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: The Journal of Cell Biology

Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

doi: 10.1083/jcb.201702058

Figure Lengend Snippet: ATF4 is activated through the ISR. (A) Western blot analysis showing the increased phosphorylation of eIF2α (Ser51) upon 6 h of treatment with the different mitochondrial stressors. Bottom, ratio between P-eIF2α and eIF2α total levels. (B and C) mRNA expression analysis of ATF4 and its target genes, CHOP (DDIT3) , ASNS , CHAC1 , PCK2 , and the ER stress marker BIP , upon 6 h of treatment with the different mitochondrial stressors and the ER stressor tunicamycin (Tn at 2.5 µg/ml) in HeLa cells, together with the inhibitor of the integrated stress response (ISRIB at 500 nM). (D) Boxplots showing an increase in basal and ATP-dependent respiration of HeLa cells treated with 500 nM of ISRIB for 24 h. OCR: oxygen consumption rate. (E) mRNA expression analysis of eIF2α kinases upon knock down with specific shRNAs. Data are presented as mean ± SEM of two independent shRNAs for each gene. Statistical differences were calculated compared with pLKO1. (F) mRNA expression analysis of ATF4 and some of its target genes upon knock down of the eIF2α kinases and 6 h of treatment with FCCP. Data are presented as mean ± SEM of two independent shRNAs for each gene. No statistical differences were found between the FCCP treated conditions. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.

Article Snippet: The following primary antibodies were used: mouse anti–β-actin (sc-47778; Santa Cruz Biotechnology, Inc.); mouse anti-HSP90 (BD Biosciences, 610418); OXPHOS antibody cocktail (mouse mAbs, ab110413; Abcam); mouse anti-HSP60 (ADI-SPA-806; Enzo Life Sciences); mouse anti-CLPP (WH0008192M1-100; Sigma-Aldrich); mouse anti-HSPA9 (ABIN361739; Antibodies online); rabbit anti-LONP1 (HPA002192; Sigma-Aldrich); rabbit anti-OTC (sc-102051; Santa Cruz Biotechnology, Inc.); mouse anti-OPA1 (BD, 612606); rabbit anti-CREB-2 (ATF4, sc-200; Santa Cruz Biotechnology, Inc.); phospho-eIF2α (Ser51) rabbit mAb (9721; Cell Signaling Technology); eIF2α rabbit mAb (9722; Cell Signaling Technology); and α tubulin (T5168; Sigma-Aldrich).

Techniques: Western Blot, Phospho-proteomics, Expressing, Marker, Knockdown

Journal: The Journal of Cell Biology

Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

doi: 10.1083/jcb.201702058

Figure Lengend Snippet: Genetic link of ATF4 and mitochondrial stress in human and mouse populations. (A and B) Multitissue correlation network analysis of transcript levels across (A) 49 human tissues in GTEx and (B) 16 mouse tissues in the BXD genetic reference population. Nodes represent the transcripts and the width of the ties among nodes indicate the probability to show a significant positive correlation in all tissues analyzed. The human network shows a tighter clustering likely caused by the higher number of tissues and samples per tissues. (C) Heatmap representing the KEGG pathway analysis of the top negative-correlated genes across 49 tissues using GTEx data for ATF4 , CEBPB , DDIT3/CHOP , and NCOR1 (used as positive control). Color key represents the negative logarithm base 10 of the p-value of each pathway obtained in the analysis. (D) Expression quantitative trait locus (eQTL) analysis of Atf4 transcript level in the prefrontal cortex. The yellow mark represents the Atf4 gene locus on chromosome 15, whereas the red mark represent the position of a trans-eQTL for Atf4 expression levels on chromosome 1 (Chr1). (E) eQTL mapping of Atf4 transcript levels across several tissues identifies a common and strong trans-eQTL on chromosome 1 (170–180 Mb). (F) Representation of the chromosome 1 locus (170–180 Mb) containing 142 genes, 6 of which have nonsynonymous substitutions, and only one gene, Fh1 , encodes a mitochondrial protein. Fh1 contains a nonsynonymous sequence variant (A296T; rs32536342 ) that segregates in the BXDs. This sequence variant regulates the expression levels of Fh1 , which in turn regulates Atf4 expression. (G) mRNA expression analysis of HeLa cells after knockdown of fumarate hydratase (shFH) and treatment with monomethyl fumarate at 2.5 mM for 24 h. Data are presented as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Enrichment score plot from GSEA using microarray data from renal cysts of mice with renal tubule specific inactivation of Fh1 ( Fh1 −/− ; GSE29988 ). The list of mitochondrial stress genes (mt-stress genes; Table S5) was used as the gene set of interest. FDR q-val: false discovery rate adjusted p-value; NES, normalized enrichment score; NOM p-val, nominal p-value. (I) Scheme summarizing our working hypothesis. Mitochondrial stress stimulates the phosphorylation of the eIF2α, which inhibits cytosolic translation and activates the ATF4 pathway. At the same time, mitochondrial stress also reduces the expression of MRPs to inhibit mitochondrial translation and protect mitochondrial function.

Article Snippet: The following primary antibodies were used: mouse anti–β-actin (sc-47778; Santa Cruz Biotechnology, Inc.); mouse anti-HSP90 (BD Biosciences, 610418); OXPHOS antibody cocktail (mouse mAbs, ab110413; Abcam); mouse anti-HSP60 (ADI-SPA-806; Enzo Life Sciences); mouse anti-CLPP (WH0008192M1-100; Sigma-Aldrich); mouse anti-HSPA9 (ABIN361739; Antibodies online); rabbit anti-LONP1 (HPA002192; Sigma-Aldrich); rabbit anti-OTC (sc-102051; Santa Cruz Biotechnology, Inc.); mouse anti-OPA1 (BD, 612606); rabbit anti-CREB-2 (ATF4, sc-200; Santa Cruz Biotechnology, Inc.); phospho-eIF2α (Ser51) rabbit mAb (9721; Cell Signaling Technology); eIF2α rabbit mAb (9722; Cell Signaling Technology); and α tubulin (T5168; Sigma-Aldrich).

Techniques: Positive Control, Expressing, Sequencing, Variant Assay, Knockdown, Microarray, Phospho-proteomics

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: 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