cell culture sk br3 cells  (ATCC)

Journal: Nature Communications

Article Title: Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

doi: 10.1038/s41467-026-72524-3

Figure Lengend Snippet: a Volcano plot presenting RNA-seq analysis of HEK293T cells cultured for 24 h in complete or cysteine-free media ( n = 3 independent replicates). Differential expression was assessed using DESeq2 by fitting gene-wise negative binomial generalized linear models; statistical significance was evaluated using two-sided Wald tests, with p -values adjusted for multiple testing using the Benjamini-Hochberg procedure (adj. p ≤ 0.01). LRRC58 transcript levels show no significant change. Dashed lines indicate thresholds at 2-fold change and p -value = 0.05. b Average LRRC58 intensities in HEK293T proteomes ( n = 4 independent replicates) after 24 h treatment with proteasome (MG132) or neddylation inhibitors (MLN4924) in complete or cysteine-free media. c Average intensities of proteins identified in HEK293T proteomes ( n = 4 independent replicates), which were detectable when cultured in 10-fold excess cysteine but undetectable in cysteine-free media. d Average intensities of LRRC58 and CDO1 in HEK293T proteomes after 24 h culture in cysteine-free media, complete media (control), or with 10-fold excess cysteine ( n = 4 independent replicates). CDO1 was undetectable after culture in cysteine-free media; LRRC58 was undetectable in the control. e Average IPTM and PEAK scores from HT-Colabfold analysis of interactions between LRRC58-EloB/C and all proteins absent in cysteine starvation proteomes, but detected with 10-fold excess cysteine for the cell lines; HEK293T, HepG2, Jurkat, HeLa, and SKBR3. f Western blot analysis of CDO1 levels in WT-HEK293T and LRRC58 CRISPR-Cas9 knockout (KO) lysates after 24 h culture in cysteine-free media. g Average intensities of LRRC58 and CDO1 in WT-HEK293T and LRRC58-KO proteomes after 24 h culture in cysteine-free media ( n = 4 independent replicates). LRRC58-KO is confirmed by the absence of LRRC58; CDO1 is detectable only in KO. h WT-HEK293T and LRRC58-KO cells were transiently transfected with Flag-LRRC58 (WT, or A266F variant (disrupts EloB/C binding)). Western blot of CDO1 levels 24 h post-transfection, followed by 24 h culture in cysteine-free or complete media. All blots are representative of n = 2 technical replicates. Flag and vinculin serve as transfection and loading controls, respectively. All error bars report standard deviation. Purple and orange asterisks indicate samples where LRRC58 or CDO1, respectively, were undetectable. Source data provided as Source Data file.

Article Snippet: Jurkat (TIB-152), HepG2 (HB-8065), and SKBR3 (HTB-30) cells were purchased from the American Type Culture Collection (ATCC).

Techniques: RNA Sequencing, Cell Culture, Quantitative Proteomics, Control, Western Blot, CRISPR, Knock-Out, Transfection, Variant Assay, Binding Assay, Standard Deviation

Journal: mAbs

Article Title: A novel insulin-like growth factor II-based masking domain for conditional activation of therapeutic antibodies

doi: 10.1080/19420862.2026.2668187

Figure Lengend Snippet: Effects of masking and protease-dependent unmasking on anti-HER2 antibody activity. (A) Size exclusion chromatography (SEC) profiles of IGF-II-s-MMP2/9-Tras and native trastuzumab (Tras). UV absorbance at 280 nm was shown as normalized peak signal versus retention time (min). (B) Reducing SDS-PAGE analysis of heavy chain (HC) and light chain (LC) for IGF-II-s-MMP2/9-Tras before (“uncut”) and after (“cut”) MMP2 cleavage, with native Tras included for comparison. (C) ELISA binding assay measuring HER2 binding by masked IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) unmasked antibody and native Tras shown for comparison. The absorbance at 450 nm (OD 450 nm) was plotted versus antibody concentration (ng/mL) with the data presented as mean values ± sd ( n = 5). (D) SK-BR-3 cell viability assay measuring inhibition of cell viability by IGF-II-s-MMP2/9-Tras, with the corresponding MMP2-cleaved (“MMP2 cut”) antibody and native Tras shown for comparison. Percent cell viability was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd. (E, F) Antibody dependent cellular cytotoxicity (ADCC) (E) and antibody dependent cellular phagocytosis (ADCP) (F) reporter assays assessing IGF-II-s-MMP2/9-Tras and Tras in promoting engagement of SK-BR-3 target cells with effector cells and activation of CD16a (ADCC)- or CD32a (ADCP)-dependent reporter gene expression. Fold induction of reporter activity was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd.

Article Snippet: SK-BR-3 cells (ATCC, HTB-30) were seeded at 10,000 cells per well in 96-well plates and treated with serial dilutions of test antibodies.

