Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A β-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

doi: 10.1016/j.jcmgh.2021.12.014

Figure Lengend Snippet: Cas9 deletion confirms a super-enhancer necessary for β-catenin-TCF-sensitive locus control. DLD1(DNTCF) cells expressing Cas9 and untargeted (clone #1) or GUCA2A-locus-targeted gRNAs harbor biallelic deletions encompassing the GUCA2A promoter (clone #2), DNase site #6 (clone #3), DNase site #7 (clone #4), or both DNase sites (clone #5). Expression of ( A ) GUCA2A protein, ( B ) GUCA2A mRNA, and ( C ) GUCA2B mRNA was quantified with (+) or without (–) 1 μg/mL DOX for 24 hours. ( D ) Hypothetical model of GUCA2A and GUCA2B promoter positioning relative to a Pol II-rich super-enhancer region upstream of GUCA2A, consisting of multiple DNase sites. ( E ) ChIP-pcr in HT29(APC) cells treated with (+) or without (–) 300 μM zinc for 24 hours reveals enrichment of TCF at the promoter of a Wnt target gene, SP5, with a TCF-specific antibody, but not with control IgG. In contrast, TCF was not detected at sites within 6kb of the GUCA2A TSS: DNase site #5 (GUCA2A promoter), site #6, site #7, or site #8. ( F ) GUCA2A mRNA expression in HT29(APC) cells stably expressing an untargeted (Ctr) or TCF-targeted shRNA, and treated with (+) or without (–) 300 μM zinc for 24 hours. GUCA2A mRNA expression is retained despite TCF knockdown, illustrated by Western blot. ( A–C ) Bars represent the average of ( A ) 2 or ( B , C ) 3 independent experiments ± SEM, and data are presented relative to noninduced cells. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. ( E ) Bars represent the mean ± SD of 3 IPs, and data are presented relative to input DNA. Significance was determined by 2-way analysis of variance. ( F ) Data points represent the average of 3 wells of cells from a single experiment, with the mean of 2 independent experiments indicated. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. Data are presented relative to noninduced cells receiving control shRNA. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Blots were blocked for 1 hour in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 10% milk, then incubated overnight at 4°C with primary antibodies at the indicated dilutions targeting GAPDH (#2118, 1:5000), APC (#2504, 1:1000), β-catenin (#8480, 1:1000), TCF (#2569, 1:1000), Histone H3 (#4499, 1:1000), or Cas9 (#19526, 1:1000) from Cell Signaling Technology (Danvers, MA), or GUCA2A (#HPA018215, 1:250) from Sigma-Aldrich.

Techniques: Control, Expressing, Stable Transfection, shRNA, Knockdown, Western Blot, Transformation Assay

Journal: Nature Communications

Article Title: Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles

doi: 10.1038/s41467-025-64200-9

Figure Lengend Snippet: a Schematic diagram illustrating the production of MuVLP Cas9 . b The levels of muscular fusogens, and Cas9 in VLPs, MuVLPs, VLP Cas9 , and MuVLP Cas9 . c ELISA quantification of Cas9 molecules per MuVLP Cas9 . The values and error bars represent the means ± s.d. of n = 3 technical replicates. d Scheme of MuVLP Cas9 or other controls cocultured with six different cell lines for detecting the specificity of MuVLP Cas9 for gene editing. e Immunofluorescence visualization of the Cas9 protein in cells cocultured with VLP Cas9 or MuVLP Cas9 . Red: anti-Cas9 antibody, green: F-actin labeled with Alexa Fluor 488 phalloidin, blue: nuclei labeled with DAPI. Scale bar, 20 μm. f Detection of exon 4 excision by genomic PCR in iC2C12 cells transfected with MuVLP Cas9 or other controls. The unedited genomic product is 883 bp long, and the gene-edited product (asterisk) is 445 bp long. g Sanger sequencing of amplicons confirmed the deletion of exon 4 and the generation of a fused intron 3/4 in genomic DNA from iC2C12 cells transfected with MuVLP Cas9 . The data are presented as the means ± s.d. of n = 3 biological replicates ( c ) or are representative of three independent experiments ( b , e – g ). Source data are provided as a Source Data file.

