Journal: Pharmacological research

Article Title: Targeting MYCN upregulates L1CAM tumor antigen in MYCN-dysregulated neuroblastoma to increase CAR T cell efficacy.

doi: 10.1016/j.phrs.2025.107608

Figure Lengend Snippet: Fig. 2. MYCN overexpression correlates with reduced L1CAM expression on neuroblastoma cells and in patient cohorts. A. MA-Plot of RNA-sequencing data using DESeq2 of fold change (log2) of MYCN induction in SK-N-AS-MYCNnon-ind versus MYCNind cells after 48 h of tetracycline treatment. Red dots represent up- or downregulated genes upon MYCN induction (n = 3 biological replicates, M (log ratio), A (mean average)). B. L1CAM cell surface expression quantified on SK-N-AS- MYCNnon-ind, -MYCNind, SK-N-SH-MYCNnon-ind, -MYCNind, IMR5/75-shMYCNamp and -MYCNtet tumor cells. Data shows L1CAM molecules per cell (n = 3 biological replicates). C. Relative expression L1CAM mRNA levels in SK-N-AS-MYCNnon-ind/ind, SK-N-SH-MYCNnon-ind/ind and IMR5/75-shMYCNamp/tet tumor cells relative to housekeeper h28S. D. Gene-expression data from two patient cohort represents log2 fold change of L1CAM expression in a cohort of 498 neuroblastoma patients, without (MYCNnon-amp n = 401) and with MYCN amplification (MYCNamp n = 92, n = 5 no MYCN status available, not included, ANOVA p = 4.53e-30) and in a cohort of 144 neuroblastoma patients, without (MYCNnon-amp n = 104) and with MYCN amplification (MYCNamp n = 40, ANOVA p = 2.63e-12) [33]. E. Patient data from Hartlieb et al. represents log2 change of L1CAM protein expression in a cohort of 34 neuroblastoma patients, without (MYCNnon-amp n = 22) and with MYCN amplification (MYCNamp n = 12, ANOVA p = 7.31e-3) [33].

Article Snippet: Proteins were detected using mouse monoclonal antibodies detecting MYCN (B8.4B; sc-53993, Santa Cruz Biotechnology), L1CAM (UJ127.11; ThermoFisher Scientific) or GAPDH (sc-32233, Santa Cruz Biotechnology) together with horseradish peroxidase-conjugated mouse IgG (diluted 1:5000, Dianova).

Techniques: Over Expression, Expressing, RNA Sequencing, Gene Expression, Amplification

Journal: Pharmacological research

Article Title: Targeting MYCN upregulates L1CAM tumor antigen in MYCN-dysregulated neuroblastoma to increase CAR T cell efficacy.

doi: 10.1016/j.phrs.2025.107608

Figure Lengend Snippet: Fig. 5. MLN8237 enhances L1CAM surface expression on MYCN-amplified tumor cell lines in vitro. A. Flow cytometry analysis representing L1CAM expression of IMR5/75, SK-N-BE(2) and SK-N-DZ cell lines treated with 80 and 800 nM MLN8237 for 72 h. Dot blots represent normalized MFI of L1CAM of IMR5/75, SK-N-BE(2) and SK-N-DZ n = 4. B. Combinational therapy of L1CAM-specific CAR T cells (L1CAM-28/ζ; E:T 1:10) and 40 nM MLN8237. Biophotonic signal of IMR5/75, SK-N-BE (2) and SK-N-DZ cells was measured to analyze cytotoxic potential of therapies alone and in combination (n = 3 biological replicates in technical replicates). mean ± SD, students T-test, ns= not statistically significant, * , p ≤0.05.

Article Snippet: Proteins were detected using mouse monoclonal antibodies detecting MYCN (B8.4B; sc-53993, Santa Cruz Biotechnology), L1CAM (UJ127.11; ThermoFisher Scientific) or GAPDH (sc-32233, Santa Cruz Biotechnology) together with horseradish peroxidase-conjugated mouse IgG (diluted 1:5000, Dianova).

