Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Diagram depicting the culture model used to generate time course samples from healthy human CD4+ memory T cells. Cells were stimulated with CD3/CD28 Dynabeads for 12 days, with fresh beads plus full media change every other day (fresh media alone added on alternate days). Samples were collected on days highlighted in bold. (B) Multiomics workflow (see for details). Experiments were performed in biological triplicate, with proteomics, phosphoproteomics, and metabolomics done on same samples. (C) ELISA-based quantification of secreted cytokines (IL-2, IFN-γ, TNF-α) on days 0, 6, 10, and 12, 3 replicates per time point. (D) Cell counts were taken each time point, and average cell division compared to previous time point calculated. Cell viability remained stable throughout at ~75%–85%. (E) Flow cytometric analysis of inhibitory receptors PD-1, TIM-3, and TIGIT at resting, day 6, and day 12 time points. Data normalized to mode for accurate population comparisons to account for differences in number of cells analyzed. The data underlying can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: Mean fluorescence intensity of PD-1 in CD4+ T cells from both HIV+ and HIV- patients (grouped due to observing same effect in both) for CD4+ cells that are CD276+ or FLT-1+. The data underlying this figure can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Fluorescence, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Causal network analysis leveraging annotated interactions (transcription factor-target, kinase-substrate, phosphatase-substrate) as a framework to evaluate protein expression changes (late exhaustion (day 12) versus resting, taking into account previous time point comparisons) to predict key nodes (regulators) impacting most changes seen across the data set. (B) Pruned version of a large subnetwork with p300 (black outline) at main hub. Orange circles mark proteins of interest mentioned in main text. (C) Depiction of experimental design of p300 inhibition studies. (D) Flow cytometric analysis of PD-1 and TIM-3 showing shifts in fluorescent intensity of cell populations in treated versus untreated (cell number normalized to mode). Cell numbers shown for each plot are 20,312 for p300 treated and 30,438 for DMSO, which corresponds to all CD4+ T cells from the sample (excluding doublets and debris, see for gating), corresponding to >95% of sample. (E) Bar chart of mean fluorescent intensity showing significant changes ( T test) in PD-1 and TIM-3 during p300 inhibition versus controls (antigen removed for 2 days, or treatment with T-cell activation inhibitor Dasatinib); *, p -value <0.05, ***, p -value <0.005. (F) Heatmap showing recruitment of cofactors (p300, class I HDACs, KAT2B) to transcription factor family DNA motifs as measured using CASCADE protein-binding microarrays. The data underlying this figure can be found in and Data (A, B), supplemental flow cytometry files uploaded to flowrepositroy.org (D), and the (E, F). Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Expressing, Inhibition, Activation Assay, Protein Binding, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: UMAPs of CD4+ subtypes and CD8+ T exhausted tumor lymphocytes showing gene expression of (A) exhaustion-associated genes, and (B) gene expression of proteins identified in the model presented by Lawton and colleagues. UMAPs made using Single Cell Portal, Tarhan and colleagues. The data from this figure and tool used to make it can be found at https://singlecell.broadinstitute.org/single_cell/study/SCP1039/a-single-cell-and-spatially-resolved-atlas-of-human-breast-cancers . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Gene Expression

Journal: BMC Gastroenterology

Article Title: Inhibition of TLR4 enhances oxaliplatin chemotherapy sensitivity in esophageal squamous cell carcinoma by suppressing inflammation and glycolysis

doi: 10.1186/s12876-026-04663-2

Figure Lengend Snippet: OXA activated TLR4 signaling in ESCC cells. The cells were treated by OXA (25 µM) for 24 h. A , B . The mRNA expressions of TLR4 and MYD88 in ESCC cell lines and normal esophageal cells were detected by qRT-PCR. C . ICC showed the immunocytochemical activity of NF-κB p65, p-NF-κB p65, COX-2, and MYD88. Scale bar: 50 μm. D . qRT-PCR analysis of mRNA levels of IL-1β, IL-6, COX-2, CXCL5, and CXCL8. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group

Article Snippet: Serum concentrations of IL-6 (JL20268, Jonin) and IL-1β (KE10003, Proteintech) were quantified using commercial ELISA kits according to manufacturers’ protocols.

