Journal: Journal of Neurochemistry

Article Title: Repeated Low‐Level Inflammatory Challenge Leads to Alterations in the TNF ‐ CXCL10 Signalling Pathway in Mouse Cerebral Endothelial Cells In Vitro

doi: 10.1111/jnc.70130

Figure Lengend Snippet: Repeated administration of a low concentration of TNF modifies the expression of genes associated with endothelial cell activation and the TNF‐CXCL10 pathway. qRT‐PCR was performed to quantify expression of the genes indicated in bEnd.3 cells in four treatment groups: Control (PBS), a single low concentration of Tumour necrosis factor (TNF) (0.5 ng/mL) for 1 h, a single cumulative concentration of TNF (2.0 ng/mL) for 1 h or repeated treatment with TNF at a low concentration (0.5 ng/mL) for 1 h on 4 consecutive days. Expression of Intercellular Adhesion Molecule 1 ( Icam1 ) (a), C‐X‐C Motif Chemokine Ligand 10 (Cxcl10) (b), Tumour Necrosis Factor (Tnf) (c), TNF Receptor‐Associated Factor 2 (Traf2) (d), Interferon Gamma (Ifng) (e) and C‐X‐C Chemokine Receptor 3 (Cxcr3) (f) was quantified 4 h and 24 h post final treatment. Data were analysed using one‐way ANOVA, followed by Tukey's post hoc test. Biological replicates ( n = 6) are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001. ns = not statistically significant.

Article Snippet: The effects of exogenous CXCL10, a pro‐inflammatory chemokine, were determined by exposing cells to recombinant mouse CXCL10 (R&D Systems; cat. no #466‐CR) or vehicle alone (PBS) for 24 h. For experiments involving repeated inflammatory challenge, cells were plated in 6‐ or 12‐well plates at a seeding density of 1 × 10 5 or 5 × 10 4 cells per well, respectively.

Techniques: Concentration Assay, Expressing, Activation Assay, Quantitative RT-PCR, Control

Journal: Journal of Neurochemistry

Article Title: Repeated Low‐Level Inflammatory Challenge Leads to Alterations in the TNF ‐ CXCL10 Signalling Pathway in Mouse Cerebral Endothelial Cells In Vitro

doi: 10.1111/jnc.70130

Figure Lengend Snippet: Repeated inflammatory challenge with TNF upregulates key proteins within the TNF‐CXCL10 pathway. bEnd.3 cells were exposed to control (phosphate buffered saline; PBS), a single low concentration of Tumour necrosis factor (TNF) (0.5 ng/mL), a single cumulative concentration of TNF (2.0 ng/mL) or repeated treatment with a low TNF concentration (0.5 ng/mL) for 1 h for 4 days. Intercellular Adhesion Molecule 1 (ICAM1) (a), C‐X‐C Motif Chemokine Ligand 10 (CXCL10) (b, c), Signal Transducer and Activator of Transcription 1 (STAT1) (d) and Phosphorylated STAT1 (pSTAT1) (e) expression were evaluated by western blotting at both 4 and 24 h post final treatment. Secreted CXCL10 protein was quantified using ELISA at both time points (c). Blots were run on the same gels and membranes, cut according to molecular weight and probed separately; therefore, the same GAPDH loading control is shown for some of these targets. Data were analysed using one‐way ANOVA, followed by Tukey's post hoc test. Biological replicates ( n = 4) are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001.

Article Snippet: The effects of exogenous CXCL10, a pro‐inflammatory chemokine, were determined by exposing cells to recombinant mouse CXCL10 (R&D Systems; cat. no #466‐CR) or vehicle alone (PBS) for 24 h. For experiments involving repeated inflammatory challenge, cells were plated in 6‐ or 12‐well plates at a seeding density of 1 × 10 5 or 5 × 10 4 cells per well, respectively.

Techniques: Control, Saline, Concentration Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Molecular Weight

Journal: Journal of Neurochemistry

Article Title: Repeated Low‐Level Inflammatory Challenge Leads to Alterations in the TNF ‐ CXCL10 Signalling Pathway in Mouse Cerebral Endothelial Cells In Vitro