Techniques: Activity Assay, Size-exclusion Chromatography, SDS Page, Comparison, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Viability Assay, Inhibition, Activation Assay, Gene Expression

Journal: mAbs

Article Title: A novel insulin-like growth factor II-based masking domain for conditional activation of therapeutic antibodies

doi: 10.1080/19420862.2026.2668187

Figure Lengend Snippet: Effects of masking on anti-VEGF antibody activity. (A) ELISA-binding assay measuring vascular endothelial growth factor (VEGF) binding by the masked antibody IGF-II-MMP2/9-Bev and native bevacizumab (Bev). The absorbance at 450 nm (OD 450 nm) was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd. (B) VEGF sequestration assay assessing depletion of free VEGF secreted by SK-BR-3 cells following treatment with IGF-II-MMP2/9-Bev or Bev. Free VEGF concentration in the culture supernatant was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd. (C) HUVEC proliferation assay measuring neutralization of VEGF-driven human umbilical vein endothelial cell (HUVEC) proliferation by IGF-II-MMP2/9-Bev and Bev. The percent cell proliferation was plotted versus antibody concentration (ng/mL). The experiments were performed in duplicate with the data presented as mean values ± sd.

Article Snippet: SK-BR-3 cells (ATCC, HTB-30) were seeded at 10,000 cells per well in 96-well plates and treated with serial dilutions of test antibodies.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Proliferation Assay, Neutralization

Journal: bioRxiv

Article Title: De novo masking domains that gate TNF-family ligand assembly and activity

doi: 10.64898/2026.04.20.719557

Figure Lengend Snippet: To evaluate the generality of the masked TNFL platform, we extended the design and screening workflow to the additional TNFL family members OX40L and 4-1BBL using expanded computational design strategies. Unless otherwise noted, protein concentrations are reported per monomeric protomer. a, Expanded computational strategies for masking-domain design. In the scaffold-guided approach, the masking domains from mTNFα7 and mTNFα8 were used as structural scaffolds to guide backbone generation against the trimerization interfaces of OX40L and 4-1BBL. In the superposition-based approach, artificial complexes were generated by superimposing TNFα and 4-1BBL monomers, followed by partial diffusion to generate new 4-1BBL masking-domain backbones using the TNFα masking domains as templates. b, OX40 binding of selected masked OX40L constructs measured by ELISA before (filled circles) and after (open circles) TEV activation. Data are from a single experiment and are shown as representative results. c, Oligomeric states of selected masked OX40L constructs assessed by analytical SEC before (left) and after (right) TEV activation. The dashed line indicates the elution volume of the trimeric OX40L control. d, Oligomeric states of selected masked 4-1BBL constructs assessed by analytical SEC after TEV activation. The dashed line indicates the elution volume of the trimeric 4-1BBL control. e, Functional activity of masked 4-1BBL (m4-1BBL6) assessed in Jurkat-Lucia h4-1BB reporter cells. Responses to the monomer (filled circles) and SEC-purified homotrimer (open circles) are shown. TEV-treated 4-1BBL_control was included as a positive control. Data are shown as mean ± s.d. from n = 3 technical replicates. f, AlphaFold2-predicted complex structures of representative masked OX40L and masked 4-1BBL constructs, highlighting designs from the scaffold-guided and superposition-based approaches.

Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).

Techniques: Generated, Diffusion-based Assay, Binding Assay, Construct, Enzyme-linked Immunosorbent Assay, Activation Assay, Control, Functional Assay, Activity Assay, Purification, Positive Control

Journal: bioRxiv

Article Title: De novo masking domains that gate TNF-family ligand assembly and activity

doi: 10.64898/2026.04.20.719557

Figure Lengend Snippet: A single masked TNFα or 4-1BBL module was fused to a one-armed, affinity-attenuated trastuzumab variant to generate an antibody format that undergoes activation-dependent homotrimerization. Homotrimerization was first validated using the masked TNFα construct (b–d), and functional consequences were then evaluated using an analogous masked 4-1BBL fusion in assays with HER2-positive SK-BR-3 cells and Jurkat-Lucia h4-1BB reporter cells (e, f). Protein concentrations are reported per molecular species (monomeric or trimeric antibody, as indicated). Where indicated, trimeric species were re-isolated by SEC before downstream assays. a, Proposed mechanism for delivery of membrane-type TNFLs. Before activation, the construct is predominantly monomeric with reduced apparent target engagement and masked TNFL function. Protease-triggered activation promotes assembly into a trimeric state with increased functional valency and the capacity to drive receptor crosslinking, thereby enhancing signaling. b, Design of antibody formats used to test the concept: OATmab_mTNFα, OATmab, and Tmab. All constructs are based on an affinity-attenuated trastuzumab variant and contain a C-terminal peptide tag on the light chain for site-selective drug conjugation . In OATmab_mTNFα, TNFα (orange) within the masked TNFα module was fused to the C-terminus of one heavy chain. One-armed formats were assembled using knob-into-hole Fc heterodimerization mutations. c, Oligomeric states of OATmab and OATmab_mTNFα assessed by analytical SEC before (black) and after (gray) TEV activation. The arrow indicates the TEV-dependent shift in the main peak consistent with homotrimer formation. d, Binding of monomeric and trimeric antibody species to HER2 (left) and TNFR2 (right) measured by ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. e, Binding of monomeric and trimeric antibody species to HER2-positive SK-BR-3 cells measured by cell-based ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. f, Functional activity of monomeric and trimeric antibody species measured in Jurkat-Lucia h4-1BB reporter cells cultured alone (dashed lines) or co-cultured with SK-BR-3 cells (solid lines). Data are shown as mean ± s.d. from n = 3 technical replicates.

Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).

Techniques: Variant Assay, Activation Assay, Construct, Functional Assay, Isolation, Membrane, Drug discovery, Conjugation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Activity Assay, Cell Culture