Techniques: Quantitative RT-PCR, Activity Assay, Control

Journal: BMC Gastroenterology

Article Title: Inhibition of TLR4 enhances oxaliplatin chemotherapy sensitivity in esophageal squamous cell carcinoma by suppressing inflammation and glycolysis

doi: 10.1186/s12876-026-04663-2

Figure Lengend Snippet: TLR4 knockout enhances the sensitivity of OXA chemotherapy in ESCC in vivo. A . Number of tumors. B . HE staining of esophageal epithelial tissues. Scale bar: 50 μm. C . Body weight of mice. D , E . the levels of serum IL-1β and IL-6 in mice from different groups were detected by ELISA. F . The immunohistochemical activities of PCNA, CK14, Cyclin D1, COX-2, S100A8 and S100A9 in the esophageal tissue were detected. Scale bar: 100 μm. G , H . The mRNA levels of inflammatory cytokines and glycolysis-related proteins in esophageal tissue were detected by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. WT + 4NQO + OXA group

Article Snippet: Serum concentrations of IL-6 (JL20268, Jonin) and IL-1β (KE10003, Proteintech) were quantified using commercial ELISA kits according to manufacturers’ protocols.

Techniques: Knock-Out, In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Quantitative RT-PCR

Journal: BMC Gastroenterology

Article Title: Inhibition of TLR4 enhances oxaliplatin chemotherapy sensitivity in esophageal squamous cell carcinoma by suppressing inflammation and glycolysis

doi: 10.1186/s12876-026-04663-2

Figure Lengend Snippet: Schematic diagram illustrating the mechanism by which TLR4 inhibition enhances oxaliplatin (OXA) chemosensitivity in esophageal squamous cell carcinoma (ESCC). OXA treatment upregulates TLR4 and its downstream adaptor protein MYD88, which activates the phosphorylation of NF-κB p65. This activation drives two parallel pathways: (1) the inflammatory response, characterized by the upregulation of pro-inflammatory factors such as IL-6, COX-2, and CXCL5; (2) the glycolytic metabolic reprogramming, mediated by the HIF-1α/GLUT1 axis and enhanced expression of glycolytic enzymes including PFKM and LDHB. These two pathways synergistically promote ESCC cell proliferation, migration, and invasion, ultimately reducing OXA chemosensitivity. Inhibition of TLR4 (via genetic knockout, shRNA knockdown, or pharmacological inhibitor TAK-242) or its downstream mediator MYD88 (via shRNA knockdown or inhibitor ST2825) blocks NF-κB p65 phosphorylation, thereby suppressing both the inflammatory response and glycolytic activity. This dual inhibition disrupts the adaptive survival mechanisms of ESCC cells, potentiating the anti-tumor efficacy of OXA

Article Snippet: Serum concentrations of IL-6 (JL20268, Jonin) and IL-1β (KE10003, Proteintech) were quantified using commercial ELISA kits according to manufacturers’ protocols.

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Expressing, Migration, Knock-Out, shRNA, Knockdown, Activity Assay

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of incubation of cell lines with rec-TF and PAR2-activation on PTEN phosphorylation. ( A ) Cells (MDA-MB-231, MCF-7, T47-D, LoVo, CaCo-2, AsPC-1 and Panc-1) were cultured in the recommended media and activated either by the addition of recombinant TF (0–1300 pg/ml) or by incubation with PAR2-agonist peptide (PAR2-AP; SLIGKV; 20 µM) and incubated for the durations shown. The cells were then lysed in electrophoresis-loading buffer and separated on a 12% (w/v) denaturing polyacrylamide gel. The proteins were then transferred to nitrocellulose membrane and blocked with TBST. The membranes were in turn probed using a rabbit anti-human phosphoSer382/Thr382/Thr383-PTEN, a polyclonal rabbit anti-human PTEN antibody, both diluted 1:2000 (v/v), or a goat anti-human GAPDH polyclonal antibody (V-18), diluted 1:4000 (v/v) in TBST. The membranes were then washed with TBST and probed with goat anti-rabbit or donkey anti-goat alkaline phosphatase-conjugated antibodies as required, diluted 1:4000 (v/v), for 90 min. Bands were then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded (micrographs are representative of 6 independent experiments; due to the number of gels the micrographs are cropped to include the main band but also to include at least three marker bands spanning the protein of interest. Full micrograph replicates are included in the ). The ratio of phospho-PTEN:Total PTEN were determined in the cell samples treated with ( B ) rec-TF (65 pg/ml) for 1 h, and ( C ) PAR2-AP for 30 min (n = 6; *p < 0.05 vs. the respective untreated samples). Similarly, the ratios of phospho-PTEN:Total PTEN were determined in the samples treated with ( D ) rec-TF (130 pg/ml) and ( E ) rec-TF (1300 pg/ml).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Incubation, Activation Assay, Phospho-proteomics, Cell Culture, Recombinant, Electrophoresis, Membrane, Western Blot, Marker

