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: 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: ImmunoTargets and Therapy

Article Title: Recombinant Antithrombin Alleviated Pulmonary Injury and Inflammation in LPS-Induced ARDS by Inhibiting IL17a/NF-κB Signaling

doi: 10.2147/ITT.S502925

Figure Lengend Snippet: rAT reduced pulmonary inflammation in LPS-induced ARDS model mice. ( A–D ) The levels of inflammatory factors, including IL-6 ( A ), TNF-α ( B ), IL-8 ( C ), and hs-CRP ( D ), were decreased in the serum of the rAT-treated group, as detected by ELISA. ( E ) Immunohistochemical staining for F4/80 revealed that rAT treatment reduced the proportion of macrophages in the lung tissue of LPS-induced ARDS model mice. ( F ) Immunohistochemical staining for MRCI, a marker of M2 macrophages, showed that rAT treatment increased the proportion of M2 macrophages in the lung tissue of LPS-induced ARDS model mice. ( G ) Immunohistochemical staining for Ly6G, a marker of neutrophils, showed that rAT treatment reduced the proportion of neutrophils in the lung tissue of LPS-induced ARDS model mice. All the data are presented as the means ± SDs of three independent experiments. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant; scale bar, 50 μm.

Article Snippet: A mouse CXCL15 ELISA Kit (E-EL-M0269) and a mouse hs-CRP ELISA Kit (E-EL-M0677) were purchased from Elabscience (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Marker

Journal: ImmunoTargets and Therapy

Article Title: Recombinant Antithrombin Alleviated Pulmonary Injury and Inflammation in LPS-Induced ARDS by Inhibiting IL17a/NF-κB Signaling

doi: 10.2147/ITT.S502925

Figure Lengend Snippet: The efficacy of rAT in mitigating lung injury, suppressing the immune response, and inhibiting the activation of the NF-κB signaling pathway in LPS-induced ARDS mice were diminished by the administration of IL-17a. ( A ) ELISA results demonstrated that the administration of IL17a inhibited the ability of rAT to reduce inflammatory factors, including IL-6, TNF-α, and IL-8, in the serum of LPS-induced ARDS mice. ( B ) The analysis of the wet/dry weight ratio of the lung tissue revealed that the administration of IL17a counteracted the ability of rAT to alleviate pulmonary exudation in LPS-induced ARDS mice. ( C ) The administration of IL17a did not significantly affect the ability of rAT to reduce the number of cells in the BALF of LPS-induced ARDS mice. ( D ) The administration of IL17a attenuated the ability of rAT to reduce the concentrations of proteins in the BALF of LPS-induced ARDS mice. ( E ) Real-time PCR results showed that the administration of IL17a blocked the ability of rAT to downregulate the expression of target genes in the IL17a/NF-κB signaling pathway. ( F ) The protein levels of the NF-κB signaling pathway were assessed by Western blotting, and gray intensity analysis of the blots showed that the administration of IL17a in LPS-induced ARDS mice counteracted the ability of rAT to suppress the phosphorylation of IκBα, IKKα/β, and P65. The data are expressed as the means ± SDs (n=3 in each group). One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, and ns not significant.

Article Snippet: A mouse CXCL15 ELISA Kit (E-EL-M0269) and a mouse hs-CRP ELISA Kit (E-EL-M0677) were purchased from Elabscience (Wuhan, China).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: Moreover, phosphorylation levels of calcium/calmodulin-dependent protein kinase II isoforms CAMK2A and CAMK2D at Thr286 (p-CAMK2A, p-CAMK2D) were quantified using the Colorimetric Cell-Based ELISA Kit (CAMK2A/CAMK2D (Phospho-Thr286), Boster Bio, #EKC2366).

Techniques: Expressing, Enzyme-linked Immunosorbent 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: 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

Journal: Drug Design, Development and Therapy

Article Title: Sinomenine Alleviates Rheumatoid Arthritis by Suppressing the PI3K-Akt Signaling Pathway, as Demonstrated Through Network Pharmacology, Molecular Docking, and Experimental Validation

doi: 10.2147/DDDT.S475959

Figure Lengend Snippet: SIN suppressed the release of inflammatory cytokines and inhibited PI3K-Akt signaling pathway in CIA mice. IL-1β ( A ), IL-6 ( B ), and TNF-α ( C ) levels in the serum of mice were determined using ELISA. Western blot detected changes in the phosphorylation of PI3K and AKT in ankle tissues of mouse models ( D – F ). The data are depicted as mean ± SEM (n=5). Symbols *, **, # , and ## Denote significance with P-values < 0.05, < 0.01 compared to the CIA and normal groups, respectively.

