Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: Whole-body p53 deletion promotes the progression of renal dysfunction in X-linked AS mouse model. (A) Glomeruli were isolated from WT or AS mice in early (10 weeks) or late (21 weeks) stage of AS. Proteins were extracted from glomeruli and p53 protein was analyzed by immunoblotting. Actin was used as loading control. Relative amount of p53 was quantified and normalized to actin (mean±SEM, n=3). **P<0.01 versus WT. (B) Urine samples were obtained from 12-week-old mice of each genotype, and urinary albumin score was measured. U-Albumin scores were normalized with urine creatinine score (mean±SEM, n=4–10). (C) Urine samples were collected at 6, 9, 12, 15, 18, 21, and 24 weeks, and protein concentration was measured. Proteinuria score was calculated based on the urine protein concentration and urine creatinine score (mean±SEM, n=3–8). Measuring proteinuria score of p53−/− AS group was terminated at 15 weeks due to the death of all mice in this group. (D) BUN score of p53+/+ AS and p53−/− AS mice was measured at 6 and 12 weeks (mean±SEM, n=5–7). (E) Survival rate of p53+/+, +/−, −/− AS mice was measured and analyzed by Kaplan–Meier method. Log-rank test was used for statistical analysis (n=3–11). *P<0.05; **P<0.01 versus p53+/+ AS; ##P<0.01 versus p53+/− AS.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Isolation, Western Blot, Control, Protein Concentration

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: p53-deficient AS mice showed enhanced renal inflammation, glomerular injury, and fibrosis. (A) Total RNA was isolated from renal tissue of 12-week-old WT, p53+/+ AS, and p53+/− AS mice. Quantitative RT-PCR was performed to analyze the expression of indicated renal injury marker genes and cytokines. Gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as internal control (mean±SEM, n=6–7). (B) Staining of renal sections from 12-week-old mice was performed using PAS or MT staining. Scale bars, 50 μm (PAS) and 100 μm (MT). (C) Glomerulosclerosis scores were calculated by counting the level of glomerular injury in PAS-stained kidney sections. (D) Tubulointerstitial fibrosis scores were evaluated by measuring the region of fibrosis in MT-stained sections. (E) Representative images of glomerular crescent formation in p53+/− AS mouse in PAS-stained kidney sections. Broken line indicates the crescent formation regions. Scale bars, 50 μm. (F) Quantitative results of glomerular crescent formation in p53+/+ or +/− AS groups. (C, D, F) mean±SEM, n=3. *P<0.05; **P<0.01 versus WT; #P<0.05; ##P<0.01 versus p53+/+ AS. n.s., not significant.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Marker, Control, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: p53 suppressed podocyte migration in vitro and positively regulated podocyte-specific genes. (A) Glomeruli were isolated from p53+/+ or −/− mice and cultured for 5 days. Podocytes migrate from glomeruli and proliferate as colonies.52 Migrating GECs were fixed with formalin, stained with WT-1, and visualized by immunofluorescence. Scale bars, 200 μm. (B) p53 expression in the glomeruli of p53+/+ and −/− mouse was confirmed by immunoblotting. Actin was used as loading control. (C) Podocyte-spreading area in (A) was quantified (mean±SEM, n=67–98). (D) MPC-5 cells were treated with 25 μM nutlin-3α or 50 μM pifithrin-α for 18 hours. After treatment, cells were fixed, stained with antibodies against p53 (red), F-actin (green), and DAPI (blue) and visualized by immunofluorescence. Scale bars, 20 μm. (E, F) Filopodia- and lamellipodia-positive cells were counted and ratio was calculated (mean±SEM, 33–55 cells per sample, n=4). (G) Differentiated MPC-5 cells were treated for 24 hours with 25 μM nutlin-3α or 50 μM pifithrin-α. After washout, in vitro scratch assay was performed, and cells were re-incubated for additional 10 hours. Acquisition of phase-contrast images at 0 and 10 hours was done using Bio-Revo. Scale bars, 300 μM. (H) The area covered by cells (blue field) was quantified and analyzed using Bio-Revo imaging and analysis software. Values shown are mean±SEM (con, n=7; nutlin-3α, n=7; pifithrin-α, n=9). (I-K) Primary GECs were isolated from p53+/+ or −/− mouse and cultured. Basal mRNA expression of the indicated genes was analyzed by quantitative RT-PCR (mean±SEM, n=3). (L) Primary GECs from p53+/+ or −/− mouse were treated with 25 μM nutlin-3α for 24 hours and protein lysate was extracted. p53 expression was analyzed by western blotting. Actin was used as a loading control. (M-O) Total RNA was extracted from p53+/+ or −/− primary GECs after treatment with 25 μM nutlin-3α for 24 hours. Expressions of the indicated genes were analyzed by quantitative RT-PCR. Gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as internal control. (mean±SEM, n=3). †P<0.1; *P<0.05; **P<0.01.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Migration, In Vitro, Isolation, Cell Culture, Staining, Immunofluorescence, Expressing, Western Blot, Control, Wound Healing Assay, Incubation, Imaging, Software, Quantitative RT-PCR