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of incubation of cell lines with rec-TF and PAR2-activation on PTEN activity and Akt inhibition. Cells (2 × 10 5 ) were treated with ( A ) rec-TF (65 pg/ml) for 1 h, or ( B ) PAR2-AP for 30 min, lysed in PhosphoSafe buffer (150 µl) and the lipid-phosphatase activity of PTEN measured using the echelon PTEN Activity ELISA kit (n = 3; *p < 0.05 vs. the respective untreated samples). Akt activity was also determined in the samples treated with ( C ) rec-TF or ( D ) PAR2-AP as above, and measured using the Akt Kinase activity assay kit according to manufacturer's instructions (n = 3; *p < 0.05 vs. the respective untreated samples). Samples of MDA-MB-231, LoVo and CaCo-2 cells were pre-incubated with a PAR2 blocking antibody (SAM-11, 20 µg/ml) or a control isotype antibody. The cells were treated with recombinant TF and ( E ) PTEN activity and ( F ) Akt activity measured as above (n = 3; *p < 0.05 vs. the respective control isotype antibody).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Incubation, Activation Assay, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Kinase Assay, Blocking Assay, Control, Recombinant

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: The influence of long-term treatment of cell lines with rec-TF on cellular PTEN antigen levels and Akt activity. Equal number of cells were lysed in PhosphoSafe buffer (150 µl) and ( A ) the level of PTEN antigen measured using a PTEN ELISA kit (n = 5; *p < 0.05 vs. the samples on first day 0). ( B ) The Akt activity was also measured using the Akt Kinase activity assay kit (n = 3; *p < 0.05 vs. the samples on first day 0). Cells (2 × 10 5 ) were cultured in the recommended media and supplemented every 2 days with rec-TF (65 pg/ml) up to 14 days. ( C ) The number of cells in the treated and untreated samples were determined using crystal violet staining (n = 3; *p < 0.05 vs. the samples on first day 0). In addition, cells were lysed in PhosphoSafe buffer and ( D ) the level of PTEN antigen and ( E ) the Akt kinase activity measured. (n = 3; *p < 0.05 vs. the samples on first day 0).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Cell Culture, Staining

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: Analysis of the proximity of PTEN and MAGI1-3 by proximity ligation assay and the influence of PAR2 activation. MDA-MB-231 cells (10 3 ) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min. The proximity between PTEN and MAGI1-3 were examined using a mouse anti-human PTEN (217702) diluted 1:100 (v/v) together with a rabbit anti-MAGI1 antibody (H-70; 2 μg/ml), a rabbit anti-MAGI2 antibody (1 μg/ml) and a rabbit anti-MAGI3 antibody (1 μg/ml). The cells were then labelled with DAPI (2 μg/ml) and Phalloidin-FITC (2 µg/ml). Images were acquired using a Ziess Axio Vert.A1 inverted fluorescence microscope with a ×40 magnification (the micrographs are representative of 10 fields of view from 4 independent experiments RED = PLA incidences; GREEN = Phalloidin; BLUE = DAPI).

