off target cell line  (ATCC)

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Off-target cytotoxicity evaluation of CAR T cells using the 3D GOC system. A) Schematic representation of the differing cytolytic mechanisms of UTD, TV-13, and IL-13 CAR T cells against IL13Rα1 + HT-1080 tumor cells. Created with BioRender.com . B) Flow cytometric analysis confirming IL13Rα1 and mCherry (reporter gene) expression on IL13Rα1 + HT-1080 tumor cells. Antigen expression (IL13Rα1 or mCherry) on viable tumor cells shown in histograms: blue for IL13Rα1 + HT-1080 tumor cells and red for control tumor cells. The values within each histogram indicate the percentage of positive cells, with the mean fluorescence intensity (MFI) shown in parentheses. C) Microfluidic evaluation of off-target toxicities of T cells. (i) Representative tile images of tumor-stroma interface stained for actin cytoskeleton (green), showing differences in migration of IL13R1 + HT-1080 tumor cells (red) within the 3D GOC model across varying densities of UTD, TV-13 CAR, and IL-13 CAR T cells. (ii) Quantification of the migration distance of the IL13Rα1 + HT-1080 tumor cells in response to varying T cell concentrations. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. (iii) Bar graph showing the difference in nuclei per field of view (FOV) across different T cell densities, used as a measure of chain migration by IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (iv) Bar graph representing the percentage of T cells positive for intracellular cytokines in the presence of IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: HT-1080 Culture : Human fibrosarcoma cells (CCL-121, ATCC or HT-1080) were used to generate an off-target cell line (IL13Rα1 + HT-1080) expressing IL13Rα1-T2A-mCherry gene, which was single-sorted for the experiments described here.

Techniques: Gene Expression, Expressing, Control, Fluorescence, Staining, Migration

Journal: Communications Chemistry

Article Title: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors

doi: 10.1038/s42004-025-01871-x

Figure Lengend Snippet: a IPD libraries were screened in a 20 h dose–response using a TEAD1 luciferase degradation assay (HiBiT-TEAD1, NCI-H2052 cells). Compounds with D max > 40% and DC 50 < 1000 nM were further selected for a follow-up 2 concentration screen for endogenous TEAD1 degradation (20 h treatment with 0.3 and 3 µM IPD, NCI-H2052 cells). Three IPDs that achieved >40% degradation of endogenous TEAD1 were selected. b Chemical structures of the three IPDs selected (ALP A232 , ALP2 A531 , and XB2 A538 ) representing different IAP binder series, linkers and exit vectors (left panel), and corresponding endogenous TEAD1 and HiBiT-TEAD1 degradation results (middle and right panel). For endogenous TEAD1, NCI-H2052 cells were treated with IPDs (0.3 or 3 µM) or DMSO control for 20 h and RIPA lysates generated. Following capillary western electrophoresis, these were probed with TEAD1 and GAPDH (loading control) antibodies. Shown is one representative capillary western image for each IPD out of n = 2 independent experiments performed. Uncropped blot images are available in Supplementary Data . Percentage endogenous TEAD1 degradation (represented as mean % values) was calculated relative to 100% value of DMSO controls. For HiBiT-TEAD1 degradation (screening assay), NCI-H2052 cells stably expressing HiBiT-TEAD1 were treated for 20 h with a dose–response of IPDs or DMSO control. Percentage TEAD1 remaining was plotted based on HiBiT luminescence normalized to CTG (HiBiT/CTG ratio) relative to vehicle control. Plotted data represent individual data points from n = 3 independent experiments. Degradation DC 50 and D max were fitted as described .

Article Snippet: One day later 0.3 × 10 6 cells of the target cell line NCI-H2052 (ATCC original lot#58033333) were seeded in 2 mL medium (RPMI, Gibco, A1049101; +10% FCS, Gibco, 26140-079) in 6 well plates.