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: Podocyte-specific p53 deletion promotes AS-induced renal dysfunction. (A) Mating procedure to generate podocyte-specific p53-deficient WT and AS mice. Each littermate group of pod-p53+/+ or −/− WT and pod-p53+/+ or −/− AS was used for the following experiments. (B) Frozen sections of renal cortex harvested from 15-week-old mice were stained for immunofluorescence with antibodies against p53 (green) and WT-1 (red) or with type IV collagen A5 (green) and WT-1 (red). Fields in yellow box are magnified in the panel below. Yellow arrows indicate p53 expression in WT-1-positive cells. Scale bars, 10 μm. (C, D) Urine samples from 15-week-old mice were assessed for (C) U-Albumin and (D) proteinuria scores. (mean±SEM, n=4–9). (E) BUN score in 15-week-old mice was measured (mean±SEM, n=4–8). *P<0.05, **P<0.01 versus pod-p53+/+ WT; #P<0.05 versus pod-p53+/+ AS.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Staining, Immunofluorescence, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: Renal pathology of AS mouse was exacerbated by podocyte-specific p53 deletion. (A) Total RNA was isolated from renal tissues of 15-week-old mice. Quantitative RT-PCR was performed to analyze the expression of the indicated renal injury marker genes and cytokines. Gapdh (glyceraldehyde-3-phosphate dehydrogenase) was used as internal control (mean±SEM, n=4–6). (B) PAS and MT staining of renal sections in 15-week-old mice were performed. Scale bars, 50 μm. (C, D) Glomerulosclerosis and tubulointerstitial fibrosis scores were quantified from the PAS and MT staining results (mean±SEM, n=4–6). *P<0.05; **P<0.01 versus pod-p53+/+ WT; #P<0.05 versus pod-p53+/+ AS. n.s., not significant.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Marker, Control, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: Microarray analysis of gene-expression pattern in glomeruli of pod-p53+/+ and pod-p53−/− mice. Total RNA was isolated from the glomeruli of 12-week-old pod-p53+/+ or −/− WT and AS mice and analyzed by 3D-Gene DNA chip microarray (TORAY, Japan). (A) Gene ontology analysis was performed for the gene data of pod-p53+/+ AS and pod-p53−/− AS groups that were up-regulated or down-regulated statistically compared with pod-p53+/+ WT group using DAVID functional annotation. Gene categories were lined up by P value (P<0.05) and the top six categories that include up- or down-regulated genes in pod-p53+/+ AS group (gray bar), or in pod-p53−/− AS group (black bar) and common category between pod-p53+/+ and −/− AS groups (highlighted in blue) were picked up. (B) Genes were classified according to the changes in expression pattern. Clusters of genes whose AS-induced alteration of expression was enhanced by pod-p53 deletion were picked up. The red text indicates genes that are up-regulated three-fold in the kidneys of pod-p53−/− AS mice compared with pod-p53+/+ WT mice as determined by quantitative PCR shown in Supplement Figure 6B.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Microarray, Gene Expression, Isolation, Functional Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: p53 modulates podocyte abnormal growth and foot process effacement in AS. (A) Kidney sections from 15-week-old pod-p53+/+ or −/− WT or AS mice were immunostained with cell proliferation marker protein, PCNA. Sections were counterstained with hematoxylin. PCNA-positive cells in the renal corpuscle (RC) were counted and distinguished by histologic localization as glomerular, PEC or total. Red arrows show PCNA-positive cells in glomerular region and black arrows show PCNA-positive cells in Bowman’s capsule region. Scale bars, 50 μm. (B) PCNA-positive cells in RC were counted and analyzed in glomerular region (Glomerular) and in Bowman’s capsule region (PEC) or counted as total PCNA-positive cells in RC (Total). PCNA count was performed in 27–90 glomeruli per sample (mean±SEM, n=4–6). (C, D) Structure of podocyte foot process in 15-week-old mice was analyzed by SEM and TEM. High-magnification micrographs for the indicated mouse genotype are shown. (C) Scale bars, 1 μm. (D) Scale bars, 4 μm (upper panels) and 0.5 μm (lower panels). Podocyte is marked with “P”. (E) Morphometric analysis of the foot process. The foot processes were manually counted in four random glomeruli in each mouse genotype. Values shown are mean±SEM **P<0.01 versus pod-p53+/+ WT. ##P<0.01 versus pod-p53+/+ AS.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Marker

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: Pod-p53 regulates the glomerular hyperplastic phenotypes in the progression of AS. Podocyte foot process structures are maintained in early stage of AS. Progressive disruption of foot process structures gradually induces renal dysfunction. Pod-p53 deletion in AS mouse enhances podocyte foot process effacement and induces aberrant filopodia formation. Pod-p53−/− AS mouse had increased number of proliferative PECs and podocytes. Gene-expression patterns were altered, and secreted type of factors that modify cell proliferation and migration were enhanced in pod-p53−/− AS mouse. These secreted factors may influence proliferation of PECs and crescent formation in AS progression. Furthermore, p53 expression is suppressed in the glomeruli of AS mouse at late stage and this contributes to the hyperplastic glomerular disorder and progression of renal dysfunction in AS.