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Proximity Ligation Assay, Activation Assay, Incubation, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

doi: 10.1038/s41598-020-77963-6

Figure Lengend Snippet: Analysis of the interaction of PTEN and MAGI1-3 and the influence of PAR2 activation. MDA-MB-231 (10 3 ) were seeded out into 35 mm-glass based μ-dishes and adapted to serum-free medium for 1 h prior to activation. The cells were then incubated with PAR2-AP (20 μM) for up to 30 min and analysed by PLA as described in Fig. . The number of red fluorescent events and nuclei were determined using ImageJ, in 10 fields of view from each assay for ( A ) the interactions of PTEN with MAGI1-3 in non-activated and at 20 min post-activation. ( B ) In addition, the interaction of PTEN and MAGI1 and 2, was measured at intervals up to 30 min (n = 3; *p < 0.05 vs. the non-activated sample). MAGI2 was immunoprecipitated from cell lysates with an anti-MAGI2 (C3; 4 µg) antibody using protein A-magnetic beads. The MDA-MB-231 lysate samples were washed five times with PBST (1 ml) and denatured in SDS-PAGE loading buffer and ( C ) examined for PTEN and MAGI2 by western blot using a mouse anti-PTEN antibody (217702) and a rabbit anti-MAGI2 antibody. ( D ) The ratio of the PTEN band densities were normalised against those of MAGI2 in the same co-immunprecipitated samples (n = 3; *p < 0.05 vs. the non-activated sample). Similarly, the LoVo lysate samples were used to immunoprecipitate MAGI2 and ( E ) examined for PTEN and MAGI2 by western blot using a mouse anti-PTEN antibody (217702) and a rabbit anti-MAGI2 antibody. ( F ) The ratio of the PTEN band densities were normalised against those of MAGI2 in the same co-immunprecipitated samples (n = 3; *p < 0.05 vs. the non-activated sample). Full micrograph replicates are included in the s .

Article Snippet: The protein concentration in the lysates was determined using the Bradford protein estimation assay and the amount of cellular PTEN was measured in lysates (adjusted to 50 μl) using the Human PTEN ELISA kit (Abcam, Cambridge, UK).