Article Snippet: Primary antibodies (GAPDH, PI3K, p-PI3K, AKT, p-AKT), secondary antibodies conjugated with horseradish peroxidase specific for rabbit IgG, and ELISA kits for mouse IL-1 beta, IL-6, and TNF Alpha were sourced from BOSTER Biological Technology, Co., Ltd in Wuhan, China.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A-C) mRNA levels of PPP2R5C are nutritionally regulated in various mouse tissues. Quantitative RT-PCR of PPP2R5C (NCBI splice variant 4) from liver (A), abdominal white adipose tissue (B) and gastrocnemius muscle (C) of control C57BL/6 (“WT”) or diabetic leptin-receptor deficient (“db/db”) 8–10 week old male mice. Mice were first starved for 16 hours (“Fasting”) and then given normal chow diet for 6 hours (“Refed”). Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (n ≥ 3).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Quantitative RT-PCR, Variant Assay, Control

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A-C) Increased glucose uptake/tolerance in mice upon liver-specific knockdown of PPP2R5C. 8–10 week CL57BL/6 male mice tail-injected with adeno-associated virus containing miRNA targeting PPP2R5C (“PPP2R5C KD”) or a scrambled control miRNA (“Control KD”). 7 weeks after knockdown mice were starved for 16 hours (“Fasting”) or starved and then given normal chow diet for 6 hours (“Refed”) prior to sacrificing. Although blood glucose levels are not altered (A), serum insulin levels are significantly reduced (B) compared to controls. (C) Glucose tolerance test performed 4 weeks after virus injection shows significantly improved tolerance in knockdown mice (2g glucose/kg body weight injected intraperitoneally, n = 12). (D) Insulin signaling, detected via AKT and GSK3 beta phosphorylation, does not drop in all feeding regimes in PPP2R5C HepKD livers compared to controls, despite PPP2R5C HepKD serum insulin levels being lower (see panel B). (E) Insulin sensitivity is increased after PPP2R5C knockdown in liver. Control C57BL/6 mice and PPP2R5C HepKD mice were virus-injected as in Fig 2. Four weeks later, mice were fasted for 6 hours, then insulin was injected 1IU/kg and 10 minutes later mice were sacrified and liver samples were taken. (F-G) Glucose consumption and lactate production are increased in Hepa 1–6 cells upon PPP2R5C knockdown. Hepa 1–6 cells infected by adenovirus carrying shRNA targeting all mouse PPP2R5C isoforms (PPP2R5C KD) or a negative-control scramble shRNA (Control KD). After 48h, glucose consumption (F) and lactate production (G) were measured in the medium for 24 hours, and normalized to total cell protein. (n = 3) (H) Glycolytic flux measured as ECAR (extracellular acidification rate) using the glycolysis stress kit from Seahorse Bioscience on the extracellular flux analyser XF96. After addition of glucose to control or PPP2R5C knockdown Hepa 1–6 cells, oligomycin is added to inhibit respiration, thereby boosting glycolytic flux. 2-deoxy-glucose is added to compete with glucose and shut down glycolysis (n = 9). (I) Acute glucose uptake is increased in Hepa 1–6 cells upon PPP2R5C knockdown. Stably transfected Hepa1-6 cell-lines carrying two independent, inducible shRNAs (PPP2R5C KD1 and KD2) were induced with 30 μg/ml cumate for 3 days, starved overnight in serum-free DMEM, and uptake of fluorescent 2-deoxy-glucose analog 2-NBDG was quantified by FACS. (n = 3) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001, †p-value<10 −4 by Wilcoxon signed-rank test (C) or student t-test (B, F-I).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Injection, Virus, Control, Phospho-proteomics, Infection, shRNA, Negative Control, Stable Transfection, Transfection, Significance Assay

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A-C) Liver-specific knockdown of PPP2R5C leads to pro-anabolic changes including increased liver lipid synthesis and secretion and reduced glycogen breakdown. As in , 7 weeks post hepatocyte-specific knockdown, liver weight (A), glycogen (B) and triglycerides (C) were quantified. (n = 5 or 6) (D-E) Cellular triglyceride levels are increased in Hepa 1–6 (D) or mouse primary hepatocytes (E) upon PPP2R5C knockdown. Cells infected as in . (n = 3) ( F-G ) Liver-specific knockdown of PPP2R5C leads to elevated serum VLDL levels. 7 weeks post hepatocyte-specific knockdown, serum triglycerides were quantified either in aggregate (F), or when fractionated by FPLC to resolve lipoprotein particles of various densities (G). (n = 5 or 6) Error bars: std. dev. *p-value<0.05, **p-value<0.01 by one-way ANCOVA with liver NEFA as a covariate (C, “Random”) or student t-test (A-F).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Infection