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Disruption, Gene Expression, Migration, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Podocyte p53 Limits the Severity of Experimental Alport Syndrome

doi: 10.1681/ASN.2014111109

Figure Lengend Snippet: Primer sequence for real-time quantitative RT-PCR

Article Snippet: 51 Blots were reacted for 2 hours with monoclonal mouse anti-p53 (#2524; Cell Signaling Technology) or for 1 hour with polyclonal goat anti-Actin (sc-1616; Santa Cruz Biotechnology) diluted at 1:1000, and with the respective HRP-conjugated secondary antibodies diluted at 1:5000.

Techniques: Sequencing

Journal: Immunity

Article Title: CRISPR screens unveil nutrient-dependent lysosomal and mitochondrial nodes impacting intestinal tissue-resident memory CD8 + T cell formation

doi: 10.1016/j.immuni.2024.09.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified naive OT-I, P14, or YopE-I cells were activated for 20 h with 5 μg/ml plate-bound anti-CD3 (2C11, Bio X Cell) and 5 μg/ml plate-bound anti-CD28 (37.51, Bio X Cell) in complete Click’s medium (catalog #9195, Irvine Scientific) containing 10% fetal bovine serum (FBS; R&D Systems), 1× penicillin–streptomycin–L-glutamine (catalog #15140122, Thermo Fisher Scientific), and 55 μM β-mercaptoethanol.

Techniques: Purification, Virus, Expressing, Mutagenesis, Recombinant, Electron Microscopy, Control, Modification, Plasmid Preparation, Cell Isolation, Transfection, Sample Prep, Reverse Transcription, SYBR Green Assay, Microarray, RNA Sequencing Assay, Knock-In, Sequencing, Software, Flow Cytometry, Microscopy, Real-time Polymerase Chain Reaction

Journal: Cell Reports

Article Title: A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs

doi: 10.1016/j.celrep.2018.12.074

Figure Lengend Snippet: Defects in DNA Damage Response in the syp-1 Phosphorylation Alleles (A) Sensitivity of L4-stage worms from the indicated strains to different doses of IR. Relative survival of offspring is shown. Data are represented as average percentage ± SD from at least four experiments with 15 worms each. ∗ p = 0.02, ∗∗ p = 0.0015, ∗∗∗ p = 0.0006, ∗∗∗∗ p < 0.0001; p values for paired t test. (B and C) Quantification of recombination marker RAD-51 foci in the indicated strains in normal conditions (B) or 20 h after 75 Gy (C). At least 15 gonads were analyzed in each condition and ten nuclei were scored in each zone (mitotic region, 1; transition zone, 2; early-mid-late pachytene regions, 3-4-5; and diplotene-diakinesis regions, 6) for at least three independent experiments. (D) Germ cell apoptosis was measured by differential interference contrast (DIC) microscopy in animals of the indicated strains at the indicated time points after IR treatment. Data are represented as average ± SD from at least ten worms for each time point of three independent experiments. ∗∗∗∗ p < 0.0001, p value for paired t test.

Article Snippet: Rabbit RAD-51 Antibody , Novus Biologicals , NB100-148.

Techniques: Marker, Microscopy

Journal: Cell Reports

Article Title: A Meiotic Checkpoint Alters Repair Partner Bias to Permit Inter-sister Repair of Persistent DSBs

doi: 10.1016/j.celrep.2018.12.074

Figure Lengend Snippet:

Article Snippet: Rabbit RAD-51 Antibody , Novus Biologicals , NB100-148.

Techniques: Recombinant, Plasmid Preparation, Peptide Microarray, Clone Assay, Sequencing, Mutagenesis, Software, Fluorescence

Journal: Frontiers in Immunology

Article Title: Guanabenz Prevents d -Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality

doi: 10.3389/fimmu.2017.00679

Figure Lengend Snippet: Guanabenz (GBZ) immunomodulatory effect on TLR4 signaling. (A) GM-CSF-derived dendritic cells (DCs) were stimulated for 8 h with Proteus mirabilis (0.1 MOI), in the presence or absence of GBZ (50 µM). Gene expression was established by Affymetrix microarray analysis and compared with the ingenuity pathway analysis (IPA) Ingenuity Pathway software. Venn diagram was used to separate DEGs in three subsets. Tables contain selected DEGs for each subset and displaying the highest difference in expression level compared to control groups. Major upstream regulators identified by IPA are presented for each subset or group. (B) GM-CSF-derived DCs were treated for 6 h with 100 ng/ml lipopolysaccharide (LPS) 055:B5, in the presence or absence of GBZ (50 µM). Protein lysates were blotted for P-eIF2α and total eIF2α. The ratio, calculated in three independent experiments, was plotted. (C) On the same samples, the level of interleukin-10 (IL-10) transcription and secretion was measured by qPCR ( n = 3) and ELISA ( n = 4) (D) . GM-CSF-derived DCs were treated for 8 h with P. mirabilis (0.1 MOI) or 10 µg/ml of poly I:C, with or without GBZ (50 µM). The secretion of IL-12 ( n = 3) and IL-6 ( n = 4) was measured by ELISA. Statistical significance was assigned using one-way ANOVA test followed by Tukey range test to assess the significance among pairs of conditions (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Rabbit polyclonal antibody against p-eIF2α (Ser51) was from Abcam.