Techniques: Activation Assay, Incubation, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: Hypoxia increased the production of S100 calcium-binding protein A8 (S100A8) in neuron and microglia and induced the release of S100A8 in SH-SY5Y cells. ( A , B ) S100A8 expression (red) were detected by immunocytochemical analysis in primary cultured neurons (NeuN, neuron marker) and cultured mixed glia (Iba1, microglial marker and GFAP, astrocyte marker) exposed to hypoxic conditions for 48 h. Scheme 25 μm. S100A8 expression was detected by western blot analysis in ( C , D ) SH-SY5Y cells and ( E , F ) BV-2 cells exposed to hypoxic conditions for 48 h. ( G , H ) S100A8 protein expression in BV-2 cells were confirmed by immunocytochemistry and ( I ) S100A8 release in SH-SY5Y was measured by enzyme-linked immunosorbent assay (ELISA) at 48 h after hypoxia. Values of * p < 0.05, ** p < 0.01, *** p < 0.001 versus control were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Cell Culture, Marker, Western Blot, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces pro-inflammatory cytokines and inflammation in BV-2 cells. BV-2 cells were stimulated with S100A8 (10 μg/mL) for 24 h. ( A ) The supernatant was collected and TNF-α and interleukin-6 (IL-6) analyzed by ELISA. ( B ) The protein and mRNA were extracted, and the expression levels of IL-1β were assessed by ELISA and RT-qPCR. ( C – E ) The protein was extracted, separated on 10% SDS-acrylamide gels (15 μg/lane) and transferred to nitrocellulose membrane. The protein expression level was detected by western blotting with anti-ERK1/2, anti-phospho-ERK1/2 (p-ERK1/2), anti-JNK and anti-p-JNK. ( F ) Cells were pre-treated with ERK inhibitor (PD98059, 20 μM), JNK inhibitor (SP600125, 10 μM) or the equivalent volume of DMSO for 1 h, then stimulated for 24 h with LPS or S100A8 for ELISA of TNF-α, IL-6. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, *** p < 0.001 versus control; ### p < 0.001 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Membrane, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces inflammasome priming by toll-like receptor (TLR)-4 receptors associated with ERK and JNK pathway in BV-2 cells. BV-2 cells were incubated for 24 h with LPS (1 μg/mL) or S100A8 (10 μg/mL) followed by Adenosine 5′-triphosphate disodium salt hydrate (ATP) (1 mM) for 1 h. ( A , B ) The NLRP3, ASC, and ( C , D ) cleaved caspase-1 were detected by western blotting. β-actin was used as an internal control. ( E , F ) BV-2 cells were lysed to whole lysates and IκB-α phosphorylation was analyzed by western blotting. ( G , H ) The translocation of nuclear factor- κB (NF-κB) was also detected by western blotting. BV-2 cells were lysed to cytosolic extracts and nucleic extracts. Lamin-B1 was used as internal controls. ( I , J ) BV-2 microglial cells were pre-treated with PD98059 (ERK inhibitor, 20 μM), SP600125 (JNK inhibitor, 10 μM), TAK-202 (TLR4 inhibitor, 10 μg/mL) or an equivalent volume of DMSO and stimulated for 24 h with LPS or S100A8. Cells harvested and lysed in RIPA buffer for western blotting of NLRP3. Results are from one experiment that is representative of at least three others. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Control, Phospho-proteomics, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 derived from neuronal cells induces NLRP3 inflammasome priming in microglia under hypoxic conditions. BV-2 cells were pre-treated with TAK-202 (TLR4 inhibitor, 10 μg/mL) for 1 h, then stimulated for 48 h in hypoxic condition with SH-SY5Y cells indirectly co-cultured in 0.4 μm pore transwell. ( A ) The protein expression level was detected by western blotting with NLRP3. β-actin was used as an internal control. ( B ) Quantitative analysis of NLRP3 levels. Data from three independent experiments are presented as the means ± S.D. Values of ** p < 0.01 versus control; # p < 0.05 versus co-cultured sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces apoptosis of neuronal cells in hypoxic condition. ( A , B ) SH-SY5Y cells incubated without or with S100A8 KD BV-2 cells for 48 h in hypoxic condition. Cleaved caspase-3 immunofluorescence images and were detected and quantitative analysis of the number of cleaved-caspase3-positive cells are shown in lower panel. ( C , D ) Representative Annexin-V/PI images were detected by flow cytometry. Quantitative analysis of the apoptotic rate of SH-SY5Y cells are shown in lower panel. ( E , F ) Primary neuron-glial mixed cells were transfected with S100A8 shRNA vector for 24 h followed by 48 h in hypoxic condition. Cells were harvested, and the expression protein levels of S100A8 and cleaved caspase-3 were analyzed by Western blotting. Data from three independent experiments are presented as the means ± S.D. Values of *** p < 0.001 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Immunofluorescence, Flow Cytometry, Transfection, shRNA, Plasmid Preparation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces the Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE 2) pathway. BV-2 cells were transfected with S100A8 shRNA or Scramble vector. After 24 h, cells were incubated in hypoxic condition for 48 h. ( A ) The mRNA and ( B ) the protein levels of S100A8 and COX-2 were detected by real-time PCR and western blotting. ( C ) Secretion of PGE 2 level analyzed by ELISA. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Cysteine‐rich protein 61 (Cyr61) levels are upregulated in plasma and bone marrow ( BM ) samples from patients with CML . A, Left panel: Levels of Cyr61 in the plasma from CML patients (n = 36) and normal plasma from age‐matched healthy individuals ( CON ; n = 66) were detected by ELISA . Right panel: Levels of Cyr61 in the BM supernatant from CML patients (n = 33) and the normal BM supernatant from age‐matched healthy transplant donors (n = 11) were detected by ELISA . B, Levels of Cyr61 in the plasma from CML patients in blast crisis ( BC ) (n = 5) and in chronic phase ( CP ) (n = 31) were detected by ELISA . Right panel: Levels of Cyr61 in the marrow from CML patients in BC (n = 4) and in CP (n = 29) were detected by ELISA . C, Relative levels of Cyr61 mRNA in a T acute lymphoblastic leukemia ( ALL ) cell line (Jurkat), B ALL cell line (Nalm‐6), and CML cell line (K562) were detected by qRT ‐ PCR , and the level of Cyr61 mRNA in Nalm‐6 cells was taken as the control to calculate the relative expression of Cyr61 in Jurkat and K562 cells. D, Levels of Cyr61 protein in Jurkat, Nalm‐6, and K562 cells were detected by western blotting. Band intensity of Cyr61 was quantified by densitometry and normalized to GAPDH . E, Concentration of Cyr61 in the culture supernatant of Jurkat, Nalm‐6, and K562 cells was detected by ELISA . Data represent mean ± SEM of at least 3 independent experiments. *P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Expressing, Western Blot, Concentration Assay