Techniques: Luciferase, Degradation Assay, Concentration Assay, Control, Generated, Western Blot, Electrophoresis, Screening Assay, Stable Transfection, Expressing

Journal: Communications Chemistry

Article Title: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors

doi: 10.1038/s42004-025-01871-x

Figure Lengend Snippet: a Molecular matched pair IAP- or TEAD-negative controls were generated by modification of the IAP BIR binding N-methylalanine group with N,N-dimethylglycine or TEAD binding central amide -NH methylation (refer Supplementary Fig. for full chemical structures of IPD negative controls). b Profiling of endogenous TEAD1 and cIAP1 degradation using capillary western electrophoresis (20 h treatment, dose titration and DMSO, NCI-H2052 cells). % Endogenous TEAD1 and cIAP1 degradation was quantified relative to DMSO samples, and dose response curves (as in c , e , g ) fitted using one-phase decay model to calculate D max and DC 50 values. c Endogenous cIAP1 auto-degradation curves in NCI-H2052 cells for ALP2 A531 (orange line) and matched IAP and TEAD negative control IPDs (black and gray lines, respectively). d Profiling of IPDs in cellular TEAD1 target engagement assay. Dose–response NanoBRET signal was measured for displacement of a fluorescent TEAD tracer from NanoLuc-TEAD1 (HEK293T cells) following treatment with ALP2 A531 (orange line) and matched IAP- and TEAD- negative IPD controls ( A557 , A423 ; black and gray lines respectively) and percentage tracer displacement plotted relative to a vehicle control. Data represent mean ± SD of n = 3 independent experiments. e Endogenous TEAD1 degradation curves for ALP2 A531 (left, orange line) and XB2 A538 (right, blue line) and matched IAP and TEAD negative IPD controls (black and gray lines respectively). f Proteasome dependency analysis for ALP2 A531 (orange) and XB2 A538 (blue). Top panel shows western blot analysis of NCI-H2052 cells treated for 16 h with DMSO, 3 µM compound ±5 µM MG132. Bottom panel shows bar graph of % endogenous TEAD1 degradation relative to DMSO-treated cells. g IAP dependency of ALP hit A531 (orange) and XB2 hit A538 (blue) in isogenic WT and IAP KO NCI-H2052 lines. Plot shows endogenous TEAD1 degradation after 20 h compound treatment (5 concentrations with ten-fold serial dilutions starting from 10 µM and DMSO vehicle control) assessed in NCI-H2052 wildtype cells (orange/blue line), cIAP1 KO (gray dashed line), XIAP KO (black dashed line) and cIAP1/XIAP DKO (black dotted line) cell lines. All data points for endogenous degradation curves represent mean ± SD of n = 2 biologically independent experiments, except n = 5 for A531 and n = 3 for A538 in ( e ) and n = 3 for ( g ). All uncropped blot images are available in Supplementary Data .

Article Snippet: One day later 0.3 × 10 6 cells of the target cell line NCI-H2052 (ATCC original lot#58033333) were seeded in 2 mL medium (RPMI, Gibco, A1049101; +10% FCS, Gibco, 26140-079) in 6 well plates.

Techniques: Generated, Modification, Binding Assay, Methylation, Western Blot, Electrophoresis, Titration, Negative Control, Drug discovery, Control

Journal: Communications Chemistry

Article Title: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors

doi: 10.1038/s42004-025-01871-x

Figure Lengend Snippet: a Immunoblots of equivalent total cell lysate (L), cytosolic fraction (C) and nuclear fraction (N) from indicated cell lines were probed for nuclear marker (Lamin B1), cytosolic marker (Hsp90), E3 ligases (cIAP1 and XIAP) and target proteins (TEAD1 and TEAD4). b Subcellular profiling of endogenous TEAD1 and cIAP1 degradation. NCI-H2052 cells were treated with dose titration of ALP hit A531 (four concentrations with 10-fold serial dilutions from 10 µM and DMSO vehicle control), nuclear and cytosolic fractions purified, and equivalent amounts run on Western blot. Antibodies against nuclear marker (Lamin B1), cytosolic marker (Hsp90), target protein (TEAD1) and E3 ligases (cIAP1 and XIAP) were used for probing the blots. All subcellular fraction experiments are performed as a single biological experiment ( n = 1), but have at least n = 2 biologically independent experiments overall for TEAD1 and cIAP1/XIAP localization in NCI-H2052 cells ( a , b ). All uncropped blot images are available in Supplementary Data .

