c57bl 6 mice female  (ATCC)

Journal: World Journal of Microbiology & Biotechnology

Article Title: The impact and safety of encapsulated nanomaterials as a new alternative against carbapenem resistant bacteria. a systematic review

doi: 10.1007/s11274-024-03894-3

Figure Lengend Snippet: The 9 included studies: main characteristics, objectives and results

Article Snippet: (Lam et al. ) , Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers , C57BL/6 mice female (10 to 14-week-old) (23.2 ± 1.7 g) , structurally nanoengineered antimicrobial peptide polymers’ (SNAPPs) , Colistin and multi-drug resistant (CMDR) A. baumannii wild-type A. baumannii (ATCC 19,606) or CMDR A. baumannii (FADDI-AB056) , Imipenem and untreated control groups , Peritonitis infection , Intraperitoneal injected (SNAPPs) (8.3 mg kg –1 /dose) , Blood bacterial counts (CFU/mL) in blood (Blood-Cleansing Treatment)) , X , X , No specific cytotoxicity tests were performed.

Techniques: Histopathology, Animal Model, Bacteria, Control, Infection, Injection, Negative Control, Saline, Positive Control, Staining, Formulation, Isolation, Derivative Assay, Activity Assay, Suspension

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: High-throughput screens reveal regulatory elements of maternal and paternal SNRPN alleles (A) Schematic of the PWS locus on chr15 with common PWS deletions and the PWS gRNA library. Each thin vertical line represents an sgRNA. Genes colored blue are maternally imprinted, those that are pink are paternally imprinted, and those that are gray are not imprinted. (B) Summary of the PWS gRNA library. (C) Schematic of experimental protocol for CRISPRa/CRISPRi screens. (D) CRISPR screen results (magnified, see Figure S1 E) displayed as −log 10 ( p adj ), where p adj is the multiple-hypothesis-corrected p value from DESeq2. Notable regions are highlighted in red. Note that genes SNORD107 and SNORD64 in the schematic are intended to help orient the reader, and due to the genes’ small size, locations are approximate and not drawn to scale. (E) qPCR of SNRPN-GFP for validations of individual gRNAs of the pat SNRPN-2A-GFP CRISPRi dCas9 KRAB screen with either dCas9 KRAB or dCas9 only (no effector) to control for steric hindrance. Fold-change values normalized to NT gRNA within either dCas9 KRAB - or dCas9-only conditions. (F) qPCR of SNRPN-GFP from individual or pooled gRNA validations of selected gRNAs in the mat1 and mat2 regions. (G) Summary of the PWS gRNA sub-library. (H) qPCR of SNRPN-GFP in mat SNRPN-GFP iPSCs with Tet1c dCas9 14 days after transduction with the indicated gRNA. For qPCR in (E), (F), and (H), fold change values are plotted as mean ± SD, but statistics were calculated on ΔΔCt values (normalized to GAPDH and empty or NT vector sample); for (E), two-way ANOVA followed by Tukey's multiple comprisons test vs. NT; for (F) and (G), one-way ANOVA, followed by Dunnett’s test vs. empty vector. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 relative to NT/empty vector. Unmarked comparisons are not significant.

Article Snippet: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

Techniques: High Throughput Screening Assay, CRISPR, Control, Transduction, Plasmid Preparation

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: Tet1c and VP64 activate maternally imprinted PWS genes in ΔPWS iPSCs (A) Schematic of chr15 in isogenic wild-type (WT) and PWS type II deletion (ΔPWS) iPSCs. (B–D) (B) qPCR of SNRPN in WT or ΔPWS iPSCs with (C) VP64 dCas9 VP64 and (D) Tet1v4 dCas9 14 days after transduction with the indicated gRNA. For both qPCR plots, fold change values are plotted as mean ± SD, but statistics were calculated on ΔΔCt values (normalized to GAPDH and WT ctrl sample); 1-way ANOVA, followed by Dunnett’s test vs. ΔPWS NT gRNA ∗∗∗∗ p < 0.0001. (D) Differential expression analysis of total RNA sequencing of VP64 dCas9 VP64 ΔPWS iPSCs, comparing mat1 g3 to NT gRNA. (E) Differential expression analysis of total RNA sequencing of Tet1v4 dCas9 ΔPWS iPSCs, comparing mat3 g5 to NT gRNA. (F and G) HCR FlowFISH assessing SNRPN (transcript variant 1) signal in (F) VP64 dCas9 VP64 and (G) Tet1v4 dCas9 iPSCs (WT or ΔPWS) with the indicated gRNA. SNRPN (transcript variant 1) signal on X axis, with TBP as a control for cell size and staining. (H) HCR FlowFISH assessing SNHG14 signal in VP64 dCas9 VP64 iPSCs (WT or ΔPWS) with the indicated gRNA. (I and J) Targeted bisulfite sequencing of WT and ΔPWS iPSCs with (I) VP64 dCas9 VP64 and (J) Tet1v4 dCas9 covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353–25200693), 2 weeks post-transduction. Data for (I) and (J) are shown as the range of the data, with the plotted point being the median; n = 3 replicates. (K) Read-level methylation analysis showing number of methylated cytosines in a CpG context per read (containing a total of 24 CpGs) in each of the indicated conditions in WT or ΔPWS iPSCs expressing Tet1v4 dCas9.