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Role of cysteine‐rich protein 61 (Cyr61) in the chemosensitivity of CML cells to imatinib mesylate ( IM ). A, K562 cells were treated with Cyr61 (125, 250, 500, 1000 ng/mL) for 24 h, and then treated with 0.5 μmol/L IM for 24 h; the percentages of apoptotic K562 cells were determined by flow cytometric analysis. Average percentage of apoptotic cells is shown. B, K562 cells were collected, incubated with Cyr61 (1000 ng/ mL ) preincubated with the antihuman Cyr61 093G9 monoclonal antibody (5000 pg/ mL ) or murine isotype‐matched antibody (Con‐IgG) (5000 pg/ mL ), and then treated with 0.5 μmol/L IM for 24 h. Cell apoptosis was determined by flow cytometric analysis. C, Cyr61 knockdown by shCyr61 or sh NC (negative control) in K562 cells. Endogenous Cyr61 expression is shown in the upper panel, whereas the secreted Cyr61 level in culture medium was determined by ELISA and shown in the lower panel. D, Ratio of apoptotic K562‐shCyr61 and K562‐sh NC cells was determined by flow cytometry at 24 h post‐treatment with or without 0.5 μmol/L IM . E, K562 cells were incubated with BM supernatants from a mixture of different CML patients (Cyr61 concentration was 243 pg/ mL ) with preincubation with 1000 pg/ mL 093G9 antibody or murine isotype‐matched antibody (Con‐IgG) for 2 h, and then treated with 0.5 μmol/L IM for 24 h. F, Human CML cell line KCL 22 cells were treated with Cyr61 (1000 ng/mL) for 24 h and then treated with 0.5 μmol/L IM for 24 h; the percentages of apoptotic cells were determined by flow cytometric analysis. G, Primary leukemic cells from three patients with CP CML were isolated and treated with exogenous recombinant human Cyr61 (1000 ng/mL) for 24 h, and then treated with 0.5 μmol/L IM for 24 h. Data represent mean ± SEM of at least 3 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Incubation, Knockdown, Negative Control, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay, Isolation, Recombinant