Techniques: Derivative Assay, Gene Expression, Microarray, Software, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34 + cell therapy to induce fetal hemoglobin for sickle cell disease

doi: 10.1101/2024.04.30.591737

Figure Lengend Snippet: ( A ) Schematic of SpCas9-3xNLS genome editing target sites in the γ-globin promoters highlighting ZBTB7A (−196) and BCL11A (−115) binding motifs in red (boxed). Blue arrow with dotted line shows the predicted SpCas9 cleavage position at −196 and −115 sites. Single guide-RNA design location shown in black line with SpCas9 protospacer adjacent motif (PAM) highlighted in blue line (NGG) for respective sites. 13nt deletion associated with hereditary persistence of fetal hemoglobin (HPFH) mutation indicated with black dotted line. ( B ) Indels for WT SpCas9 and HiFi SpCas9 (R691A) determined by NGS after editing at −196 and −115 γ-globin promoter targets using Lonza 4D nucleofector under optimal conditions. Scatter plots shown as mean with error line. ( C ) Percent of HbF estimated in erythroid differentiated cultures at Day 21 by ion exchange high-performance liquid chromatography (HPLC). Scatter plots show the results as a mean, adjusted P values for all samples are <0.0001**** compared to controls (ordinary one-way ANOVA, Dunnett’s multiple comparison tests). Adjusted P values for −196 vs −115 γ-targets with WT and HF are 0.0002*** and 0.0024** (2way ANOVA, Šídák’s multiple comparisons test). ( D ) On-target indels determined by NGS after editing at day 4 and 17 weeks of post-transplantation for different targets BCL11A enhancer, −196 γ-globin promoter, and −115 γ-globin promoter (‘ns’ represents statistically not significant for input and 17 weeks, P values 0.374, 0.991, 0.347 and −115 HF γ-globin promoter (P value <0.0001****) (2way ANOVA, Šídák’s multiple comparisons test). Each dot represents an independent mouse from 17 weeks (n = 5). ( E ) Scatter plot of %HbF in human erythroid cells for control or edited CD34 + cells, measured by HPLC at 17 weeks of xenotransplantation. Data shown as mean with error line and adjusted P value <0.0001**** (n=5) compared to controls as each value shown as dots (Ordinary one-way ANOVA, Dunnett’s multiple comparison tests) and adjusted P values for BCL11A enhancer vs −115 with WT, −196 vs −115 with WT, and −115 with WT vs HF are 0.0169*, <0.0001****, and 0.0001*** (Ordinary one-way ANOVA, Tukey’s multiple comparison tests).

Article Snippet: Probed with primary antibodies rabbit anti α-globin from ResGen (Invitrogen Corporation) and mouse anti γ-globin (Santa Cruz, Sc-21756, 51-7) at dilutions 1:1500 and 1:10 followed by secondary antibodies Donkey anti-rabbit IgG NorthernLights557 (R&D Systems, NL004) and Donkey anti-mouse-Alexa Fluor647 (Fischer, A31571) at 1:40 dilution. scWest chips were scanned by InnoScan710 micro array scanner and 16-bit TIFF images were analyzed by scout software.

Techniques: Binding Assay, Mutagenesis, High Performance Liquid Chromatography, Comparison, Transplantation Assay, Control

Journal: bioRxiv

Article Title: Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34 + cell therapy to induce fetal hemoglobin for sickle cell disease

doi: 10.1101/2024.04.30.591737

Figure Lengend Snippet: Analysis of CD235a + cells from transplanted mouse BM derived from three SCD donors at 17 weeks. ( A ) Representative scatter plots of F-expressing cells by flow cytometry for control and Cas9 edited cells. Percentages were shown as mean (SD), with P values are <0.0001**** and significant for all three donors (2way ANOVA, Šídák’s multiple comparisons test). ( B ) Scatter plots of α-globin (shown on x-axis) and γ-globin (shown on x-axis) comparing between control and Cas9-3xNLS by single cell western analysis. ( C ) Kernel density plots showing the distribution of the percentage of HBG transcripts ( HBG1 + HBG2 / HBG1 + HBG2 + HBB ) in Cas9 edited erythroblasts compared to unedited controls. ( D ) UMAP plot showing annotated cell clusters at different stages of erythroid maturation from pooled erythroid samples derived from mice BM. Terminal erythroid differentiation stages classified as proerythroblast/basophilic erythroblast (ProE/BasoE), early & late stages of polychromatophilic erythroblast (PolyE), and early & late stages of orthochromatic erythroblast (OrthoE). ( E ) Scatter plot showing the percentage of each cluster at different stages of erythroid differentiation between control and Cas9. Differences between control and Cas9 were not significant (i.e., ns) (2way ANOVA, Šídák’s multiple comparisons test). ( F ) Volcano plot showing differentially expressed genes. Blue dot indicates the expressed genes with FDR ≤ 0.01, red dot indicates upregulated genes with both FDR ≤ 0.01 and log 2 FC ≥ 1, and grey dot (ns) indicates not significant.