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) Schematic diagram of “BioID”—proximity-dependent biotinylation of proteins—to identify PPP2R5C substrates interacting transiently with the phosphatase complex. Biotin ligase (BirA) was fused to either the N-terminus (Myc-BirA-PPP2R5C) or C-terminus (PPP2R5C-BirA-HA) of PPP2R5C (NCBI variant 1). As negative controls, only BirA-HA or Myc-BirA were used. A mutation in the active center of the catalytic domain was introduced (PP2A-C D85N) as a substrate-trapping mutation. Biotinylated proteins from Hepa 1–6 cells were purified by cell lysis and streptavidin pulldown, and subsequently detected by immunoblotting. (B-B’) BioID identifies AMPK beta 1, HIF1 α , STAT3 and S6K as PPP2R5C interacting proteins. Proteins interacting with PPP2R5C in vivo in Hepa 1–6 cells were biotinylated and purified as in (A), and probed by immunoblotting. LXR, PPAR alpha, SREBP-1, Actin, and Tubulin were included as negative control proteins which were not detected in the pulldowns. (Lysates shown at same exposure as biotin pulldowns.) (C) AMPK phosphorylation and activity increase upon PPP2R5C knockdown in Hepa 1–6 cells. PPP2R5C was knocked-down using two independent, inducible shRNAs as in . AMPK phosphorylation increases on T172 upon PPP2R5C knockdown. AMPK activity determined via phosphorylation of two substrates, ACC1 and TBC1D1. (D) HIF1α phosphorylation increases upon PPP2R5C knockdown in Hepa 1–6 cells. HIF1α phosphorylation is detected as reduced mobility (up) on a 25 μM PhosTag gel (8% gel), which is abolished upon treatment of cell lysates with calf intestinal phosphatase (CIP) prior to gel electrophoresis (right panel). PPP2R5C knockdown was performed by generating stable cell lines carrying two independent inducible shRNA constructs (KD1 and KD2) to exclude off-target effects. (E) Expression of HIF1 α target genes increases in primary hepatocytes upon PPP2R5C knockdown. (n = 4) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (E).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Variant Assay, Mutagenesis, Purification, Lysis, Western Blot, In Vivo, Negative Control, Phospho-proteomics, Activity Assay, Knockdown, Nucleic Acid Electrophoresis, Stable Transfection, shRNA, Construct, Expressing

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) PPP2R5C knockdown leads to upregulation of genes enriched for PPARA and SREBP-1 targets. Genes either up- or down-regulated upon PPP2R5C knockdown in mouse primary hepatocytes were analyzed using TFactS software to identify transcription factors putatively misregulated upon PPP2R5C knockdown. FDR (False Discovery Rate) rate was controlled using the Benjamini-Hochberg procedure. (B-C) Expression of bona-fide SREBP-1 target genes is increased upon PPP2R5C knockdown in primary hepatocytes in culture (B) or in mouse liver in vivo (C). PPP2R5C was knocked-down in mouse primary hepatocytes using adenovirus and in vivo using adeno-associated virus as in . SREBP-1 target genes quantified by Q-RT-PCR, normalized to TBP. (D) Upon PPP2R5C knockdown in liver, SREBP-1 protein levels are elevated. (E) Expression of ChREBP target genes is increased upon PPP2R5C knockdown in mouse liver in vivo. PPP2R5C was knocked-down in vivo using adeno-associated virus as in . ChREBP target genes quantified by Q-RT-PCR, normalized to TBP. (F) Graphical representation of the metabolic changes induced upon PPP2R5C knockdown in mouse liver. Livers with reduced PPP2R5C have increased glucose uptake, increased TAG synthesis, and increased VLDL secretion. Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (B-C,E) (n = 4 for mouse primary hepatocytes, and 5 or 6 for mouse liver).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Software, Expressing, In Vivo, Virus, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) Expression of PPP2R5C is elevated in livers of diabetic patients. Quantitative RT-PCR of Human PPP2R5C in liver of healthy controls (n = 40) or type 2 diabetic patients (n = 26), normalized to 18S rRNA. (B) Expression of PPP2R5C is elevated in non-diabetics with visceral obesity. People enrolled in the analysis were divided into 3 subgroups according to their adiposity: Lean (n = 12), Subcutaneous (“SC”) Obesity (n = 21) and Visceral (“VIS”) Obesity (n = 7). (C) Expression of PPP2R5C inversely correlates with insulin sensitivity. Pearson correlation analysis was done for PPP2R5C expression levels and glucose infusion rate (GIR) during hyperinsulemic-euglycemic clamp. **p-value<0.01, ***p-value<0.001 by student t-test (A-B).