Article Snippet: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

Techniques: Transduction, Quantitative Proteomics, RNA Sequencing, Variant Assay, Control, Staining, Methylation Sequencing, Methylation, Expressing

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: Tet1c and VP64 alter chromatin accessibility and/or DNA methylation at the PWS locus (A) Browser tracks of ATAC-seq (reads per kilobase per million mapped reads [RPKM]-normalized BigWig) of WT and ΔPWS iPSCs with VP64 dCas9 VP64 and NT or mat1 g3 gRNA. (B) Quantification of ATAC-seq reads (counts per million [CPM]) at the peak at the mat1 g3 binding site (dashed line in A). ∗∗∗ p < 0.001, 1-way ANOVA followed by Tukey’s test. (C) H3K4me3 CUT&RUN (CPM-normalized BigWig) of WT and ΔPWS iPSCs with VP64 dCas9 VP64 and NT or mat1 g3 gRNA, magnified and shown in full in Figure S4 C. n = 2 replicates as shown. (D) Quantification of CUT&RUN reads (CPM) shown in (C) at the annotated peak adjacent to the mat1 g3 binding site. (E) Browser tracks of ATAC-seq (RPKM-normalized BigWig) of WT and ΔPWS iPSCs with Tet1v4 dCas9 and NT or mat3 g5 gRNA. n = 2 or 3 replicates as shown. (F) Quantification of ATAC-seq reads (CPM) at each of 2 peaks within the PWS-IC. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; Tukey’s test following 2-way ANOVA. N = 2 or 3 as shown in Figure 3 E. (G) H3K4me3 CUT&RUN (CPM-normalized BigWig) of WT and ΔPWS iPSCs with Tet1v4 dCas9 and NT or mat3 g5 gRNA, magnified at PWS-IC and shown in full in Figure S4 E. n = 2 replicates as shown in figure. (H) Quantification of CUT&RUN reads (CPM) shown in (G) at the annotated peak at the PWS-IC.

Article Snippet: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

Techniques: DNA Methylation Assay, Binding Assay

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: Transient expression of Tet1v4 dCas9 in ΔPWS iPSCs stably activates maternal PWS genes (A) Schematic of experimental protocol for transient delivery of Tet1v4 dCas9 plasmid and PWS gene expression analysis. (B) qPCR of dCas9 and SNRPN in WT and ΔPWS iPSCs after transient delivery of Tet1v4 dCas9 on day 0. Two-way ANOVA on ΔCt values (normalized to GAPDH ), followed by Dunnett’s test, compared to ΔPWS + NT gRNA; ∗ p < 0.05; ∗∗ p < 0.0001; ns, not significant. Data shown as mean ± SD. (C) qPCR of PWS genes in iPSC-derived neurons. Data plotted as mean fold change ± SD, but statistics computed on ΔΔCt (normalized to GAPDH and WT + NT). Two-way ANOVA followed by Dunnett’s test, compared to ΔPWS + NT gRNA; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. Data shown as mean ± SD. (D) Targeted bisulfite sequencing of ΔPWS iPSC-derived neurons ∼21 days post-differentiation, covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353–25200693). Data shown as median ± range; n = 3 replicates. (E) Read-level methylation analysis showing number of methylated cytosines in a CpG context per read (containing a total of 24 CpGs) in each of the indicated conditions in WT or ΔPWS iNs expressing Tet1v4 dCas9. For all data shown in the figure, n = 2 replicates for WT NT; all other conditions, n = 3.