Article Snippet: One day later 0.3 × 10 6 cells of the target cell line NCI-H2052 (ATCC original lot#58033333) were seeded in 2 mL medium (RPMI, Gibco, A1049101; +10% FCS, Gibco, 26140-079) in 6 well plates.

Techniques: Western Blot, Marker, Titration, Control, Purification

Journal: Communications Chemistry

Article Title: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors

doi: 10.1038/s42004-025-01871-x

Figure Lengend Snippet: a Chemical structure of ALP2 series IPD A536 incorporating a spirocyclic linker and matched IAP- and TEAD- negative controls ( A558 and A560 , respectively). b Profiling of degradation of HiBiT-TEAD1 (NCI-H2052 cells) following 20 h dose–response treatment with ALP2 IPD ( A536 , green line) or matched IAP negative control ( A558 , black line) or TEAD1 negative control ( A560 , gray line) IPDs. HiBiT signal was normalized to CTG reading, and the HiBiT/CTG ratio was compared to a vehicle control to plot percentage of TEAD1 remaining. Plotted data represent individual data points from three independent biological experiments. Degradation DC 50 and D max were fitted as described . c Capillary-based western profiling of endogenous TEAD1 degradation (left panel) and cIAP1 auto-degradation (right panel) in NCI-H2052 cells (dose response, 20 h) by ALP2 IPD A536 , its matched IAP negative control, A558 and TEAD negative control, A560 (color scheme as in b ). Uncropped blot images are available in Supplementary Data . Dose response curves are represented with each concentration denoting mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \pm $$\end{document} ± SD of n = 2 biologically independent experiments. d IAP cellular target engagement. A cellular IAP target engagement assay was developed based on the displacement of a fluorescent IAP tracer B678 from NanoLuc-tagged cIAP1 184–618 F616A or XIAP 124–497 V461E (HEK293T cells) and nanoBRET signal measured for IAP binders or IPDs treated in dose–response (left panel, refer Supplementary Fig. and Synthetic Chemistry methods for synthesis of B678 ). Percentage tracer occupancy (based on nanoBRET signal, normalized to DMSO vehicle) was measured for live cells (cIAP1 and XIAP) or cells permeabilized by pre-treatment with digitonin (cIAP1 only). For cIAP1, a cellular Availability Index (AI) was determined by first comparing the fitted IC 50 values in live and permeabilized modes to obtain a Relative intracellular availability (RBA) value, then normalizing this to ASX series IAP ligand A255 , selected as a cell-permeable control compound with high affinity to BIR3 of cIAP1 and XIAP . cIAP1 was used for AI determination as most compounds tested have potent cIAP1 binding. Larger AI values represent lower intracellular availability relative to the permeable control A255 . Right panel: tabulated IC 50 , RBA and AI values for ASX series IAP binder reference A255 and IPDs ALP1 A232 , ALP2 A531 , XB2 A538 and ALP2 A536 . e Cellular ternary complex formation. Cellular IAP/IPD/TEAD1 ternary complex formation was measured by treating NCI-H2052 cells co-expressing NanoLuc-tagged cIAP1 184–618 F616A /Halo-TEAD1 or NanoLuc-tagged XIAP 124–497 V461E /Halo-TEAD1 with a dose response of IPDs (ALP1 IPD A531 and corresponding IAP or TEAD negative controls A557 and A423 , or XB2 IPD A538 , or ALP2 IPD A536 ), alongside NanoGlo Substrate and HaloTag 618 ligand. Measured nanoBRET signals relative to background were fitted to a Gaussian distribution model to calculate E max and EC max values for ternary complex formation with TEAD1 and either cIAP1 or XIAP. Data represent mean ± SD for n = 3 biologically independent experiments.

