Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GSPT1-specific protein degradation is effective in preclinical models of chemoresistant MYCN -amplified neuroblastoma

doi: 10.1186/s13046-026-03647-0

Figure Lengend Snippet: GSPT1 degradation in NB organoids. A Western blot analysis of GSPT1 protein levels in NB organoids (LU-NB-1, LU-NB-2, LU-NB-3R) after treatment with CTX-56 for 24 h. Quantification shows the fold change expression compared with DMSO control presented as the mean of 3 biological replicates; B ) Cell viability and cell death in NB organoids following GSPT1 degradation with CTX-56. Organoids were treated for 72 h. 3 biological replicates (x3 technical replicates) were performed for each model; C ) Western blot analysis of GSPT1 protein levels in NB organoids after treatment with CTX-18 for 24 h. Quantification shows the fold change expression compared with DMSO control presented as the mean of 3 biological replicates; D ) Cell viability and cell death in NB organoids following GSPT1 degradation with CTX-18. Organoids were treated for 72 h. 3 biological replicates (x3 technical replicates) were performed for each model; E ) Cell viability of NB organoids LU-NB-1, LU-NB-2, LU-NB-3R, and HEK293 cells after CTX-18 treatment for 72 h. 3 biological replicates were performed; F ) Western blot analysis of GSPT1 levels following co-treatment with CTX-18 and siCRBN in the LU-NB-1 model. Quantification shows the fold-change expression compared with DMSO control presented as the mean of 3 biological replicates; G ) Western blot analysis of GSPT1 levels following co-treatment with CTX-18 and proteasome inhibitor MG132 in LU-NB-1 organoids. Cells were pre-treated with 5 µM MG132 for 1 h, followed by 6-hour co-treatment with CTX-18 at 10 nM and 100 nM. Quantification shows the fold change in expression compared with DMSO control presented as the mean of 3 biological replicates; H ) Analysis of compound selectivity in the AlphaLISA-based ternary complex formation assay. Luminescence observed for the neosubstrate-CRBN system at 10 µM of tested MGs (CC-90009, CTX-56, CTX-18) was normalized to signal observed for the reference compound at the same concentration. The normalized response is expressed as a percentage of the reference value (mean ± SD): 0–25% (green), 26–75% (orange), 76–100% (red). NA – not analyzed; BLD – below limit of detection; I ) Western blot analysis of CRBN-neosubstrate levels in LU-NB-1 organoids treated with CTX-18 for 24 h. Quantification shows the fold change in expression compared with DMSO control presented as the mean of 3 biological replicates. Samples were run on 2 separate gels, and appropriate loading controls were used for each gel (representative loading control is shown); J ) Proteomic analysis showing downregulation of proteins in LU-NB-1 organoids after treatment with 10 nM CTX-18 for 6 h (left) or 24 h (right). ~6770 proteins were detected; downregulated proteins are marked in red. A p -value < 0.01 was considered significant. The data show the results from 4 biological replicates; K ) Proteomic analysis showing downregulation of proteins in LU-NB-1 organoids after treatment with 1 µM CTX-18 for 6 h (left) or 24 h (right). ~6770 proteins were detected; downregulated proteins are marked in red, downregulated NB-related proteins are marked in orange. A p -value < 0.01 was considered significant. The data show the results from 4 biological replicates

Article Snippet: 24 h later, organoids were treated with either 100 nM or 1 μM MG or a control amount of DMSO and were allowed to grow for further 48 h. Organoids were then dissociated, resuspended in AnnexinV binding buffer (Invitrogen, #00-0055-43) and stained with Annexin V/propidium iodide (AnnexinV: Invitrogen, #11-8005-72, PI; Invitrogen, #00-6990-50) for 15 min. Flow cytometry was performed using FACS MelodyTM Cell Sorter (BD Biosciences), and data were analyzed using FlowJo 10.8.1 software.