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS Genetics

Article Title: PPP2R5C Couples Hepatic Glucose and Lipid Homeostasis

doi: 10.1371/journal.pgen.1005561

Figure Lengend Snippet: (A) Hyperglycemia in db/db mice is decreased upon hypatocyte-specific PPP2R5C knockdown with adeno-associated virus. After 5 weeks knockdown, db/db mice were sacrificed under ad libitum feeding with normal chow diet. Blood glucose was monitored at each week, week 0 was one day before virus injection (n = 6). (B) Insulin tolerance test shows improved insulin sensitivity in PPP2R5C knockdown db/db mice at week 4 after virus injection (1.5IU/kg insulin was tail-injected after 6 hour fasting) (n = 6). (C-F) PPP2R5C HepKD in db/db mice increases body weight (C), whole body fat content (D), and liver weight (E), without changing abdominal adipose tissue weight (Abd.WAT) (F) (n = 6). Error bars: std. dev. *p-value<0.05 and **p-value<0.01 by student t-test (D-E). p-value in the (A-C) was calculated by two-way ANOVA.

Article Snippet: Human PPP2R5C mRNA expression was measured by quantitative real-time RT-PCR in a fluorescent temperature cycler using TaqMan assay-on-demand kits (Hs00604899_g1, Applied Biosystems, Darmstadt, Germany), and fluorescence was detected on an ABI PRISM 7000 sequence detector (Applied Biosystems, Darmstadt, Germany).

Techniques: Knockdown, Virus, Injection

Journal: Oxidative Medicine and Cellular Longevity

Article Title: A Nonpolar Blueberry Fraction Blunts NADPH Oxidase Activation in Neuronal Cells Exposed to Tumor Necrosis Factor- α

doi: 10.1155/2012/768101

Figure Lengend Snippet: Nonpolar blueberry fraction inhibits association of p67 phox into plasma membranes without disrupting phosphorylation of p40 phox . Serum free cultures of SH-SY5Y cells were incubated with nonpolar (NP BB ) and polar (PO BB ) blueberry fractions (5 μ g/mL each, 1 h) prior to insult with 200 ng/mL TNF α or 400 ng/mL PMA (30 min). (a) After cell lysis and subcellular fractionation, equal amounts of total plasma membrane protein were subjected to SDS gel electrophoresis followed by western blotting, immunoreactivity against p67 phox (colorimetric detection) was quantified by densitometry (ImageJ64), and all values were normalized to control. TNF α and PMA (filled bars, resp.) induced significant association of p67 phox in plasma membrane fractions compared to control (open bar). Preincubation of SH-SY5Y cells with NP BB prevented p67 phox plasma membrane association regardless of stimulus (TNF α or PMA) whereas preincubation with PO BB was ineffective. Note that incubation of SH-SY5Y cells with PO BB alone but not NP BB resulted in a significant increase in p67 phox plasma membrane association. (b) Immunoreactivity against phosphorylated p40 phox was determined in whole cell lysates (ELISA). All data was adjusted total cellular protein (BCA assay) and normalized to controls. Exposure to 400 ng/mL PMA significantly stimulated phosphorylation of p40 phox (solid bar), necessary for NOX activity, compared to control (CON, open bar). Importantly, pretreatment of SH-SY5Y cells with 5 μ g/mL NP BB had no effect on the extent of p40 phox phosphorylation (grey bar). All data represent the mean of at least four independent experiments ± standard error of the mean (SEM), and statistical significance was determined at * P < 0.05 (ANOVA and Tukey's post hoc analysis).

Article Snippet: BCA Protein assay kit and 1-step NBT/BCIP were from Pierce (Rockford, IL).

Techniques: Clinical Proteomics, Phospho-proteomics, Incubation, Lysis, Fractionation, Membrane, SDS-Gel, Electrophoresis, Western Blot, Control, Enzyme-linked Immunosorbent Assay, BIA-KA, Activity Assay