Journal: JCI Insight

Article Title: RAG1 splicing mutation causes enhanced B cell differentiation and autoantibody production

doi: 10.1172/jci.insight.148887

Figure Lengend Snippet: ( A ) The skin lesions on the lower limbs of the patient. ( B ) Pedigree of the family. ( C ) Reverse transcription PCR analysis of RAG1 mRNA splicing and expression. First strand cDNA from BMMNCs of the patient (P) and HC and from human pre-B cell line Nalm6 was amplified as described in Methods. As a positive control, the first strand cDNA was also amplified for ACTIN expression. ( D ) Schematic illustration of the genomic structure of WT (upper) and mutant (lower) RAG1 genes. The location of the authentic translation initiation site (ATG) is shown by a blue arrow. The mutant base (G) is shown in red, and the cryptic splice donor site is shown by a red arrow.

Article Snippet: Nalm6 human pre-B cells (RCB1933, RIKEN Bioresource Research Center) were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and 5 × 10 −5 M 2-mercaptoethanol.

Techniques: Reverse Transcription, Expressing, Amplification, Positive Control, Mutagenesis

Journal: Scientific Reports

Article Title: Plasma-derived exosomal miR-326, a prognostic biomarker and novel candidate for treatment of drug resistant pediatric acute lymphoblastic leukemia

doi: 10.1038/s41598-023-50628-w

Figure Lengend Snippet: Isolation and characterization of the exosomes derived from acute lymphoblastic leukemia cell lines. ( A ) Representative histogram and a video image of particle concentration and size distribution of exosomes isolated from cell culture supernatant using Nanoparticle Tracking Analysis (NTA). Exosomes were extracted from the conditioned media of RN95 and Nalm6 cell lines using ultracentrifuge. The incubation time for RN95 and Nalm6 cells were 48h and 24h, respectively. The Orange error bar area indicates ± standard error of the mean, n = 5. ( B ) Confirmation of the presence of common exosomal markers on the extracted leukemia cell lines exosomes. Evaluation of CD63 (positive) marker and GM130 (negative) marker using semi quantitative traditional western blotting. Automated western blot analysis indicated the positive expression levels of CD81, FLOT1, and HSP70 in the isolated exosomes. CD63, Lysosome-associatd membrane protein-3; GM130, Cis-Golgi marker; CD81, Tetraspanin, non-specific transmembrane protein member 8; FLOT1, Flotillin-1; HSP70, Heat Shock Protein 70. ( C ) Field-emission scanning and transmission electron micrographs of the isolated exosomes from RN95 and Nalm6 conditioned media.

Article Snippet: Nalm6 cell line was purchased from the cell bank of Pasteur Institute in Iran.

Techniques: Isolation, Derivative Assay, Concentration Assay, Cell Culture, Incubation, Marker, Western Blot, Expressing, Membrane, Transmission Assay

Journal: Scientific Reports

Article Title: Plasma-derived exosomal miR-326, a prognostic biomarker and novel candidate for treatment of drug resistant pediatric acute lymphoblastic leukemia

doi: 10.1038/s41598-023-50628-w

Figure Lengend Snippet: Expression of miR-326 transcript in exosomes isolated from the conditioned media of RN95 and B-ALL Nalm6 cell lines. Bar graph representing the expression level (2 −Δct ) of exosomal miR-326 transcript in the cell culture supernatant of RN95 and Nalm6 cell lines, respectively. ΔCt value is normalized against Cel-miR-39 as exogenous control. Exosomal miR-326 is expressed in two conditioned media with a high level in RN95 compared with Nalm6 media.

Article Snippet: Nalm6 cell line was purchased from the cell bank of Pasteur Institute in Iran.

Techniques: Expressing, Isolation, Cell Culture, Control

Journal: Scientific Reports

Article Title: Plasma-derived exosomal miR-326, a prognostic biomarker and novel candidate for treatment of drug resistant pediatric acute lymphoblastic leukemia

doi: 10.1038/s41598-023-50628-w

Figure Lengend Snippet: Viability assessment of B-ALL cell lines after treatment with autocrine and exocrine exosomes. ( A ) RN95 and ( B ) Nalm6 cells were seeded into 96-well plates and treated with exosomes extracted from 44 ml RN95 conditioned media. Treated cells were incubated at 37℃ for 48h and cell viability was assessed using MTT assay. ( C ) Nalm6 exosomes with identical concentration to RN95 exosomes in A, were subjected to RN95 cells. Percent viability was not significantly different from that of untreated cells or cells treated with exosome-free media (depicted as 0). Values are mean ± SEM of 3 independent experiments in triplicates. * P < 0.05, ** P < 0. 005, *** P < 0.001, *** P < 0.0001. ( D ) Internalization and uptake of exosomes by B-ALL cells. DAPI-labeled B-ALL cells were incubated (24h) with DiI-labeled exosomes (originated from lymphoblastic cells). Related images were captured at the end of the incubation time using fluorescence microscope and a representative example of a triplicate experiment is shown. Scale bar = 20 µm.

Article Snippet: Nalm6 cell line was purchased from the cell bank of Pasteur Institute in Iran.

Techniques: Incubation, MTT Assay, Concentration Assay, Labeling, Fluorescence, Microscopy