Article Snippet: Probed with primary antibodies rabbit anti α-globin from ResGen (Invitrogen Corporation) and mouse anti γ-globin (Santa Cruz, Sc-21756, 51-7) at dilutions 1:1500 and 1:10 followed by secondary antibodies Donkey anti-rabbit IgG NorthernLights557 (R&D Systems, NL004) and Donkey anti-mouse-Alexa Fluor647 (Fischer, A31571) at 1:40 dilution. scWest chips were scanned by InnoScan710 micro array scanner and 16-bit TIFF images were analyzed by scout software.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Control, Single Cell Western

Journal: bioRxiv

Article Title: Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34 + cell therapy to induce fetal hemoglobin for sickle cell disease

doi: 10.1101/2024.04.30.591737

Figure Lengend Snippet: ( A ) Schematic representation of designated ddPCR probes and primers at HBG1 and HBG2 gene promoters. Simultaneous Cas9-induced DNA DSBs (red arrows) results in loss of 4.9kbp deletion represented in blue dotted lines. PacBio long-range PCR primers for 14kbp region shown as purple dotted lines. ( B ) Measurement of 4.9kbp deletion by ddPCR in edited human CD34 + bulk HSPCs (Pre-ER#1-3, n=3, plerixafor mobilized normal donors)) at day 5. ( C ) Coverage of large deletions evaluated by long-range PCR-based PacBio sequencing in edited human HSPCs at day 5. ( D ) Manhattan plots of CHANGE-seq (n=3) detected on- and off-target sites. Intended on-target site highlighted with arrow (pink) and possible off-target sites shown as bar heights. X-axis represents chromosome location and y-axis represents number of CHANGE-seq read counts. ( E ) Alignment of intended target site (top line) with CHANGE-seq detected on- and off-target sites (top to bottom by read count) for Cas9:sgRNA (RNP) complex. Mismatches column indicates the number of mismatches relative to intended target site, where 0 indicated the on-target identified by CHANGE-seq. The coordinates column indicates the genomic coordinate, for the on- and off-target sites identified by CHANGE-seq. Note: output is truncated to the top sites. ( F ) Genome-wide off-target activity evaluated by multiplex targeted sequencing (rhAmp-seq, IDT) based on on- and off-target sites detected by CHANGE-seq. X-axis represents chromosomal location and y-axis indicates indels for control and Cas9 (Pre-ER#1-3, n=3) (multiple paired t-tests compared indel frequencies between edited and unedited cells using two-tailed paired t-test, controlling for false discovery rate using the procedure of Benjamini, Krieger, and Yekutieli). ( G ) Circos plots representing genomic re-arrangements for −115 γ-globin promoter and −115 γ-globin promoter + BCL11A enhancer as a control by UDiTaS method.

Article Snippet: Probed with primary antibodies rabbit anti α-globin from ResGen (Invitrogen Corporation) and mouse anti γ-globin (Santa Cruz, Sc-21756, 51-7) at dilutions 1:1500 and 1:10 followed by secondary antibodies Donkey anti-rabbit IgG NorthernLights557 (R&D Systems, NL004) and Donkey anti-mouse-Alexa Fluor647 (Fischer, A31571) at 1:40 dilution. scWest chips were scanned by InnoScan710 micro array scanner and 16-bit TIFF images were analyzed by scout software.

Techniques: Long Range PCR, PacBio Sequencing, Genome Wide, Activity Assay, Multiplex Assay, Sequencing, Control, Two Tailed Test

Journal: iScience

Article Title: Integrated stress response regulates GDF15 secretion from adipocytes, preferentially suppresses appetite for a high-fat diet and improves obesity

doi: 10.1016/j.isci.2021.103448

Figure Lengend Snippet:

Article Snippet: Anti-phospho-eIF2α (Ser51) , Cell Signaling Technology , Cat# 9721, RRID: AB_330951.

Techniques: Recombinant, Liposomes, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Gene Expression, Microarray, CRISPR, Plasmid Preparation, Software

Journal: Cancer cell

Article Title: p53 is a master regulator of proteostasis in SMARCB1 -deficient malignant rhabdoid tumors

doi: 10.1016/j.ccell.2019.01.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-phospho-eIF2alpha (Ser51) (D9G8) XP , Cell Signaling Technology , Cat#3398, RRID:AB_2096481.

