Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 4. A - NAC can reverse the transfer of NFATc3 from nucleus to cytoplasm caused by As4S4 combined with radiotherapy. RMS A-673 cells were treated with 2 Gy radiation, 1 μM As4S4, and combination therapy，respectively. The expressions of NFATc3 was detected by Western blot 24 h later. B - shC3-1 and shC3-2 were effectively knocked down, Western blot assay were used to evaluate expression levels of NFATc3, RAG1, and γ-H2AX. C - Western blot analysis of γ-H2AX expression changes in RMS A-673 cells after transfection with (shC3-1 + shRAG1-1), shC3-1, and shRAG1-1, respectively.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: Western Blot, Expressing, Transfection

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 3. As4S4 enhances radiosensitivity via activating NFATc3-RAG1 mediated DSB in RMS cells. All samples were run in triplicate. A - RMS cells were treated with radiotherapy, As4S4, and combination therapy, respectively. Western blot assay and qPCR were used to evaluate expression levels of NFATc3 and RAG1. B - RMS cells were treated with radiotherapy, As4S4, and combination therapy, respectively. Western blot assay and qPCR were used to evaluate expression levels of p53 and γ-H2AX. C - RMS cells were treated with radiotherapy, As4S4, and combination therapy, respectively. Immunofluorescence assays for the expression of γ-H2AX. Left, γ-H2AX (red); middle, DAPI (blue); right, γ-H2AX and DAPI merged.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: Western Blot, Expressing, Immunofluorescence

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 6. A, B - IHC images (NFATc3 and RAG1) in RMS tissues of in vivo xenograft models (40X). C, D - Histograms of NFATc3 and RAG1 in qPCR experiments.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: In Vivo

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 7. NFATc3 and RAG1 are independent prognostic factors for RMS patients. A, B - IHC images of NFATc3 and RAG1 in RMS tissues (40X). C - The Kaplan-Meier survival curves of the overall survival of two groups defined as low or high expression (NFATc3 and RAG1) in 59 RMS patients. D - Boxplots of expression (NFATc3 and RAG1) between tumor and adjacent normal tissues. E - Nomogram of the prediction model. F. ROC curves predicting 5-year survival of NFATc3-RAG1 based prediction model, TNM staging, and Risk level.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques: Expressing

Journal: Chemico-biological interactions

Article Title: Arsenic Sulfide Enhances Radiosensitivity in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB).

doi: 10.1016/j.cbi.2024.111149

Figure Lengend Snippet: Fig. 8. Schematic figure describing that As4S4 enhances radiosensitivity in RMS via activating NFATc3-RAG1 mediated DSB.

Article Snippet: Antibodies were listed as follows, rabbit NFATc3 antibody (ABclonal, China), rabbit RAG1 antibody (Novus, USA), rabbit p53 antibody (CST, USA), rabbit phospho-histone H2AX antibody (ABclonal, China), rabbit β-Tubulin antibody (Novus, USA), rabbit PCNA antibody (Novus, USA), and rabbit GADPH antibody (CST, USA).

Techniques:

Journal: Signal transduction and targeted therapy

Article Title: Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

doi: 10.1038/s41392-023-01544-y

Figure Lengend Snippet: Fig. 1 Base-edited monkeys were generated using the CBE4max system. a Schematic illustrating the process of generating base-edited monkeys. b Specific gRNAs designed to target RAG1 and IL2RG genes. The gRNA target sites are highlighted in red, while the edited bases are indicated in green. c The status of the embryos after CBE4max systemic injection. d T7E1 assay for base-edited embryos. e Following the microinjection of 152 embryos, 35 embryos in the developmental stage were transferred to surrogate female monkeys. Subsequently, five recipient monkeys were confirmed to be pregnant, resulting in the successful acquisition of six newborn monkeys. f The editing efficiency in the peripheral blood of the edited animals was assessed using Sanger sequencing-based EditR software. g The genotype of the base-edited monkeys (mutation) was determined. Red boxes indicate the presence of stop codons after the substitution, while the asterisk (*) denotes a substituted base. The gRNA sequencing map of the RAG1 gene is derived from monkey NO.1, while the gRNA sequencing map of the IL2RG gene is from monkey NO.3. h The frequencies of base substitution in deceased base-edited monkeys determined through targeted deep sequencing

Article Snippet: Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies.

Techniques: Generated, Injection, Microinjection, Sequencing, Software, Mutagenesis, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

doi: 10.1038/s41392-023-01544-y

Figure Lengend Snippet: Fig. 2 Phenotypic and histopathological changes in base-edited monkeys. a, b Comparison of body weights, body lengths, and head circumferences between mutant monkeys and age-matched WT monkeys while being subjected to the same feeding environment. c, d Analysis of thymus and spleen in base-edited monkeys. e H&E staining depicting the development of thymus and spleen in base-edited monkeys. Scale bars: 50 µm. f–k Evaluation of RAG1 and IL2RG protein expression in the thymus and spleen of mutant monkeys. Western blot analysis illustrating RAG1 and IL2RG protein expression levels and quantification in the thymus, spleen, liver, and lung of mutant and WT monkeys (f, g). Immunohistochemistry depicting the expression and quantification of RAG1 and IL2RG in the thymus and spleen of mutant and WT monkeys (h–k). Statistical analysis was performed using an unpaired two-tailed t-test to compare the two groups. P < 0.05 was considered statistically significant. Three replicates were performed for each analysis. Scale bars: 20 µm

Article Snippet: Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies.

Techniques: Comparison, Mutagenesis, Staining, Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Signal transduction and targeted therapy

Article Title: Generation of inactivated IL2RG and RAG1 monkeys with severe combined immunodeficiency using base editing.

doi: 10.1038/s41392-023-01544-y

Figure Lengend Snippet: Fig. 4 Off-target analysis in base-edited monkeys. a Whole-genome sequencing analysis was conducted to identify single-nucleotide variations (SNVs) and indels in three base-edited monkeys (NO.1, NO.2, and NO.3) as well as three wild-type monkeys (WT1, WT2, and WT3). The height of the bars represents the average number of mutations in each group, while black dots indicate the number of mutations in each sample. b The distribution of mutation types in base-edited and wild-type monkeys. The number within each cell indicates the proportion of a specific mutation type among all mutations. c The genomic feature regions of mutations in base-edited and wild-type monkeys. A large proportion of SNVs were detected in intronic and intergenic regions, and no difference was observed between base-edited and wild-type monkeys. d Evaluation of the potential for mutations in base-edited and wild-type monkeys to impact gene function. No difference was observed between base-edited and wild-type monkeys. e Distribution of indel fragment lengths. f, g Assessment of Off-target base editing frequency in the RAG1 (f) and IL2RG (g) genes in base-edited monkeys generated by the CBE4max system. Potential off-target sites (OT1–OT9) were detected by deep sequencing and predicted using Cas-OFFinder. Deep sequencing was employed to determine the frequency of substitutions at predicted target sites in the spleen (NO.1 and NO.3) and peripheral blood (NO.2) of three deceased base-edited animals

Article Snippet: Antibodies specific to RAG1 (Cell Signaling Technology, 3968 S), Vinculin (Sigma, MAB3574), and IL2RG (Invitrogen, PA5-80730) were used as primary antibodies.

Techniques: Sequencing, Mutagenesis, Generated

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Rag and Lamtor antibody sampler kit , Cell signaling , Cat#8665; RRID: AB_.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis