Journal: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: PPAR Research

Article Title: Activation of PPAR γ Protects Obese Mice from Acute Lung Injury by Inhibiting Endoplasmic Reticulum Stress and Promoting Mitochondrial Biogenesis

doi: 10.1155/2022/7888937

Figure Lengend Snippet: Activation of PPAR γ attenuated LPS-induced apoptosis and inflammation in alveolar epithelial cells. (a) Relative mRNA expressions of TNF- α and IL-1 β in A549 epithelial cells; (b) Levels of TNF- α and IL-1 β in cell culture supernatants; (c) Western blotting and relative protein expression of cleaved-caspase3/caspase3 in A549 alveolar epithelial cell (cleaved-caspase3 was an activated form of caspase3); and (d) flow cytometry was used to detect the apoptosis of A549 alveolar epithelial cells and the percentage of apoptosis of A549 alveolar epithelial cells. Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 vs. Control, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. LPS, +++ p < 0.001, ++++ p < 0.0001 vs. LPS-RSG.

Article Snippet: Proteins were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against PPAR γ (2435, Cell Signaling Technology), PGC-1 α (ab191838, Abcam), NRF-1 (ab175932, Abcam), TFAM (ab47517, Abcam), MFN2 (ab124773, Abcam), GRP78 (ab21685, Abcam), CHOP (ab11419, Abcam), Caspase12 (ab8118, Abcam), Caspase3 (9662, Cell Signaling Technology), APN (21613-1-AP, Proteintech), and β -actin (20536-1-AP, Proteintech).

Techniques: Activation Assay, Cell Culture, Western Blot, Expressing, Flow Cytometry, Control

Journal: PPAR Research

Article Title: Activation of PPAR γ Protects Obese Mice from Acute Lung Injury by Inhibiting Endoplasmic Reticulum Stress and Promoting Mitochondrial Biogenesis

doi: 10.1155/2022/7888937

Figure Lengend Snippet: Activation of PPAR γ inhibited LPS-induced apoptosis in lung tissue of obese mice. (a) Western blotting and relative protein expression of cleaved-caspase3/caspase3 in lung tissue (cleaved-caspase3 was an activated form of caspase3). (b) TUNEL staining was used to detect apoptosis and apoptotic index of alveolar epithelial cells (original magnification, ×200). Data are presented as mean ± SD. ∗∗∗∗ p < 0.0001 vs. DIO-DMSO; #### p < 0.0001 vs. DIO-LPS-DMSO; ++++ p < 0.0001 vs. DIO-LPS-RSG.

Article Snippet: Proteins were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against PPAR γ (2435, Cell Signaling Technology), PGC-1 α (ab191838, Abcam), NRF-1 (ab175932, Abcam), TFAM (ab47517, Abcam), MFN2 (ab124773, Abcam), GRP78 (ab21685, Abcam), CHOP (ab11419, Abcam), Caspase12 (ab8118, Abcam), Caspase3 (9662, Cell Signaling Technology), APN (21613-1-AP, Proteintech), and β -actin (20536-1-AP, Proteintech).