Techniques: Luciferase, Virus, Plasmid Preparation, Expressing, Mutagenesis, Recombinant, Purification, Flow Cytometry, Microarray, shRNA, Sequencing, Software

Journal: iScience

Article Title: Neuropilin-1 cooperates with PD-1 in CD8 + T cells predicting outcomes in melanoma patients treated with anti-PD1

doi: 10.1016/j.isci.2022.104353

Figure Lengend Snippet: NRP1 is expressed in activated CD8 + T cells and controls their antitumoral function in mice (A) NRP1 expression and cell trace intensity analyzed by flow cytometry in OT1 murine CD8 + T cells activated during 24, 48, 72, and 96 h with OVA 257 peptide pulsed on dendritic cells (SIINFEKL, 10 −9 mg/mL). Data are representative of 5 independent experiments. (B) Flow cytometry analysis of NRP1 expression in OT1 murine CD8 + T cells, 72 h after activation with OVA peptide (SIINFEKL, 10 −9 mg/mL). Expression is shown according to different effector CD8 + T cell / antigen-presenting cell (DC) ratios (E/A ratio: 1/1, 2/1, 4/1, and 8/1). p value (p = 0.0006) was determined by one-way ANOVA. Data are representative of 2 independent experiments. Expression was assessed by flow cytometry and data are presented as the mean ± SEM of percentage of NRP1 on CD8 + T cells (c) Expression of NRP1 in CD8 + T cells from B6 mice, after intramuscular immunization with AAV-OVA vector. Expression was assessed by flow cytometry and data are presented as the mean ± SEM of percentage of NRP1 on iTAg Tetramer/PE - H-2 Kb OVA, at day 7, 14, 21, 28, and 35 after immunization. Data are representative of 5 independent experiments. (D) NRP1 expression profiles in H2-Db GP33-specific CD8 + T cells according to in vivo infection in mice with LCMV Armstrong (n = 16), LCMV clone 13 (n = 16), or naïve CD44 low CD8 + T cells from controls (n = 4) at days 6, 8, 15, and 30. Raw transcriptomic data were from Doering et al. (2012) microarray experiments [32]. Data are presented as the mean ± SEM p value was determined by two-way ANOVA(p = 0.0008). (E) Flow cytometry analysis of NRP1 expression (blue line curve) on iTAg Tetramer/PE - H-2 Kb OVA CD8 + TILs collected from C57BL/6 mice bearing a B16-OVA tumor at day 14 post-immunization with ovalbumin and poly-IC. Data are representative of 3 independent experiments. (F) Flow cytometry analysis of NRP1 and PD1 expression in OT1 CD8 + T cells activated with OVA 257 peptide (SIINFEKL, 10 −9 M) at 24, 48, 72, and 96 h post-activation. Data are representative of 3 independent experiments. (H) Mice were pre-immunized (immunized) or not pre-immunized (control) with ovalbumin and poly-IC. B16-OVA tumor volume was assessed at day 0, 8, 11, 14, and 18 post-immunization in CD8Nrp1KO (KO) and control C8Cre (WT) mice. Data are presented as mean ± SEM. p values were determined by using student t- test ∗∗∗p < 0.001, ∗p < 0.05. Data are representative of 3 independent experiments. (G) CD8Nrp1KO (KO) and control (WT) mice were injected in the right flank with 1 × 10 5 TC1 lung tumor cells subcutaneously. Data are presented as mean ± SEM p values was determined by using student t- test ∗∗p < 0.01, ∗p < 0.05. Data are representative of 3 independent experiments (I) CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumin and poly-IC. Tumor volume was assessed 21 days after immunization. Data are presented as mean ± SEM p values was determined by using student t- test (p = 0.0012). Data are representative of 3 independent experiments. (J) Number of CD8 + TILs per fields with highest CD8 + T cells infiltration from CD8Nrp1KO mice (KO) or controls (WT) assessed by confocal microscopy at day 21 post immunization with ovalbumin and poly-IC. Data are presented as mean ± SEM. p values (p < 0.0001) was determined by using student t- test. Data are representative of 3 tumors per group. (K) Percentages of Tetramer/PE - H-2 Kb OVA CD8 + TILs in B16-OVA tumors of four different mice group assessed at day 14 post-immunization by flow cytometry from CD8Nrp1KO (KO) and control (WT) mice immunized or not immunized (control) with ovalbumin and poly-IC. Data are presented as the mean percentage of CD8 + TILs Tetramer positive ± SEM. p values were determined by using student T test ∗∗p < 0.01, ∗p < 0.05. Data are representative of 2 independent experiments. (L) Ex vivo TILs proliferation was analyzed by flow cytometry 72 h post-activation with anti-CD3 and anti-CD28. TILs were collected from B16-OVA tumors at day 21 post-immunization from 3 mice. Data are presented as the mean percentage of divided CD8 + T cells ± SEM. p values was determined by using student t- test ∗∗∗p < 0.001. Data are representative of 2 independent experiments.

Article Snippet: anti-mouse CD28 (37.51) , BioXcell , Cat# BE0015-1, RRID: AB_1107624.

