human colon adenocarcinoma cell line  (ATCC)

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Adaptable expression ratios of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 3xHA -P2A-K n -MCT12 2xmyc construct. (B) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (C) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 5). Data are represented as mean ± SEM. (D) Immunoblot analysis of CRT1 3xHA -P2A-K n -MCT12 2xmyc . (E) Ratio of MCT12 2xmyc and CRT1 3xHA expression as determined by immunoblotting ( n = 4). Data are represented as mean ± SEM. See also .

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Western Blot, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet: Relative cell surface expression of the creatine transporters CRT1 and MCT12 in HEK293 SLC6A8 −/− cells (A) Scheme of the CRT1 eGFP -P2A-K 0 -MCT12 mCherry (a) and MCT12 mCherry -P2A-K 0 -CRT1 eGFP construct (b). (B) Ratio of mCherry and eGFP expression in eGFP-P2A-K 0 -mCherry (K 0 , as a control), CRT1 eGFP -P2A-K 0 -MCT12 mCherry , and MCT12 mCherry -P2A-K 0 -CRT1 eGFP constructs as determined by flow cytometry ( n = 6). Data are represented as mean ± SEM. (C) Live-cell confocal microscopy of HEK293 SLC6A8 −/− cells expressing CRT1 eGFP -P2A-K 0 -MCT12 mCherry and MCT12 mCherry -P2A-K 0 -CRT1 eGFP . The plasma membrane was stained with CellMask Deep Red. Scale bars, 20 μm. (D) Analysis of cell surface expression of CRT1 eGFP and MCT12 mCherry . n = 30 (CRT1 eGFP -P2A-K 0 -MCT12 mCherry ); n = 28 (MCT12 mCherry -P2A-K 0 -CRT1 eGFP ).

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Expressing, Construct, Control, Flow Cytometry, Confocal, Microscopy, Clinical Proteomics, Membrane, Staining, Analysis

Journal: iScience

Article Title: A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by MCT12

doi: 10.1016/j.isci.2026.117719

Figure Lengend Snippet:

Article Snippet: Human: HEK293 Lenti-X , Takara , Cat # 632180.

Techniques: Cell-Signaling, Virus, Recombinant, Clinical Proteomics, Membrane, Staining, Cloning, Software

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Expression of NOD2 is elevated in the lung tissue of patients with COPD The expression of NOD2 was evaluated in lung tissue samples from healthy subjects and patients with COPD. ( A ) Schematic diagram of the experimental workflow for analyzing human lung tissue. ( B ) Quantitative real-time PCR (qRT-PCR) analysis of NOD2 mRNA. ( C ) Representative images of immunohistochemical (IHC) staining for NOD2 protein. ( D ) Quantification of IHC staining. ( E ) Representative images of fluorescence in situ hybridization (FISH) for NOD2 mRNA (green), with nuclei counterstained with DAPI (blue). ( F ) Quantification of FISH signal. ( G ) Representative multicolor immunofluorescence images showing CD66b-positive neutrophils (pink) and NOD2 (green), with nuclei counterstained with DAPI (blue). Enlarged views of the boxed regions are shown on the right. Scale bars: 20 μm. ( H ) Immunoblotting analysis and quantification of NOD2 protein levels in differentiated human HL-60-derived neutrophil-like cells and primary mouse neutrophils following CSE stimulation for 0, 2, 4, or 8 h, with untreated cells serving as the 0-h controls. ( I) qRT-PCR analysis of NOD2 mRNA expression in the corresponding human and mouse neutrophil models. Data are presented as mean ± SD. Data in B, D, and F were analyzed using an unpaired two-tailed Student’s t-test; data in H and I were analyzed separately for each cell model using one-way ANOVA with Tukey’s test. ** p < 0.01 versus the corresponding healthy-subject or CSE 0-h control group

Article Snippet: The human promyelocytic leukemia cell line HL-60 (American Type Culture Collection (ATCC), Manassas, VA, USA; catalog no. CCL-240) was maintained in Iscove’s Modified Dulbecco’s Medium (IMDM), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS).