Article Snippet: One day later 0.3 × 10 6 cells of the target cell line NCI-H2052 (ATCC original lot#58033333) were seeded in 2 mL medium (RPMI, Gibco, A1049101; +10% FCS, Gibco, 26140-079) in 6 well plates.

Techniques: Negative Control, Control, Western Blot, Concentration Assay, Drug discovery, Binding Assay, Expressing

Journal: Communications Chemistry

Article Title: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors

doi: 10.1038/s42004-025-01871-x

Figure Lengend Snippet: a Endogenous TEAD1 and TEAD4 degradation profiling in NCI-H2052 cells with 20 h treatment with ALP2 IPD A531 , XB2 IPD A538 and ALP2 IPD A536 . With compound concentrations represented in increasing concentration from DMSO to 10 µM, Top panel shows representative capillary western blot of TEAD4 and loading control GAPDH; middle panel, blot of TEAD1 and GAPDH and bottom panel shows degradation dose response curves with each data point representing mean ± SD of n = 2 biologically independent experiments, except for n = 5 for A531 (TEAD1) and n = 3 for A538 (TEAD1). Uncropped blot images are available in Supplementary Data . b HiBiT assay measuring D max of ALP2 IPD A531 , XB2 IPD A538 and ALP2 IPD A536 after 18 h treatment of HiBiT-TEAD1–4 NCI-H226 transgenic cell lines, normalized to CTG viability assay. Compound dTAG-13 (heterobifunctional degrader of the FKBP12 F36V sequence incorporated in the TEAD1–4 transgenic constructs) was used as positive control. Data are representing mean ± SD of n = 4 biologically independent experiments, each with two technical replicates. c Global proteomic analysis of XB2 hit A538 specificity in NCI-H2052 cells, treated with compound A538 (0.5 µM, 16 h) or DMSO ( n = 5 biological replicates). Volcano plots show relative protein abundance (log2 fold change) vs significance (−log10 p -value) of quantified proteins. Proteins significantly altered lie above the horizontal dashed line (adjusted p -value “or” FDR ≤ 0.05) and beyond vertical cut off lines (left, 1.25 times downregulated; right, 1.25 times upregulated in A538 treated cells). A complete protein list is provided in Supplementary Data .

Article Snippet: One day later 0.3 × 10 6 cells of the target cell line NCI-H2052 (ATCC original lot#58033333) were seeded in 2 mL medium (RPMI, Gibco, A1049101; +10% FCS, Gibco, 26140-079) in 6 well plates.

Techniques: Concentration Assay, Western Blot, Control, Transgenic Assay, Viability Assay, Sequencing, Construct, Positive Control, Quantitative Proteomics

Journal: Communications Chemistry

Article Title: Expanding the toolbox to develop IAP-based degraders of TEAD transcription factors

doi: 10.1038/s42004-025-01871-x

Figure Lengend Snippet: a Viability of Hippo pathway-dependent mesothelioma cells ZL55, NCI-H226, NCI-H2052 and Hippo pathway-independent NCI-H520 cells was assessed post dose titrations of indicated IPDs for 7 days. IC 50 (plotted as bars) and maximum inhibition % E max (plotted as dot symbols) values were calculated from dose response curves (see Supplementary Data ) for indicated cell lines. Data represent mean ± SD of n = 4 biologically independent experiments. b Effect of IPDs on transcript levels of TEAD-dependent genes. qPCR analysis of CTGF expression level (top panel) from indicated cell lines treated with 5 concentration points (1:10 dilution factor from 10 µM and DMSO) for 48 h. IC 50 and E max values were calculated as mean ± SD of n = 3 biologically independent experiments ( n = 2 in the case of ZL55 cells). IC 50 values marked >1000 and >10000 nM indicate the upper concentration limit for dose response fitting, as detailed in “Materials and Methods”.

Article Snippet: One day later 0.3 × 10 6 cells of the target cell line NCI-H2052 (ATCC original lot#58033333) were seeded in 2 mL medium (RPMI, Gibco, A1049101; +10% FCS, Gibco, 26140-079) in 6 well plates.

Techniques: Inhibition, Expressing, Concentration Assay