomes upon Piezo1 shRNA KD (one shRNA) or Piezo2 shRNA KD (two shRNAs) at day 4 post-KD selection. All scale bars are 10 μm. g, Off target, Piezo1 and 2 pKO C2C12 cells at day 1 post selection and 45 min after release of G2/M border synchronization with RO-3066. Cells were imaged by IF for α-tubulin (green), γ-tubulin (magenta) and DNA (Hoechst dye, blue), shown at prometaphase and cytokinesis. Quantitative analysis of mitotic cell populations from the IF experiments shown in . Statistical significance between an experimental group and the control group was assessed by 2-tailed t-test. ***, ** and * denote p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Control, Western Blot, Selection, Comparison, Quantitative RT-PCR, shRNA, Luciferase

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Validation of the NLS-GCaMP6 reporter stably expressed in C2C12 cells by ionomycin (1 μM), BAPTA-AM (10 μM), or Yoda1 (10 μM) treatment. b , Quantitative analysis of maximal signals for cells imaged in (a). AFU denotes arbitrary fluorescence units. c, Phase and fluorescence snapshots of C2C12 cells expressing GCaMP6 reporter at interphase and different stages of mitosis (green). Concentrated Ca 2+ signals that represent centrosome locations are marked by white arrowheads. d , Phase and fluorescence snapshots of C2C12 cells at interphase and different stages of mitosis, imaged live at 45 min after treatment with the Ca 2+ dye Fluo4-AM (9 μM, green). Concentrated Ca 2+ signals that represent centrosome locations are marked by white arrowheads. e, Quantitative analysis of maximal GCaMP6 signals at centrosomes as shown in , revealing reductions to 43% and 34% in comparison to Rosa26 pKO control values in Piezo1 and 2 pKO cells, respectively. f, GCaMP6 expression by anti-GFP IF (green) in NLS-GCaMP6 expressing C2C12 cells at day 1 post-KO selection of Rosa26 pKO, Piezo1 pKO or Piezo2 pKO. IF for γ-Tubulin (magenta) and Hoechst dye staining of DNA (blue) are also shown, exhibiting equal GCaMP6 expression regardless of Piezo1 or 2 pKO. g , Quantitative analysis of maximal Ca 2+ signal indicated by GCaMP6 fluorescence intensity at centrosomes and in the cytosol as shown in , showing that there was a similar increase or decrease of local Ca 2+ concentration upon Yoda1 activation or GsMTx4 inhibition relative to the untreated control value both in the cytosol and at the centrosomes. Images are maximum intensity Z projections and all scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments. Statistical significance between an experimental group and the control was assessed by 2-tailed t-test with *** and ** for p < 0.0001 and 0.001, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Biomarker Discovery, Stable Transfection, Fluorescence, Expressing, Comparison, Control, Selection, Staining, Concentration Assay, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, HNMR Spectra of caged-Yoda1 and hydroxy-Yoda1 synthesis. The compounds were synthesized by WuXi AppTec with purity determined by LC-MS of 98.2% and 78.6%, respectively. For hydroxy-Yoda1, the compound was clean based on the HNMR spectrum but might not be stable in LC-MS and therefore a lower purity was reported. b, A table describing the top hits from the yeast two-hybrid screen performed for Piezo2 CTD.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Synthesized, Liquid Chromatography with Mass Spectroscopy, Two Hybrid Screening

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Validation of the Snx5 interaction with the Piezo2 C-terminal domain (CTD, last 70 aa) using an alternative yeast two-hybrid assay. The Piezo2 CTD was fused to the LexA DNA binding domain and full-length Snx5 was fused to a B42 activation domain. Positivity is indicated by yeast turning dark blue in the presence of the LacZ reporter X-gal (+++) and by yeast growth in the absence of leucine. Negativity is indicated by yeast staying white (-) and no growth in the absence of leucine. Phox homology (PX) domain for lipid binding and Bin/Amphiphysin/Rvs (BAR) domain for membrane-curvature sensing are shown. b, Western blot analysis of Ni-NTA pulldown of human Snx5 by human Piezo2 CTD. Piezo2-CTD-His-Myc was co-expressed with GFP-Snx5 or GFP control in HEK293T cells. The His-tagged protein was precipitated using Ni-NTA beads (lane 1&2) or as a control with NTA beads without Ni (lane 3). Lanes 4 and 5 are cell lysates. Precipitated Piezo2-His-Myc was detected with anti-Myc antibody and GFP-hSnx5 was detected with anti-GFP antibody. c, IF images of C2C12 cells (top panel) and Neuro-2A cells (bottom panel) stained for Piezo2 (green), Snx5 (olive), ɣ-Tubulin (magenta) and DNA (blue). Co-localization among Piezo2, Snx5 and ɣ-Tubulin is shown by arrowheads and all scale bars are 10 μm. d, Western blot analysis of fractionation of isolated centrosomes. Centrosomes isolated from C2C12 cells were fractionated on a sucrose gradient, and blotted with the following antibodies: anti-Piezo1, anti-Piezo2, anti-γ-Tubulin, anti-Pericentrin and anti-Snx5.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Biomarker Discovery, Y2H Assay, Binding Assay, Activation Assay, Membrane, Western Blot, Control, Staining, Fractionation, Isolation

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a-b, IF of C2C12 cells, fixed after 3h cold treatment, 2 h treatment with Parthenolide or Taxol and stained with Piezo1 (a) or Piezo2 (b), ⍺-Tubulin (green) and Hoechst (Blue). Untreated cells were used as a control. Centrosomes are marked with white arrowheads. c , Western blots of C2C12 cells at different cell cycle stages using anti-Piezo1, Piezo2, PLK1 and GAPDH (loading control) antibodies. While Piezo2 is highly expressed at S and G2, Piezo1 expression is relatively constant. d , Cell cycle analysis by PI staining for C2C12 cells treated with PLK1 inhibitor BI-6727. Optimal concentration for BI-6727 treatment in C2C12 was determined as 100 nM. Images are maximum intensity Z projections and all scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments. Statistical significance between an experimental group and the control was assessed by 2-tailed t-test with ***, ** and * for p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining, Control, Western Blot, Expressing, Cell Cycle Assay, Concentration Assay