Techniques: Expressing, Flow Cytometry, Activation Assay, Plasmid Preparation, In Vivo, Infection, Microarray, Control, Injection, Confocal Microscopy, Ex Vivo

Journal: iScience

Article Title: Neuropilin-1 cooperates with PD-1 in CD8 + T cells predicting outcomes in melanoma patients treated with anti-PD1

doi: 10.1016/j.isci.2022.104353

Figure Lengend Snippet: NRP1 modulates PD1 activity at the synapse between CD8 + T cells and tumor cells (A) Illustrative image of phalloidin (yellow), CD8 (pink), CFP from EL4 (purple), and NRP1 (red) labeling in the synapse model between activated OT1 CD8 + T cells and EL4-CFP tumor cells bearing OVA 257 (SIINFEKL), observed by ImageStream. The synapse is the high phalloidin labeling zone. Bright field image is in white (scale bar = 7μm). Data are representative of 4 independent experiments from 2 synapse models. (B) Quantification by ImageStream of NRP1 expression (mean pixel intensity/MPI) in an allogeneic synapse model between activated CD8 + T cells and cell tracer violet labeled A20 cells. NRP1 expression was analyzed in activated CD8 + T cells at the synapse junction (high phalloidin labeling zone). Data are presented as the mean MPI ± SEM. p value (p < 0.0001) was determined by Wilcoxon matched pairs test. Data are representative of 4 independent experiments from 2 synapse models. (C) Illustrative image of phalloidin (yellow), phospho-ZAP70 (green), CD8 (red), cell tracer violet labeled A20 tumor cells (purple), and NRP1 (white) between activated NRP1 high or NRP1 low CD8 + T cells and cell tracer violet labeled A20 tumor cells observed by ImageStream. The synapse is the high phalloidin labeling zone. Bright field image is in white (scale bar = 7μm). Data are representative of 2 independent experiments. (D) Quantification by Imagestream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labeling zone) between activated NRP1 high or NRP1 low CD8 + T cells and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments. (E) Left panel: CD8 (green) NRP1 (red), PD1 (blue), and NRP1/PD1 merge (purple) expression observed by confocal microscopy in CD8 + TILs from control mice (WT) at day 21 post-activation (x63 oil objective, scale bar = 10 μm). Data are representative of 3 tumors. Right panel: Colocalization of NRP1 and PD1 was assessed by the calculation of Pearson coefficient. Data from 10 CD8 + TILs analyzed are presented as mean ± SEM. (F) Phospho-ZAP70 signal according to its localization within the synapse between the CD8 + T cells and the tumor cells. Illustrative image of phalloidin (yellow), phospho-ZAP70 (green), CD8 (red), and Cell Tracer (A20 cells, purple) labeling in the synapse model between activated CD8 + T cells from CD8Nrp1KO mice (KO) or controls (WT) and allogeneic A20 tumor cells, by ImageStream. Bright field image is in white (scale bar = 7μm). Data are representative of 2 independent experiments. (G) Phospho-ZAP70 signal according to its localization within the synapse between the CD8 + T cells and the tumor cells. Quantification by Image stream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labeling zone) between activated CD8 + T cells from CD8Nrp1KO mice (KO) or control mice (WT), and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments. (H) Proximity of NRP1 and PD1 proteins demonstrated by Duolink assay on in vitro activated CD8 + T cells from C57BL/6J mice. Upper panel: Left: Negative control experiments performed using anti-IRAP and anti-NRP1 antibodies (PLA-Duolink). Right: NRP1/PD1 complexes (anti-NRP1 and anti-PD1 antibodies with PLA-Duolink). The red spots indicate less than 40nm proximity between cellular-bound antibodies. Nuclei are stained with DAPI (blue). Images have been observed by confocal microscopy (x63 oil objective, scale bar = 10μm). Data are representative of 5 independent experiments. Lower panel: Comparison of number of PLA plots per cell. Data are presented as mean ± SEM. (I) NRP1 and PD1 interaction was demonstrated by CoIP experiments performed in splenocytes from C57BL/6J mice activated with anti-CD3 and anti-CD28 antibodies. NRP1 and PD1 immunoblot (IB) detection is shown in total lysate (TL) as control, in eluate from IgG Control (ctl) IP, and from NRP1 IP (N = 1 experiment). NRP1/PD1 Co-IP was also observed after PD1 IP (N = 2 experiment). Data are representative of 3 independent experiments. (J) Quantification by Imagestream of PD1 expression (MPI) in the synapse junction (high phalloidin labeling zone) between activated CD8 + T cells from CD8Nrp1KO mice (KO) or controls (WT), and allogeneic A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.

Article Snippet: anti-mouse CD28 (37.51) , BioXcell , Cat# BE0015-1, RRID: AB_1107624.