Article Snippet: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Gene Expression, Derivative Assay, Methylation Sequencing, Methylation

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet:

Article Snippet: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

Techniques: DNA Methylation Assay, Sequencing, Recombinant, Plasmid Preparation, Expressing, Software

Journal: Cell reports

Article Title: The C9ORF72 repeat expansion alters neurodevelopment

doi: 10.1016/j.celrep.2023.112983

Figure Lengend Snippet: (A) Representative western blot image of endogenous LRRC47 co-immunoprecipitated with GFP, untransfected control, AP(50)-GFP, or PR(50)-GFP from HEK293T cells using an anti-GFP antibody. (B) Quantification of western blot analysis of LRRC47 levels co-immunoprecipitated with GFP, untransfected control, AP(50)-GFP, or PR(50)-GFP using an anti-GFP antibody. Data are shown as the amount of LRRC47 co-immunoprecipitated normalized to the amount of GFP immunoprecipitated for each sample. Data points for each group consist of 3 independent transfections with 3 technical replicates for each transfection. LRRC47 levels were normalized to total protein for each sample. Mean ± SEM. Unpaired t test. (C) Representative western blot image of endogenous RPS6 co-immunoprecipitated with a LRRC47-FLAG-mCherry fusion protein in HEK293T cells overexpressing GFP or AP(50)-GFP. Co-immunoprecipitation with FLAG-mCherry was used as a control for any potential interaction between RPS6 and FLAG-mCherry. (D) Quantification of western blot analysis of the RPS6 levels co-immunoprecipitated with either LRRC47-FLAG-mCherry or FLAG-mCherry in HEK293T cells overexpressing GFP or AP(50)-GFP. Data points for each group consist of 3 independent transfections. Each independent differentiation was used as a data point. RPS6 levels were normalized to total protein for each sample. Mean ± SEM. One-way ANOVA with Tukey’s correction. (E) Representative images of homopropargylglycine (HPG)-Alexa Fluor 594 incorporation into control MACS-purified PSA-NCAM+ cells after transduction with GFP or AP(50)-GFP lentiviruses and further culture for 5 days with EGF and FGF. Scale bar, 10 μm. (F) Quantification of HPG incorporation into newly synthesized proteins in control MACS-purified PSA-NCAM+ cells. Data points consist of 2 independent differentiations from 4 different control lines with 2 technical replicates per line per condition. Each independent differentiation was used as a data point. Mean ± SEM. Unpaired t test. (G–I) Representative immunocytochemistry images (G), relative LRRC47 mRNA levels after ASO treatment (H), and quantification (I) of the percentage of PAX6+ cells in control line cultures after MACS purification of PSA-NCAM+ cells and treatment with scrambled or LRRC47 -suppressing ASOs on day 10 of neural differentiation and 5 additional days of culture with EGF and FGF. Mean ± SEM. The data points for each group include 2 independent differentiations per line per condition (4 control lines). Each independent differentiation was used as a data point. One-way ANOVA with Dunnett’s correction. Scale bars, 25 μm. (J and K) Representative immunocytochemistry images (J) and quantification (K) of the percentage of PAX6+ cells in control or C9ORF72 FTD/ALS cultures after MACS purification of PSA-NCAM+ cells and transduction with mCherry- or LRRC47-mCherry-encoding lentiviruses on day 10 of neural differentiation and 5 additional days of culture with EGF and FGF. Mean ± SEM. The data points for each group include 2 independent differentiations with 2 technical replicates per line per condition. Each independent differentiation was used as a data point. Unpaired t test. Scale bars, 15 μm. (L and M) Representative immunocytochemistry images (L) and quantification (M) of the percentage of PAX6+ cells in control line cultures after MACS purification of PSA-NCAM+ cells and transduction with lentiviruses encoding AP(50)-GFP and either LRRC47-mCherry, mCherry alone, or no additional lentivirus on day 10 of neural differentiation and 5 additional days of culture with EGF and FGF. The data points for each group include 2 independent differentiations per line per condition. Each independent differentiation was used as a data point. Mean ± SEM. One-way ANOVA with Tukey’s correction. Scale bars, 25 μm.

Article Snippet: LRRC47 (Rabbit IgG) , Proteintech , Cat# 23217-1-AP; RRID: AB_2879234.

Techniques: Western Blot, Immunoprecipitation, Control, Transfection, Purification, Transduction, Synthesized, Immunocytochemistry

Journal: Cell reports

Article Title: The C9ORF72 repeat expansion alters neurodevelopment

doi: 10.1016/j.celrep.2023.112983

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LRRC47 (Rabbit IgG) , Proteintech , Cat# 23217-1-AP; RRID: AB_2879234.