Techniques: Activation Assay, Western Blot, Expressing, TUNEL Assay, Staining

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 3. Fn facilitates anti-PD-1 efficacy by systematically activating CD8+ TILs in germ-free immunohumanized mice bearing subcutane- ous MSS CRC xenografts and SPF mice bearing orthotopic MSS CRC allografts (A) Model design for germ-free, CD34+-humanized mice bearing subcutaneous HT29 xenografts and experimental groups (n = 6/group). Mice received Fn, Streptococcus mutans UA159 (UA159), or PBS by gavage 3 times a week. Related results can be found in B–F. (B) Engraftment of human immune cells in CD34+-humanized mice were evaluated 14 weeks post implantation (n = 6/group). (C) Representative tumor morphology, weight, and volume (n = 6/group). Red circles indicate the location of subcutaneous tumors. (D) Ki-67, PCNA, and TUNEL staining of HT29 xenografts (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti-PCNA, or TUNEL kit. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. (E) CD8 and PD-1 co-immunofluorescence staining (n = 4/group) and PD-1 IHC in HT29 xenografts (n = 6/group). Tumor tissues were stained using DAPI, Alexa Fluor 647 anti-human CD8, anti-mouse PD-1, and Alexa Fluor 488 anti-Rabbit IgG H&L antibodies for immunofluorescence staining, and anti-PD-1 for IHC. For immunofluorescence staining, red: CD8+ cells, green: PD-1+ cells, blue: DAPI+ cells, yellow: PD-1+ CD8+ cells. For PD-1 IHC staining, blue: hematoxylin+ cells, brown: PD-1+ cells. (F) Flow cytometry analyses of infiltration of CD8+ T cells in HT29 xenografts, and PD-1, IFN-g, TNF-a, and GZMB expression in CD8+ TILs (n = 6/group). (G) Model design for SPF BALB/c mice bearing orthotopic CT26 allografts and experimental groups. Mice were gavaged with Fn conditioned medium (CM), UA159 CM (UACM), or broth Ctrl daily, with anti-PD-1 mAb (aPD-1) or IgG twice a week (n = 8–12/group). Red circles indicate the location of orthotopic tumors. Related results can be found in (H) and (I). (H) Representative tumor morphology, weight, and volume of CT26 allografts (n = 8–12/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: TUNEL Assay, Staining, Immunohistochemistry, Flow Cytometry, Expressing

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 4. Fn decreases PD-1 expression in CD8+ T cell through its small-molecule metabolites (A) Effects of Fn CM fractions on decreasing PD-1 expression in CD8+ T cell after co-culture for 18 h, as determined by flow cytometry (n = 6/group). Fn CM PK: Fn CM treated with proteinase K; >100 kDa: Fn CM > 100 kDa fraction; 10–100 kDa: Fn CM 10–100 kDa fraction; 3–10 kDa: Fn CM 3–10 kDa fraction; <3 kDa: Fn CM < 3 kDa fraction. (B) Workflow to define candidate metabolites from Fn culture medium that suppress PD-1 expression in CD8+ T and promote CD8+ T cell activation in vitro. (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot based on non-targeted metabolomic analysis of Fn CM < 3 kDa vs. control (left) and Fn gavage vs. PBS gavage germ-free mouse feces (right) (n = 6/group). (D) Venn diagram of enriched metabolites in Fn CM < 3 kDa vs. control CM, and Fn gavage vs. PBS gavage in germ-free mouse feces, and metabolomic heatmap of metabolites enriched after Fn gavage vs. PBS gavage germ-free mouse feces as determined by untargeted metabolomics (n = 6/group). (E) Targeted metabolomic analysis of short-chain fatty acids (SFCAs) in Fn- and PBS-gavaged germ-free mouse feces (left). Butyric acid quantification in CM and feces from germ-free mice (right) (n = 6/group). (F) Effect of sodium butyrate (NaB) on the expression of PD-1 and GZMB in splenic CD8+ T cells from tumor-bearing mice (n = 6/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Expressing, Co-Culture Assay, Cytometry, Activation Assay, In Vitro, Control

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 5. Fn boosts response to anti-PD-1 therapy through its metabolite butyric acid (A) Workflow for the construction of FN0271 mutant Fn strain deficient in butyric acid biosynthesis. PCR products of FN0271 were analyzed by agarose gel electrophoresis to confirm the insertion mutant of Fn (left). GC-MS was conducted to detect butyric acid levels in Fn CM and FN0271 mutant (Mut) Fn CM (right). (B) Effect of Fn CM, Mut Fn CM, and NaB on PD-1 and GZMB expression in splenic CD8+ T cells (n = 6/group). (C) Evaluation of T cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) assay and flow cytometry (n = 6/group). (D) Workflow to assess Fn CM and Mut Fn CM on anti-PD-1 mAb efficacy in vitro using CD8+ TILs from CT26 tumor-bearing mice. (E) Effect of Fn CM and Mut Fn CM in combination with anti-PD-1 mAb (aPD-1) on PD-1, GZMB, and IFN-g expression in CD8+ TILs (n = 6/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S6.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Gas Chromatography-Mass Spectrometry, Expressing, CFSE Assay, Cytometry, In Vitro