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Cysteine‐rich protein 61 (Cyr61) activates Bcl‐2 transcription in CML cells. A, Left panel: Bcl‐2, Bcl‐ xL , XIAP and Survivin mRNA expression in K562 cells treated by 1000 ng/mL Cyr61 for 8 h was detected by real‐time PCR . Right panel: Bcl‐2, Bcl‐ xL , XIAP and Survivin mRNA expression in K562‐shCyr61 cells and K562‐sh NC cells was detected by real‐time PCR . B, Left panel: Bcl‐2 protein in K562 cells stimulated by 1000 ng/mL Cyr61 for 48 h was detected by western blotting. Right panel: Bcl‐2 protein in K562‐shCyr61 cells and K562‐sh NC cells was detected by western blotting. The band intensity of Bcl‐2 was quantified by densitometry and normalized to GAPDH . C, K562 cells were treated with Cyr61 (1000 ng/mL), ABT 199 (1 μmol/L) (specific Bcl‐2 inhibitor), Cyr61 + ABT 199, or ABT 199 for 24 h, and then treated with 0.5 μmol/L imatinib mesylate ( IM ) for 24 h. Percentages of apoptotic K562 cells were determined by flow cytometric analysis. Data represent the mean ± SEM of at least 3 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Cysteine‐rich protein 61 (Cyr61) inhibits imatinib mesylate ( IM )‐induced apoptosis through the nuclear factor kappa B ( NF ‐κB) signaling pathway. A, Effect of the inhibitors of signaling pathways on Cyr61 decreased CML cell apoptosis induced by IM . K562 cells were treated with 20 μmol/L LY 294002, 1 μmol/L PD 98059 or 4 μmol/L PDTC in combination with Cyr61 (1000 ng/mL) for 24 h and then treated with 0.5 μmol/L IM for 24 h; the percentages of apoptotic K562 cells were determined by flow cytometric analysis. B, NF ‐κB phosphorylation was detected by western blotting. Lane 1: stimulation of K562 cells with 0.5 μmol/L IM for 10 min; lane 2: stimulation of K562 cells with 1000 ng/ mL Cyr61 + 0.5 μmol/L IM for 10 min. C, K562 cells were treated with 1000 ng/ mL Cyr61 in combination with or without 4 μmol/L PDTC for 24 h, and then treated with 0.5 μmol/L IM for 24 h. Protein levels of Bcl‐2 in K562 cells were detected by western blotting. D, K562‐shCyr61 cells and K562‐sh NC cells were treated with 0.5 μmol/L IM for 24 h. Left panel: NF ‐κB phosphorylation was detected by western blotting. Right panel: Bcl‐2 protein levels in K562 cells were detected by western blotting. Band intensity of Bcl‐2 was quantified by densitometry and normalized to GAPDH . Data represent the mean ± SEM of at least 3 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Inhibition of cysteine‐rich protein 61 (Cyr61) restores the chemosensitivity of CML cells to imatinib mesylate ( IM ) in vivo. NOD / SCID mice bearing s.c. K562‐shCyr61 or control K562‐sh NC cell xenografts (n = 6) were injected i.p. with IM or normal saline ( NS ) daily from 10 d after inoculation with 1.0 × 10 7 tumor cells for 20 d, and then the mice were killed. A, Representative images of tumors are shown. B, Tumor weight is shown. C, Average percentage of tumor volume is shown. * P < 0.05

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Inhibition, In Vivo, Control, Injection, Saline

Journal: Cancer Science

Article Title: Cysteine‐rich protein 61 regulates the chemosensitivity of chronic myeloid leukemia to imatinib mesylate through the nuclear factor kappa B/Bcl‐2 pathway

doi: 10.1111/cas.14083

Figure Lengend Snippet: Proposed signaling pathway by which cysteine‐rich protein 61 (Cyr61) reduces imatinib mesylate ( IM )‐induced CML cell apoptosis. Increased Cyr61 in the bone marrow from CML patients stimulates nuclear factor kappa B ( NF ‐κB) phosphorylation, then upregulates Bcl‐2 production, finally leading to decrease of IM ‐induced CML cell apoptosis and insensitivity to IM

Article Snippet: Concentrations of Cyr61 in the plasma and BM from CML patients were quantitated using the human Cyr61 ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: A multivalent peptide-polymer conjugate material mimics STING to therapeutically activate innate immune signaling

