Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Volcano plot of AraTM screening assay of Screening Set v2010 (8482 compounds) from the Chemical Biology Consortium Sweden ( www.cbcs.se ). Fold change was calculated against vehicle (DMSO) and hits were defined as compounds resulting in greater than ±0.5 fold change in GFP/OD 630 signal without affecting OD 630 by greater than 0.3-fold across three independent runs (red dots). P values were calculated by one-way ANOVA ( N = 3). Compound Div17E5 is indicated. ( B ) Chemical structure of Div17E5. ( C ) Dose response of Div17E5 in the AraTM assay of p75 NTR TMD in comparison with unrelated TMDs from α2β3 integrin and Matrix-2 protein (M2) from the viral envelope of influenza A virus. Results are plotted as mean ± SD ( N = 3). ( D – F ) Comparison of wild-type (WT) p75 NTR TMD and I252A, P253G and V254A mutants in the AraTM assay in response to increasing doses of Div17E5. GFP/OD 630 signal for WT TMD without any drug was set at 100% and all other measurements are relative to that. Results are plotted as mean ± SD ( N = 3). * P = 0.03: ** P = 0.006 (one-way ANOVA followed by Tukey’s multiple comparisons). ( G ) Live cell homo-FRET anisotropy in response to Div17E5 of full-length, wild-type human p75 NTR expressed in COS cells. Shown are representative traces of average anisotropy change after addition of Div17E5 (10 μM) or vehicle. ( H ) Live cell homo-FRET anisotropy in response to Div17E5 of full-length, wild-type human p75 NTR in comparison to I252A p75 NTR TMD mutant. Shown are representative traces of average anisotropy change after addition of Div17E5 (10 μM). .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Screening Assay, AraTM Assay, Comparison, Virus, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Expression of p75 NTR in A875 control (NT, non-targeting) and knock-down (shp75) cells. ( B ) Dose response analysis of cleaved PARP induction by Div17E5 in A875 control (NT) and knock-down (shp75) cells. ( C ) Western blot analysis of cleaved PARP in A875 melanoma cells in response to Div17E5 (10 µM) in the presence or absence of pan-caspase inhibitor Q-VD-pOH (5 µM). Reprobing for GAPDH was used as loading control. The experiment was repeated three times with comparable results. ( D ) Dose-dependent cell viability of A875 control (NT) and knock-down (shp75) cells in response to Div17E5. Results are plotted as mean ± SD ( N = 3). * P = 0.04; ** P = 0.006 (one-way ANOVA followed by Tukey’s multiple comparisons). .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Expressing, Control, Knockdown, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Chemical structure of Np75-4A22. ( B ) Dose response of Div17E5 and Np75-4A22 in the AraTM assay of p75 NTR TMD. Results are plotted as mean ± SD ( N = 3). ** P = 0.007 (one-way ANOVA followed by Tukey’s multiple comparisons). ( C – E ) Comparison of wild-type p75 NTR TMD and I252A, P253G and V254A mutants in the AraTM assay in response to increasing doses of Np75-4A22. GFP/OD 630 signal without any drug added was set at 100%. Results are plotted as mean ± SD ( N = 3). * P = 0.035; ** P = 0.008 (one-way ANOVA followed by Tukey’s multiple comparisons). ( F – H ) Specific interaction between Np75-4A22 (4 mM) and p75 NTR TMD (1 mM) in bicelles that mimic a lipid bilayer. ( F ) 1 H- 15 N TROSY-HSQC spectra of p75 NTR TMD in DMPC-DH6PC bicelles ( q = 0.5) in the absence (red) and presence (blue) of 4 molar excess Np75-4A22. Cross-peaks undergoing significant chemical shift are indicated with arrows and the corresponding residues labeled. ( G ) The same as ( F ) for the TMD of TrkB receptor tyrosine kinase, showing mostly non-specific interaction. ( H ) Same as ( F ) for the TMD of TNF receptor 2 (TNFR2), showing essentially no interaction. .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: AraTM Assay, Comparison, Labeling

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Representative Western blot analysis (IB) of fascin in p75 NTR immunoprecipitates (IP) and whole cell lysates (WCL) of A875 melanoma cells after overnight treatment with NGF or Np75-4A22 as indicated. The histogram on the right shows quantification expressed as average of three independent experiments, each performed in triplicate ± SD. # P = 0.0091 vs. control (first bar); ** P = 0.0088 vs. NGF alone (second bar) (one-way ANOVA followed by Tukey’s multiple comparisons). ( B ) Representative Western blot analysis of fascin in Triton-insoluble extracts of A875 melanoma cells after overnight treatment with NGF or Np75-4A22 as indicated. The histogram on the right shows quantification expressed as average of three independent experiments, each performed in triplicate ± SD. # P = 0.0412 vs. control (first bar); ** P = 0.0082 vs. NGF alone (second bar) (one-way ANOVA followed by Tukey’s multiple comparisons). ( C ) Photomicrographs of cultured A875 control (NT) and knock-down (shp75) cells treated with NGF or Np75-4A22 as indicated and stained with fluorophor-conjugated phalloidin (green), to reveal actin filaments, and DAPI (blue). White arrows denote some of the filopodia in NGF-treated cells. Scale bar, 5 μM. ( D , E ) Quantification of filopodia per cell in A875 NT ( D ) and A875 shp75 ( E ) cells treated with 100 ng/ml NGF or 1 or 5 μM Np75-4A22 as indicated. Average of three independent experiments, each performed in triplicate, are shown ± SEM. ### P = 0.0004 vs. vehicle (first bar); *** P = 0.0001 vs. NGF alone (second bar). .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Western Blot, Control, Cell Culture, Knockdown, Staining

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: p75 NTR immunostaining (red) in lung metastasis induced by A875-NT ( A ) or shp75-A875 ( B ) cells counter-stained for human nucleolin (green) and DAPI (white). Scale bar, 100 μM.

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Immunostaining, Staining