Techniques: Magnetic Beads, Virus, Recombinant, Knock-Out, Protease Inhibitor, Lysis, Derivative Assay, Control, CRISPR, Southern Blot, Software

Journal: Cell reports

Article Title: The C9ORF72 repeat expansion alters neurodevelopment

doi: 10.1016/j.celrep.2023.112983

Figure Lengend Snippet: (A) Representative western blot image of endogenous LRRC47 co-immunoprecipitated with GFP, untransfected control, AP(50)-GFP, or PR(50)-GFP from HEK293T cells using an anti-GFP antibody. (B) Quantification of western blot analysis of LRRC47 levels co-immunoprecipitated with GFP, untransfected control, AP(50)-GFP, or PR(50)-GFP using an anti-GFP antibody. Data are shown as the amount of LRRC47 co-immunoprecipitated normalized to the amount of GFP immunoprecipitated for each sample. Data points for each group consist of 3 independent transfections with 3 technical replicates for each transfection. LRRC47 levels were normalized to total protein for each sample. Mean ± SEM. Unpaired t test. (C) Representative western blot image of endogenous RPS6 co-immunoprecipitated with a LRRC47-FLAG-mCherry fusion protein in HEK293T cells overexpressing GFP or AP(50)-GFP. Co-immunoprecipitation with FLAG-mCherry was used as a control for any potential interaction between RPS6 and FLAG-mCherry. (D) Quantification of western blot analysis of the RPS6 levels co-immunoprecipitated with either LRRC47-FLAG-mCherry or FLAG-mCherry in HEK293T cells overexpressing GFP or AP(50)-GFP. Data points for each group consist of 3 independent transfections. Each independent differentiation was used as a data point. RPS6 levels were normalized to total protein for each sample. Mean ± SEM. One-way ANOVA with Tukey’s correction. (E) Representative images of homopropargylglycine (HPG)-Alexa Fluor 594 incorporation into control MACS-purified PSA-NCAM+ cells after transduction with GFP or AP(50)-GFP lentiviruses and further culture for 5 days with EGF and FGF. Scale bar, 10 μm. (F) Quantification of HPG incorporation into newly synthesized proteins in control MACS-purified PSA-NCAM+ cells. Data points consist of 2 independent differentiations from 4 different control lines with 2 technical replicates per line per condition. Each independent differentiation was used as a data point. Mean ± SEM. Unpaired t test. (G–I) Representative immunocytochemistry images (G), relative LRRC47 mRNA levels after ASO treatment (H), and quantification (I) of the percentage of PAX6+ cells in control line cultures after MACS purification of PSA-NCAM+ cells and treatment with scrambled or LRRC47 -suppressing ASOs on day 10 of neural differentiation and 5 additional days of culture with EGF and FGF. Mean ± SEM. The data points for each group include 2 independent differentiations per line per condition (4 control lines). Each independent differentiation was used as a data point. One-way ANOVA with Dunnett’s correction. Scale bars, 25 μm. (J and K) Representative immunocytochemistry images (J) and quantification (K) of the percentage of PAX6+ cells in control or C9ORF72 FTD/ALS cultures after MACS purification of PSA-NCAM+ cells and transduction with mCherry- or LRRC47-mCherry-encoding lentiviruses on day 10 of neural differentiation and 5 additional days of culture with EGF and FGF. Mean ± SEM. The data points for each group include 2 independent differentiations with 2 technical replicates per line per condition. Each independent differentiation was used as a data point. Unpaired t test. Scale bars, 15 μm. (L and M) Representative immunocytochemistry images (L) and quantification (M) of the percentage of PAX6+ cells in control line cultures after MACS purification of PSA-NCAM+ cells and transduction with lentiviruses encoding AP(50)-GFP and either LRRC47-mCherry, mCherry alone, or no additional lentivirus on day 10 of neural differentiation and 5 additional days of culture with EGF and FGF. The data points for each group include 2 independent differentiations per line per condition. Each independent differentiation was used as a data point. Mean ± SEM. One-way ANOVA with Tukey’s correction. Scale bars, 25 μm.

Article Snippet: Western blots were performed with an anti-LRRC47 antibody (Proteintech, cat. no. 23217–1-AP, 1:500) to detect LRRC47-FLAG-mCherry and an anti-RPS6 antibody (Thermofisher, cat. no. MA5–15123, 1:500) to detect endogenous RPS6.

Techniques: Western Blot, Immunoprecipitation, Control, Transfection, Purification, Transduction, Synthesized, Immunocytochemistry

Journal: Cell reports

Article Title: The C9ORF72 repeat expansion alters neurodevelopment

doi: 10.1016/j.celrep.2023.112983

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western blots were performed with an anti-LRRC47 antibody (Proteintech, cat. no. 23217–1-AP, 1:500) to detect LRRC47-FLAG-mCherry and an anti-RPS6 antibody (Thermofisher, cat. no. MA5–15123, 1:500) to detect endogenous RPS6.

Techniques: Magnetic Beads, Virus, Recombinant, Knock-Out, Protease Inhibitor, Lysis, Derivative Assay, Control, CRISPR, Southern Blot, Software