Techniques: Expressing, Tissue, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Analysis, Immunohistochemical staining, Immunohistochemistry, Fluorescence, In Situ Hybridization, Immunofluorescence, Western Blot, Derivative Assay, Two Tailed Test, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Knockdown of NOD2 inhibits CSE-induced NET formation, ROS production, and mitophagy in human neutrophils The role of NOD2 in cigarette smoke extract (CSE)-induced Neutrophil Extracellular Trap (NET) formation, reactive oxygen species (ROS) accumulation, and lysosome–mitochondrion spatial relationships were evaluated in differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the in vitro experimental design and treatment groups. ( B ) Representative DAPI-stained images of extracellular DNA structures and quantification of NET formation based on DAPI-stained nuclear morphology. Scale bars: 20 μm. ( C ) Extracellular DNA levels in cell culture supernatants determined using a PicoGreen dsDNA assay. ( D ) Myeloperoxidase (MPO) levels in cell culture supernatants determined by ELISA. ( E ) Neutrophil elastase (NE) levels in cell culture supernatants determined by ELISA. ( F ) Representative flow-cytometry histograms and quantitative analysis of intracellular ROS levels measured using DCFH-DA staining. ( G ) Representative immunofluorescence images showing the spatial relationship between LAMP1-positive lysosomes (green) and TOM20-positive mitochondria (red), with nuclei counterstained with DAPI (blue). The colocation efficients for LAMP1 and TOM20 was determined by ImageJ and shown on the right. Scale bars: 20 μm. Data are presented as mean ± SD. Quantitative data were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 vs. Control group; ## p < 0.01 vs. CSE + sh-NC group

Article Snippet: The human promyelocytic leukemia cell line HL-60 (American Type Culture Collection (ATCC), Manassas, VA, USA; catalog no. CCL-240) was maintained in Iscove’s Modified Dulbecco’s Medium (IMDM), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS).

Techniques: Knockdown, Derivative Assay, In Vitro, Staining, Morphology, Cell Culture, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Immunofluorescence, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: ZDHHC5-mediated S-palmitoylation is essential for NOD2 stability and signaling The association of ZDHHC5 with NOD2 and the contribution of NOD2 S-palmitoylation to its stability, localization, and downstream signaling were investigated in differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the proposed mechanism by which ZDHHC5-mediated S-palmitoylation promotes NOD2 membrane localization. ( B ) Representative immunofluorescence images showing NOD2 (red) in cells with or without CSE stimulation. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. ( C ) Acyl-biotin exchange (ABE) assay evaluating Flag-NOD2 S-palmitoylation in cells with or without CSE stimulation. HAM, hydroxylamine. ( D ) Immunoblotting analysis and quantification of NOD2 protein levels following treatment with increasing concentrations of the palmitoylation inhibitor 2-bromopalmitate (2BP) or with 100 µM 2BP for the indicated durations. ( E ) Representative immunofluorescence images showing NOD2 (red) in cells treated with vehicle or 2BP. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. ( F ) Co-immunoprecipitation analysis of the association between endogenous ZDHHC5 and NOD2. ( G ) Representative immunofluorescence images showing ZDHHC5 (green) and NOD2 (red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm. ( H ) Schematic of human NOD2 showing its functional domains and the positions of the C395S and C1033S mutations. ( I ) Immunoblotting analysis of wild-type (WT) and mutant Flag-NOD2 in total-cell lysates and membrane fractions during a cycloheximide (CHX) chase, with quantification of the relative NOD2 protein remaining shown on the right. GAPDH and Na + /K + -ATPase were used as loading controls for the total-cell lysates and membrane fractions, respectively. ( J ) ABE assay comparing the S-palmitoylation levels of WT Flag-NOD2, the C395S and C1033S single mutants, and the C395S/C1033S double mutant. ( K ) Immunoblotting analysis and quantification of p65 and p38 phosphorylation in cells expressing wt Flag-NOD2 or the C395S/C1033S double mutant following CSE stimulation. Data are presented as mean ± SD. Data in D and K were analyzed using one-way ANOVA followed by Tukey’s test. Data in I were analyzed using two-way ANOVA followed by Tukey’s test. ** p < 0.01 versus the corresponding 0 µM, 0 h, or wt control group; ## p < 0.01 versus the CSE + Flag-NOD2 wt group

Article Snippet: The human promyelocytic leukemia cell line HL-60 (American Type Culture Collection (ATCC), Manassas, VA, USA; catalog no. CCL-240) was maintained in Iscove’s Modified Dulbecco’s Medium (IMDM), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS).