Techniques: Expressing, Amplification, In Vitro, Flow Cytometry

Journal: Nature communications

Article Title: The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis.

doi: 10.1038/s41467-024-49871-0

Figure Lengend Snippet: Fig. 2 | Runx1t1 loss reverses MYCN-mediated sustained hyperplasia and induces ganglia neurite extension. a The percentage neuroblast hyperplasia scored from homozygous Th-MYCN (+/+) mice or littermate mice lacking the MYCN transgene (−/−), with either wild-type (+/+) or heterozygous loss (+/−) of Runx1t1. Scoring of N = 3–8 independent mice was performed for each genotype and timepoint. All data points were N = 3, except for +/+, +/+ week 1 and week 2 (N = 4); +/+, +/−day 0 (N = 8) and week 4 (N = 4); −/−, +/−day 0 (N = 6), week 1 (N = 4) and week 4 (N = 5). The graph is mean ± SEM. b Representative histology of RUNX1T1 staining in ganglia from mice homozygous for the Th-MYCN transgene, and either wild-type or heterozygous for Runx1t1 from day 0 and 4 weeks of age.

Article Snippet: Samples were embedded in paraffin, sectioned, and stained with H&E or for MYCN (rabbit polyclonal antibody, 10159-2-AP, Proteintech, 1:1000) and RUNX1T1 (as described above).

Techniques: Staining

Journal: Molecular cell

Article Title: The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2024.04.007

Figure Lengend Snippet: Figure 4. Exosome function controls the dynamics of the MYCN/MAX/MXD network (A) Immunoblot of IMR-32 cells stably expressing an shRNA targeting EXOSC10 treated for 72 h with Dox or EtOH as control. ACTB was loading control. Blot is representative of n = 3 independent replicates. (B) Read distribution of MYCN ChIP-Rx, MAX, MNT, and SIN3A cleavage under targets & release using nuclease (CUT&RUN) at the CDK5RAP3 and PHB genes. Cells treated as in (A) (n = 3 for MYCN ChIP-Rx; n = 2 for all C&Rs).

Article Snippet: 3 mg of each, MAX (Proteintech), MNT (Thermo Fisher Scientific), or SIN3A (Novus Biologicals) antibody were added per sample and incubated with shaking (800 rpm) at 4 C overnight.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control

Journal: bioRxiv

Article Title: Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for receptor binding

doi: 10.1101/2020.04.16.045096

Figure Lengend Snippet: VAR2CSA and its deletion constructs expressed in HEK293-F cells and SDS-PAGE of the purified recombinant VAR2CSA proteins. (A) Schematic indicating the locations of DBL domains for constructs I-V. For each construct, the numbers at either end denote the beginning and ending amino acid sequence numbers. I, II and IV contain a non-cleavable C-terminal cMyc tag and hexahistidine tag, as coded in the commercial vector. For the III and V constructs, the C-terminal tag is replaced with a TEV-cleavable 3X-FLAG tag and a hexahistidine tag, as defined in Table S2, GBLOCK2. In addition, P1 contains a glycine and threonine residue inserted between native residues 58 and 59 as a cloning artefact. (B) Purified proteins (each 2 μg/lane), electrophoresed using 4-20 % gradient gels (1 mm thick) under reducing conditions and (C) non-reducing conditions are shown. In each gel, the lanes are: M, Molecular weight markers; I, NTS-DBL6ε (~310 kDa); II, DBL1x-ID2a (~125 kDa); III, ID2b-DBL6ε (~200 kDa); IV, DBL3x-DBL6ε (~180 kDa); V, DBL4ε-DBL6ε (~130 kDa). The molecular mass marker (kDa) standards are indicated in the left margin.

Article Snippet: cMyc mAb (Cat NB600-302, Novus Biologicals, CO) and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 and incubated for 30 min on ice.

Techniques: Construct, SDS Page, Purification, Recombinant, Sequencing, Plasmid Preparation, FLAG-tag, Residue, Cloning, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for receptor binding

doi: 10.1101/2020.04.16.045096

Figure Lengend Snippet: VAR2CSA is an asymmetric molecule with the C-terminus located within the smaller lobe. (A) Micrograph of NTS-DBL6ε. The white bar is 50 nm. (B) Subset of 2D classes obtained during initial 2D classification of NTS-DBL6ε. (C) Single 2D class showing the layers (L1, L2, L3), separated by broken white lines, defined in the text. (D) Micrograph of NTS-DBL6ε bound to anti-cMyc antibody. (E) Representative 2D classes of unbound NTS-DBL6ε. (F) Representative 2D classes of unbound anti-cMyc antibody. (G) Representative 2D classes of NTS-DBL6ε:anti-cMyc-antibody complex. The letter A indicates the location of the cMyc antibody at the tip of the smaller lobe of NTS-DBL6ε.

Article Snippet: cMyc mAb (Cat NB600-302, Novus Biologicals, CO) and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 and incubated for 30 min on ice.

Techniques: