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: BMC Genomic Data

Article Title: An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse

doi: 10.1186/s12863-022-01041-1

Figure Lengend Snippet: pCYB5Aalt Nalm 6 showed a distinct transcriptional program and resistance to Venetoclax induced cell death. A CYB5Aalt- ORF is stably overexpressed in NALM-6 cells. NALM-6 cells were transfected with pCYB5Aalt (+) or pEmpty (−). RT-PCR was used to confirm overexpression of CYB5Aalt -ORF mRNA. Two representative samples of each are shown. GAPDH was used as a control. NTC = non-template control. Full length electrophoretic gel is displayed in Additional file . B pCYB5Aalt NALM-6 show a distinct transcriptional program compared to empty vector controls. Samples were grouped according to the 500 most variably expressed genes between samples. Columns indicate pCYB5Aalt clones and empty vector controls, rows represent gene expression in rlog for each sample. Colour of the heatmap indicates relative expression strength according to the deviation from the mean of all samples in rlog. C Viability of pCYB5Aalt NALM-6 cells and pEmpty upon Venetoclax treatment ( n = 3). Cells were incubated with different concentrations of Venetoclax (0 μM, 0.2 μM, 0.5 μM, 5 μM, 10 μM) for 72 h. Viability was assessed photometrically after 3 h incubation time with WST-1. Absorbance values were normalised to untreated control samples and shown as viability (%). Bars represent mean viability, error bars ±standard deviation of three independent experiments each with five technical replicates per concentration. Mann-Whitney-U-Test, *** P -value< 0.001, ns = non-significant. D Cell death of pCYB5Aalt NALM-6 and pEmpty upon Venetoclax treatment ( n = 4). Cells were incubated at different concentrations of Venetoclax (0 μM, 0.2 μM, 0.5 μM, 5 μM, 10 μM, 20 μM) for 48 h. Cell death was assessed by staining cells with PI. Bars represent percentage of PI positive cells, error bars ±standard deviation of four independent experiments each with technical duplicates for each concentration. Mann-Whitney-U-Test, *** P -value< 0.001, ** P -value< 0.01, * P -value< 0.05, ns = non-significant

Article Snippet: pCYB5Aalt NALM-6 and empty vector controls were treated with Venetoclax (SelleckChem, München, Germany) and seeded in quintets for each concentration of Venetoclax (0, 0.2, 0.5, 1, 5, 10 μM).

Techniques: Stable Transfection, Transfection, Reverse Transcription Polymerase Chain Reaction, Over Expression, Control, Plasmid Preparation, Clone Assay, Gene Expression, Expressing, Incubation, Standard Deviation, Concentration Assay, MANN-WHITNEY, Staining

Journal: Science immunology

Article Title: CD19 CAR antigen engagement mechanisms and affinity tuning

doi: 10.1126/sciimmunol.adf1426

Figure Lengend Snippet: (A and F) CAR expression analyses for FMC63 (A) and SJ25C1 (F) CAR T cells. Data are from two independent experiments using different T cell donors. (B and G) Binding activity for FMC63 (B) and SJ25C1 (G) CAR T cells. Data for FMC63 CAR T cells are from two independent experiments using different T cell donors (one replicate in each experiment). Data for SJ25C1 CAR T cells are from two independent experiments using different T cell donors (two replicates in each experiment). (C to E and H to J) CAR T cell cytotoxic activity against NALM6/WT (~27,000 CD19 molecules; C), NALM6/12–39 (~2000 CD19 molecules; D), or NALM6/12–4 (~200 CD19 molecules; E) target cells. In (C) to (E), data are means ± SEM (n = 3). In (H) to (J), data are means ± SEM (n = 5, with three replicates from T cell donor 1 and two replicates from T cell donor 2). (K) Illustration of the trogocytosis assay. (L and M) Percentage of FMC63 (L) and SJ25C1 (M) CAR T cells (both CD4 and CD8 cells) with CD19-mCherry signal after coculture with CD19-mCherry–expressing NALM6 cells. Unpaired t test P value for FMC63 CAR T cells (L) or SJ25C1 CAR T cells (M). Data are representative of two independent experiments using two different T cell donors, with each experiment performed with three replicates (n = 3). Error bars are SEM.

Article Snippet: Cell lines GFP-firefly-luciferase (FFL)-expressing NALM6 cells (NALM6/WT and 19-mCherry fusion-expressing NALM6/WT ( 23 ), NALM6/12–4 and NALM6/12–39 ( 21 ) were cultured in RPMI 1040 medium supplemented with 10% fetal bovine serum (HyClone), 10 mM HEPES (Invitrogen), 2 mM L-glutamine (Invitrogen), 1X NEAA (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 50 μM β-mercaptoethanol, 10U/ml Penicillin, 10 μg/ml Streptomycin (Gibco), and 2.5 μg/ml Plasmocyn (InvivoGen).

Techniques: Expressing, Binding Assay, Activity Assay, Trogocytosis Assay