Techniques: Stability, Derivative Assay, Membrane, Immunofluorescence, Western Blot, Analysis, Co-Immunoprecipitation Assay, Functional Assay, Mutagenesis, Phospho-proteomics, Expressing, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: ZDHHC5 overexpression partially counteracts the effects of NOD2 knockdown in human neutrophil-like cells The ability of ZDHHC5 overexpression to counteract the effects of NOD2 knockdown was evaluated in CSE-stimulated, differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the in vitro experimental design and treatment groups. ( B ) Representative DAPI-stained images of extracellular DNA structures and quantification of NET formation based on DAPI-stained nuclear morphology. Scale bars: 20 μm. ( C ) Extracellular DNA levels in cell culture supernatants determined using a PicoGreen dsDNA assay. ( D ) MPO levels in cell culture supernatants determined by ELISA. ( E ) NE levels in cell culture supernatants determined by ELISA. ( F ) Representative flow cytometry histograms and quantitative analysis of intracellular ROS levels measured using DCFH-DA staining. ( G ) Representative immunofluorescence images showing LAMP1-positive lysosomes (green) and TOM20-positive mitochondria (red), with nuclei counterstained with DAPI (blue). The colocalization coefficients for LAMP1 and TOM20 were determined by ImageJ and shown on the right. Scale bars: 20 μm. Data are presented as mean ± SD. Data were analyzed using two-way ANOVA followed Tukey’s test. ** p < 0.01 versus the CSE + sh-NC + Vector group; ## p < 0.01 versus the CSE + sh-NOD2 + ZDHHC5 group

Article Snippet: The human promyelocytic leukemia cell line HL-60 (American Type Culture Collection (ATCC), Manassas, VA, USA; catalog no. CCL-240) was maintained in Iscove’s Modified Dulbecco’s Medium (IMDM), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS).

Techniques: Over Expression, Knockdown, Derivative Assay, In Vitro, Staining, Morphology, Cell Culture, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Immunofluorescence, Plasmid Preparation

Journal: Advanced Science

Article Title: 3D Bioprinted Blood–Brain–Tumor Barrier Spheroid‐Based In Situ Evaluation of Anticancer Compounds Against Glioblastoma: ROS‐Producing Ruthenium as a Potential Therapeutic Candidate

doi: 10.1002/advs.78150

Figure Lengend Snippet: Cytotoxicity profiling and Transwell permeability assessment of test compounds across hCMEC/D3 and U‐87 MG cell models. (A1–A4) Cell viability of hCMEC/D3 human cerebral microvascular endothelial cells following 24 h treatment with Rh123 (A1), Ru (A2), Osi (A3), or TMZ (A4), assessed by CellTiter‐Glo luminescent cell viability assay. (B1–B3) Cell viability of U‐87 MG glioblastoma cells following 48 h treatment with Ru (B1), Osi (B2), or TMZ (B3), assessed by MTT assay. (C1–C4) Cumulative apical‐to‐basolateral transport profiles of Rh123 (C1), Ru (C2), Osi (C3), and TMZ (C4) across hCMEC/D3 monolayers in the presence (Ver+) or absence (Ver−) of the P‐glycoprotein inhibitor verapamil (Ver, 100 µM), measured at 15‐min intervals over 60 min. (D1–D4) Apparent permeability coefficients (P app ) of Rh123 (D1), Ru (D2), Osi (D3), and TMZ (D4) in the presence (Ver+) or absence (Ver−) of verapamil. ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Rh123, Rhodamine‐123; Ru, ruthenium compound; Osi, osimertinib; TMZ, temozolomide; Ver, verapamil. Data in (A1–A4) and (B1–B3) are presented as mean ± SD of n = 3 replicate wells per concentration seeded in parallel; data in (C1–C4) and (D1–D4) are presented as mean ± SD of n = 3 Transwell inserts seeded in parallel per condition. Statistical significance was assessed by ordinary one‐way ANOVA followed by Tukey's multiple‐comparisons test, using a two‐sided alpha of 0.05. Analyses were performed using GraphPad Prism 11.0.2.

Article Snippet: Human cerebral microvascular endothelial cells (hCMECs/D3; Cedarlane, Canada; RRID: CVCL_0M40) were cultured in Endothelial Growth Medium 2 (EGM2; Lonza Bioscience, Switzerland), containing human epidermal growth factor, hydrocortisone, GA 1000, FBS, vascular endothelial growth factor (VEGF), human fibroblastic growth factor (hFGF‐B), R 3 IGF‐I, ascorbic acid, and heparin (Lonza).

Techniques: Profiling, Permeability, Cell Viability Assay, MTT Assay, Ruthenium, Concentration Assay