doi: 10.1111/jnc.70130

Figure Lengend Snippet: CXCL10 influences apoptosis and proliferation of bEnd.3 cells. (a) bEnd.3 cells were treated for 24 h with the indicated C‐X‐C Motif Chemokine Ligand 10 (CXCL10) concentrations and caspase 3/7 activity measured, with percentage apoptosis calculated from the number of fluorescent cells relative to total cell number. (b) Cell nuclei were stained with Hoechst following CXCL10 treatment, and cell number was quantified and normalised to control. (c–h) Cells treated with CXCL10‐targeting small interfering RNA (siRNA) or scrambled control siRNA were repeatedly exposed to Tumour necrosis factor (TNF) (0.5 ng/mL). Gene and protein expression of CXCL10 and ICAM1 was measured using RT‐qPCR and western blotting. (i) Representative light and fluorescent images (10× magnification) of cells treated with either CXCL10‐targeting siRNA or scrambled control siRNA and subsequently exposed to TNF (0.5 ng/mL), showing apoptotic (green) cells. Scale bar = 300 μm. (j) Representative images of Hoechst staining (blue) for quantification of cell number in the same treated cells. Scale bar = 750 μm. (k) The number of apoptotic cells per image was calculated as a percentage of total cell number. (l) The mean cell number was calculated for each treatment group. Data were analysed using one‐way ANOVA, followed by Tukey's post hoc test. Biological replicates ( n = 4–6) are shown as mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 and **** p < 0.0001.

Article Snippet: The effects of exogenous CXCL10, a pro‐inflammatory chemokine, were determined by exposing cells to recombinant mouse CXCL10 (R&D Systems; cat. no #466‐CR) or vehicle alone (PBS) for 24 h. For experiments involving repeated inflammatory challenge, cells were plated in 6‐ or 12‐well plates at a seeding density of 1 × 10 5 or 5 × 10 4 cells per well, respectively.

Techniques: Activity Assay, Staining, Control, Small Interfering RNA, Expressing, Quantitative RT-PCR, Western Blot

Journal: Clinical Cancer Research

Article Title: XMT-2056, a HER2-Directed STING Agonist Antibody–Drug Conjugate, Induces Innate Antitumor Immune Responses by Acting on Cancer Cells and Tumor-Resident Immune Cells

doi: 10.1158/1078-0432.CCR-24-2449

Figure Lengend Snippet: XMT-2056 elicits potent antigen-dependent and Fc-R–mediated activation of monocytes in cocultures. A, Schematic of the XMT-2056 mechanism, which includes HER2-dependent ADC uptake into tumor cells and Fcγ-R–expressing myeloid cells. B, Chemical structure of XMT-2056. C, Cytokine induction as measured by a multiplex Luminex assay from supernatants of fresh human WBCs treated for 6 (IFN-β) or 24 (CXCL10, IL-6, and TNF-a) hours. Bars represent mean value of n = 2 data points shown as symbols. D, Competition with trastuzumab by biolayer interferometry (Octet): trastuzumab was loaded onto the sensor chip, and HER2 ECD or HT19 antibody associations are indicated by blue arrows. Additional binding by HT19 indicates noncompetitive binding. E, Competition with trastuzumab by cell-based flow cytometry. HT19 hIgG1 or mIgG2a formats detected with either Alexa Fluor anti-hIgG1 (h647) or Alexa Fluor anti-mIgG2a (m647) secondary antibodies in the presence or absence of trastuzumab–mIgG2a. Each point represents the mean and SD ( n = 3). F, Binding of XMT-2056 or HT19 antibody to HCC1954 cells showing fluorescence intensities measured by flow cytometry. Each point represents the mean and SD ( n = 2). G, CXCL10 cytokine induction in HCC1954 monocultures treated for 24 hours with XMT-2056, nonbinding control ADC, HT19, or the free payload. Each point represents the mean and SD ( n = 2). H, Binding of XMT-2056 or Fc-mutant XMT-2056, HT19, and nonbinding control ADC to SKOV3 cells showing fluorescence intensities measured by flow cytometry. Each point represents the mean and SD ( n = 3). I and J, IRF3 reporter activity of THP1 cells in coculture with SKOV3 cells ( I ) or cultured on recombinant HER2 antigen-coated plates ( J ) treated for 24 hours with XMT-2056 or Fc-mutant XMT-2056, nonbinding control ADC, or STING agonist payload. Each point represents the mean and SD ( n = 3). When noted, EC 50 and B max values represent mean of two independent experiments. gMFI, geometric mean fluorescence intensity; RLU, relative light units. ( A, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/s16c825 ; G, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/s16c825 ; I and J, Created in BioRender. Cetinbas, N. [2025], https://BioRender.com/s16c825 .)

Article Snippet: Culture supernatants were collected, and CXCL10 levels were measured by ELISA using Mouse CXCL10/IP-10 DuoSet ELISA Kit (R&D Systems, Cat. DY466) following the manufacturer’s recommended procedures.

Techniques: Activation Assay, Expressing, Multiplex Assay, Luminex, Binding Assay, Flow Cytometry, Fluorescence, Control, Mutagenesis, Activity Assay, Cell Culture, Recombinant