Techniques: Activity Assay, Labeling, Expressing, MANN-WHITNEY, Confocal Microscopy, Control, Activation Assay, In Vitro, Negative Control, Staining, Comparison, Western Blot, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: Neuropilin-1 cooperates with PD-1 in CD8 + T cells predicting outcomes in melanoma patients treated with anti-PD1

doi: 10.1016/j.isci.2022.104353

Figure Lengend Snippet: Targeting both NRP1 and PD1 has a synergistic effect in human and mouse CD8 + T cells immune response (A) Flow cytometry analysis of NRP1 expression according to cell trace on human CD8 + T cells 96 h after activation with anti-CD3 and anti-CD28, or on nonactivated cells. Data are representative of 5 independent experiments. (B) Flow cytometry analysis of NRP1 and PD1 expression in human CD8 + T cells 96 h after in vitro activation with anti-CD3 and anti-CD28 or non-activated cells. Data are representative of 3 independent experiments. (C) Flow cytometry analysis of NRP1 and PD1 expression in CD8 + TILs. Data are representative of 3 independent experiments in human endometrial, kidney, and ovarian cancer. (D) Flow cytometry analysis of phospho-ZAP70 in PD1 + CD8 + TILs according to NRP1 expression. Data from one experiment in human endometrial cancer. (E) Flow cytometry analysis of percentage of divided CD8 + T cells from a patient bearing an NRP1 haploinsufficiency (patient) or from controls (N = 5), respective to SEB superantigen concentration (0, 1, 10, or 100 ng/mL), in the presence or not of anti-PD1 antibody. Activation was performed during 72 h. Data are presented as the mean ± SEM. Data representative of 1 experiment. (F) Flow cytometry analysis of CD25 expression in CD8 + T cells from a patient bearing an NRP1 haploinsufficiency (patient) or from controls (N = 5), respective to SEB superantigen concentration (0, 1, 10, or 100 ng/mL), in the presence or not of anti-PD1 antibody. Activation was performed during 72 h. Data are presented as the mean % of CD25 expression ± SEM. Data representative of 1 experiment. (G) NRP1/PD-1 complexes detection by Proximity-Ligation-Assay (PLA) technology on CD8 + TILs in human colon cancer. Left panel: Representative area of tumor tissue observed. Acquisition with NDPI view software. Middle panel: Tumor infiltrating NRP1 + PD1 + CD8 + T cells (pink): Merge of green CD8 staining (FITC) and orange NRP1/PD-1 spots (TRITC). Acquisition with NDPI view software: zoom in x20. Right panel: CD8 + NRP1/PD1 positive cell (Pink) and CD8 + NRP1/PD1 negative cells (green). Acquisition with NDPI view software: zoom in x40. Nuclei are stained with DAPI (blue). Images have been observed by confocal microscopy ×20 oil objective. Data are representative of 10 independent experiments. (H) CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumin and poly-IC and treated or not with anti-PD1 antibody in vivo . B16-OVA tumor volume was assessed until 35 days after immunization. Data are presented as the mean ± SEM and as Kaplan Meyer curve. p values were determined by two-way ANOVA test ∗∗∗p < 0.001 ∗∗p < 0.01. Data are representative of 5 experiments. (I) CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumin and poly-IC and treated or not with anti-PD1 antibody in vivo . Overall survival was assessed until 50 days after immunization. Data are presented as the mean ± SEM and as Kaplan Meyer curve. p values were determined by Log rank test ∗∗∗p < 0.001. Data are representative of 5 experiments. (J) Analysis of overall survival of patients with metastatic melanoma treated with anti-PD1, according to RNA NRP1 expression (low or high expression: groups have been determined according to ROC curve analysis) assessed in the tumor before anti-PD1 treatment. Data from transcriptomics analysis of metastatic melanoma tumors were available from Hugo et al. ( Hugo et al., 2016 ) Data are presented as Kaplan Meyer curve. p value (p = 0.040) was determined by Log-rank test (n = 25 patients). (K) Analysis of relapse free survival of patients with metastatic melanoma treated with anti-PD1 and reached at least a partial response, according to NRP1 expression (NRP1 -/low compared with NRP1 +/high ) in CD8 + TILs assessed by immunohistochemistry before starting therapy. Blind analysis has been performed to assess NRP1 expression. Data are presented as the Kaplan Meyer curve. p value (p = 0.042) was determined by Log-rank test (n = 15 patients).

Article Snippet: anti-mouse CD28 (37.51) , BioXcell , Cat# BE0015-1, RRID: AB_1107624.

Techniques: Flow Cytometry, Expressing, Activation Assay, In Vitro, Concentration Assay, Proximity Ligation Assay, Software, Staining, Confocal Microscopy, Control, In Vivo, Immunohistochemistry

Journal: iScience

Article Title: Neuropilin-1 cooperates with PD-1 in CD8 + T cells predicting outcomes in melanoma patients treated with anti-PD1

doi: 10.1016/j.isci.2022.104353

Figure Lengend Snippet:

Article Snippet: anti-mouse CD28 (37.51) , BioXcell , Cat# BE0015-1, RRID: AB_1107624.

Techniques: Affinity Purification, In Situ, Virus, Recombinant, Transfection, Membrane, Software