Article Snippet: Quantification was performed using the FastScanTM Cas9 ( S. pyogenes ) ELISA Kit (29666 C, Cell Signaling Technology, MA, USA) according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling, Transfection, Sequencing

Journal: Nature Communications

Article Title: Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles

doi: 10.1038/s41467-025-64200-9

Figure Lengend Snippet: a Therapeutic scheme. B10-Dmd-KO mice were administered four intravenous injections of MuVLP Cas9 or other controls. b The grip strength of the B10-Dmd-KO mice injected with MuVLP Cas9 ( n = 5) or other controls ( n = 3) was measured using a grip strength meter. Grip strength was measured in grams and normalized to body weight. Wild-type (WT) mice were used as positive controls. c Treadmill exercise was performed to assess the time to exhaustion in the mice injected with MuVLP Cas9 ( n = 5) or other controls ( n = 3). d ELISA quantification of serum CK levels in the mice injected with MuVLP Cas9 ( n = 5) or other controls ( n = 3). e Detection of exon 4 excision by genomic PCR in skeletal muscle and nonmuscle tissues of the B10-Dmd-KO mice injected with MuVLP Cas9 or other controls. The unedited genomic product is 883 bp long, and the gene-edited product (asterisk) is 445 bp long. f Dystrophin protein in the skeletal muscle of the B10-Dmd-KO mice injected with MuVLP Cas9 or other controls. For MuVLP Cas9 group, the five lanes correspond to individual biological replicates (samples from five independent B10-DMD-KO mice). g Representative immunofluorescence staining of dystrophin (red) in the skeletal muscle of the B10-Dmd-KO mice injected with MuVLP Cas9 or VLP Cas9 . Blue: DAPI (nuclei). Scale bar: 100 μm. h Therapeutic scheme. B10-Dmd-KO mice were administered four intravenous injections of 5.5 × 10 10 , 1.1 × 10 11 , or 1.7 × 10 11 MuVLP Cas9 ( n = 3). i The grip strength of the B10-Dmd-KO mice injected with 5.5 × 10 10 , 1.1 × 10 11 , or 1.7 × 10 11 MuVLP Cas9 ( n = 3) was measured using a grip strength meter. Grip strength was measured in grams and normalized to body weight. j Treadmill exercise was performed to assess the time to exhaustion in the mice injected with 5.5 ×10 10 , 1.1 × 10 11 , or 1.7 × 10 11 MuVLP Cas9 ( n = 3). k Exon4-deleted transcripts in muscles quantified by Taqman. Data plotted for individual mice ( n = 3). l H&E staining of diaphragms from the mice injected intravenously with 5.5 × 10 10 , 1.1 × 10 11 , or 1.7 × 10 11 MuVLP Cas9 ( n = 3). Representative images are shown, along with quantification of centrally nucleated myofibers in the diaphragms from wild-type mice and B10-Dmd-KO mice treated with MuVLP Cas9 or other controls. Scale bar, 100 µm. m Masson’s trichrome staining of diaphragms from the mice injected intravenously with 5.5 × 10 10 , 1.1 × 10 11 , or 1.7 × 10 11 MuVLP Cas9 ( n = 3). Representative images are shown, along with quantification of the blue trichrome area (indicating fibrosis). Scale bar, 100 µm. The data are shown as the means ± s.d. of n = 3 – 5 biologically independent mice ( b – d , i – k ) or are representative of three independent experiments ( e – g , l , m ). Statistical data were analyzed by one-way ANOVA with Tukey’s multiple comparison test ( b-d ), two-sided Student’s t test ( i – m ). **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns indicates no significant difference. Source data are provided as a Source Data file.

Article Snippet: Quantification was performed using the FastScanTM Cas9 ( S. pyogenes ) ELISA Kit (29666 C, Cell Signaling Technology, MA, USA) according to the manufacturer’s protocol.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Muscles, Comparison

Journal: Microbiology and Immunology

Article Title: Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A, a carbohydrate antigen of Streptococcus pyogenes , in recurrent tonsillitis

doi: 10.1111/1348-0421.12213

Figure Lengend Snippet: Summary of clinical data and analyzed results in 12 cases of recurrent tonsillitis

Article Snippet: Three rats were immunized with boiled S. pyogenes (strain: J17A4, serotype: M6, inoculum size: 4 × 10 6 CFU per rat, supplied by Eiken Chemical, Nogi, Tochigi, Japan) emulsified with Freund's incomplete adjuvant (Difco Laboratories; Detroit, MI, USA).

Techniques:

Journal: Microbiology and Immunology

Article Title: Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A, a carbohydrate antigen of Streptococcus pyogenes , in recurrent tonsillitis

doi: 10.1111/1348-0421.12213

Figure Lengend Snippet: Number and percentage of plasma cells with anti‐Strep A reactivity in the axillary and popliteal lymph nodes of S. pyogenes ‐immunized and non‐immunized rats

Article Snippet: Three rats were immunized with boiled S. pyogenes (strain: J17A4, serotype: M6, inoculum size: 4 × 10 6 CFU per rat, supplied by Eiken Chemical, Nogi, Tochigi, Japan) emulsified with Freund's incomplete adjuvant (Difco Laboratories; Detroit, MI, USA).

Techniques: Clinical Proteomics