Techniques: Western Blot, Expressing, Control, Tube Formation Assay, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GSPT1-specific protein degradation is effective in preclinical models of chemoresistant MYCN -amplified neuroblastoma

doi: 10.1186/s13046-026-03647-0

Figure Lengend Snippet: GSPT1 degradation induces caspase-mediated apoptosis in NB organoids. A Western blot analysis of PARP/cleaved PARP levels in LU-NB-1 organoids following 24 h treatment with CTX-18. Quantification shows the cleaved PARP/PARP to actin ratio presented as fold change compared with DMSO control (mean of 3 independent experiments); B ) Flow cytometry analysis of Annexin V/PI-positive LU-NB-1 cells 48 h after treatment with CTX-56 at 100 nM and 1 µM concentrations; C ) Morphological changes (brightfield imaging, upper panel) and activity of caspase 3 (brightfield + fluorescence, lower panel) in LU-NB-1 organoids 48 h after treatment with 1 µM CTX-56. 50 μm (upper panel) and 100 μm (lower panel) scale were used; D ) Cell death and cell viability of LU-NB-1 organoids after co-treatment with 100 nM CTX-56 and the pan-caspase inhibitor Z-VAD, compared with CTX-56 treatment alone. 4 biological replicates were performed. t-test with Welch correction was used for statistical analysis

Article Snippet: 24 h later, organoids were treated with either 100 nM or 1 μM MG or a control amount of DMSO and were allowed to grow for further 48 h. Organoids were then dissociated, resuspended in AnnexinV binding buffer (Invitrogen, #00-0055-43) and stained with Annexin V/propidium iodide (AnnexinV: Invitrogen, #11-8005-72, PI; Invitrogen, #00-6990-50) for 15 min. Flow cytometry was performed using FACS MelodyTM Cell Sorter (BD Biosciences), and data were analyzed using FlowJo 10.8.1 software.

Techniques: Western Blot, Control, Flow Cytometry, Imaging, Activity Assay, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: GSPT1-specific protein degradation is effective in preclinical models of chemoresistant MYCN -amplified neuroblastoma

doi: 10.1186/s13046-026-03647-0

Figure Lengend Snippet: GSPT1 degradation leads to MYCN suppression in chemoresistant MYCN -amplified neuroblastoma. A Gene Set Enrichment Analysis (GSEA) of PDX1 tumors showing pathways upregulated (red) and downregulated (blue) following treatment with CTX-18 (30 mg/kg for 1 or 5 days) relative to control tumors. Pathways with p < 0.05 were considered statistically significant; B ) Changes in mRNA expression of genes associated to cycling persisters, non-cycling cells, and stress response-related genes following treatment with CTX-18 in vivo. Statistical significance was assessed using a two-tailed t -test; C ) Western blot analysis of MYCN protein levels in LU-NB-1 organoids following CTX-18 treatment for 24 h. The quantification is a mean of 3 biological replicates presented as fold-change levels compared with DMSO; D ) Western blot analysis of MYCN protein levels in PDX1 tumors from mice treated with vehicle (Ctrl2, Ctrl3) or CTX-18 (30 mg/kg) for 1 day (1, 3) or 5 days (3, 4); E ) mRNA levels of MYCN and MYCN -associated core regulatory circuitry genes in PDX1 tumors following treatment with CTX-18 in vivo (1 day and 5 days treatment combined). Statistical significance was assessed using a two-tailed t -test

Article Snippet: 24 h later, organoids were treated with either 100 nM or 1 μM MG or a control amount of DMSO and were allowed to grow for further 48 h. Organoids were then dissociated, resuspended in AnnexinV binding buffer (Invitrogen, #00-0055-43) and stained with Annexin V/propidium iodide (AnnexinV: Invitrogen, #11-8005-72, PI; Invitrogen, #00-6990-50) for 15 min. Flow cytometry was performed using FACS MelodyTM Cell Sorter (BD Biosciences), and data were analyzed using FlowJo 10.8.1 software.

Techniques: Amplification, Control, Expressing, In Vivo, Two Tailed Test, Western Blot

Journal: STAR Protocols

Article Title: High-yield isolation of pure fetal endothelial colony forming cells and mesenchymal stem cells from the human full-term placenta

doi: 10.1016/j.xpro.2022.101354

Figure Lengend Snippet:

Article Snippet: For sorting collection, prepare sterile 5 mL FACS tubes or 1.5 mL Eppendorf tubes filled with 500 μL -1 mL filtered heat-inactivated FBS, labeled ECFC (CD45 NEG CD34 POS CD31 LO ) and MSC (CD45 NEG CD34 POS CD31 NEG ).

Techniques: Staining, Control, Isolation, Recombinant, Molecular Weight, Cell Culture, Sterility, Transferring, Cell Counting, Flow Cytometry