doi: 10.64898/2026.03.24.712780

Figure Lengend Snippet: ( A ) HEK293T reporter cells were treated with 6.0 μg/mL STING or Scr conjugate using TransIT-X2 as a vehicle, cells were imaged by confocal microscopy at 6 h post treatment to examine conjugate colocalization with target TBK1 (representative of N = 3 biological replicates). Scale bar is 10 μm. ( B ) HEK293T reporter cells were treated with 8.3 μg/mL STING or Scr conjugate using TransIT-X2 as a vehicle, Western blot was performed at 6 h post treatment to examine TBK1 and IRF3 phosphorylation (representative of N = 3 biological replicates). ( C ) IRF3 reporter signal relative to buffer treatment for HEK293T reporter cells pretreated for 6 h with TBK1 inhibitor MRT67307 (TBK1i) and then treated with 8.3 μg/mL STING or Scr conjugate delivered using TransIT-X2, measured 24 h post treatment (N = 3 biological replicates). ( D ) Western blot of STING and β-actin expression in ovarian cancer cell lines KURAMOCHI and A2780. ( E-F ) KURAMOCHI and A2780 ovarian cancer cell lines were treated with 5.0 μg/mL STING or Scr conjugate using TransIT-X2 as a vehicle or 50 μM STING agonist ADU-S100. ( E ) CXCL10 and ( F ) IFN-β in supernatant was measured by ELISA 24 h post treatment (N = 3 biological replicates). Replicates where analyte was below the limit of detection (LOD) are labeled as not detected (ND), no summary statistics were computed if any replicate was ND. ( G-I ) KURAMOCHI cells were treated with 5.0 μg/mL STING or Scr conjugate using TransIT-X2 as a vehicle or 50 μM ADU-S100, mRNA sequencing was performed at 6 h post treatment (N = 4 biological replicates). ( G ) Plot of Log2 fold change of STING conjugate or ADU-S100 treatment compared to Buffer, showing high correlation between treatments. Plot of Log2 fold change of Scr conjugate or ADU-S100 treatment compared to Buffer is displayed below as a control, showing greatly reduced correlation. The coefficient of determination R 2 for line of best fit is displayed. ( H ) Gene set enrichment analysis was performed on MSigDB Hallmark gene set, normalized enrichment and adjusted P value are displayed for the 10 gene sets significantly enriched ( P < .05) when comparing STING to Scr conjugate. Normalized enrichment and adjusted P values for the same 10 gene sets are displayed for the comparison of ADU-S100 to Buffer ( I ) Heatmap of gene expression for selected genes. Replicates where a given gene was not detected are labeled ND. Data represented as geometric mean ± SD.

Article Snippet: DuoSet ELISA kits (R&D Systems) for human CXCL10 (#DY266), human IFN-β (#DY814), mouse CXCL10 (#DY466), mouse IFN-β (#DY8234), mouse IL-6 (#DY406), mouse TNF-α (#DY410), and mouse IFN-γ (#DY485) were used with 1-Step TMB ELISA Substrate Solution (Thermo Scientific) following vendor instructions.

Techniques: Confocal Microscopy, Western Blot, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Labeling, Sequencing, Control, Comparison, Gene Expression

Journal: bioRxiv

Article Title: A multivalent peptide-polymer conjugate material mimics STING to therapeutically activate innate immune signaling

doi: 10.64898/2026.03.24.712780

Figure Lengend Snippet: ( A-B ) Mice were dosed with 20 μg of STING conjugate delivered by LNP IP and serum was collected at 0, 1, 3, 6, 10, 24, and 50 h. Serum was analyzed to measure ( A ) STING conjugate concentration by Cy5 fluorescence (showing one phase exponential decay fit to data) and ( B ) CXCL10 concentration by ELISA (N = 3 mice). Conditions where analyte was below the limit of detection (LOD) are labeled as not detected (ND). ( C ) Mice were inoculated with 3×10 6 BPPNM cells IP and dosed with 20 μg of STING or Scr conjugate (N = 3 mice) delivered by LNP IP at 14 days after inoculation. ( D ) Omental tumor, ( E ) ascites, and ( F ) serum were collected 6 h after dosing. Concentrations of CXCL10, IFN-β, IL-6, TNF-α, and IFN-γ were measured by ELISA and are reported relative to total protein concentration in tumor and ascites ( D-E ) or relative to volume in serum ( F ). Conditions where analyte was below the LOD are labeled as ND. Average fold change increases in cytokine concentration for STING conjugate treatment compared to Scr conjugate are displayed. Where cytokine level was ND, a lower bound on the fold change was computed by setting all ND replicates as the LOD. Data represented as mean ± SD.

Article Snippet: DuoSet ELISA kits (R&D Systems) for human CXCL10 (#DY266), human IFN-β (#DY814), mouse CXCL10 (#DY466), mouse IFN-β (#DY8234), mouse IL-6 (#DY406), mouse TNF-α (#DY410), and mouse IFN-γ (#DY485) were used with 1-Step TMB ELISA Substrate Solution (Thermo Scientific) following vendor instructions.

Techniques: Concentration Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Protein Concentration