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 6. Fn boosts anti-PD-1 mAb tumor-killing effects through its metabolite butyric acid in autologous co-culture system of PBMCs with tumor organoids from patients with MSS CRC (A) Schematic diagram for the evaluation of Fn CM and metabolites on anti-PD-1 therapy-mediated killing of CRC organoids with autologous peripheral blood mononuclear cell (PBMC)-derived CD8+ T cells. (B) Representative images of anti-PD-1 (aPD-1) therapy combined with Fn CM, FN0271 Mut Fn CM, or NaB on tumor killing efficiency. MSS CRC organoids (red) were labeled with CellTrace Far Red and apoptotic cells were labeled with green caspase-3/7 probe (n = 6/group). Black circle: organoid, small dot: T cell, red: CellTrace far red+ cells, green: caspase-3/7+ cells. (C) Apoptosis in CRC organoids, as determined by flow cytometry (n = 6/group). (D) Flow cytometry of PD-1, GZMB, and IFN-g in autologous CD8+ T cells (n = 3/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S7 and Table S2.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Co-Culture Assay, Derivative Assay, Labeling, Cytometry, Flow Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 7. Fn-derived butyrate facilitates anti-PD-1 therapy in MSS CRC in a CD8+ T cell-dependent manner (A) CT26 subcutaneous allografts tumor-bearing BALB/c mice received NaB in drinking water, together with anti-PD-1 mAb (aPD-1), anti-CD8a mAb, or IgG twice a week (n = 6–10/group). Related results can be found in B–H. (B) Representative tumor gross morphology, tumor weight, and tumor volume. NaB potentiated with anti-PD-1 to suppress tumor growth, an effect abolished by the depletion of CD8+ T cells by anti-CD8a (n = 6–10/group). Red circles indicate the location of subcutaneous tumors. (C) Tumoral butyrate among different groups (n = 6/group). (D) TUNEL, (E) Ki-67, and PCNA IHC in CT26 allografts to analyze cell apoptosis and proliferation (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti- PCNA, or TUNEL kit. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. (F) Infiltration of CD8+ TILs in CT26 allografts, as determined by flow cytometry and (G) IHC (n = 6/group). Tumor tissues were stained using anti-CD8. Blue: hematoxylin+ cells, brown: CD8+ cells.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 8. Fn metabolite butyric acid regulates CD8+ T cell through an epigenetic mechanism leading to upregulation of TBX21 (T-bet) (A) CD8+ T cells treated with Fn CM, FN0271 Mut Fn CM, and control CM were analyzed by RNA sequencing. Principal component analysis (PCA) and heatmap analysis of differentially expressed genes, and validation by qPCR (n = 3/group). qPCR data were normalized using Actb as an internal control.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Control, RNA Sequencing, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Levels of poliovirus infection in GBM cell lines at 24 h post-infection (h.p.i.). U-251 MG or DBTRG-05MG cell lines maintained in DMEM or Plasmax media were infected with poliovirus at MOI 1 for 6 ( a ) and 24 h ( a , b ), and virus infection was monitored by assessment of TCID50 ( a ) or by immunostaining ( b ) using primary antibodies to poliovirus and FITC-conjugated secondary antibodies. TCID50 values ( a ) are presented as means ± SD, * p ≤ 0.05 by two-way ANOVA with Tukey post hoc test.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Virus, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Polyamine levels in GBM cells with and without poliovirus infection, expressed as nmol/mg, measured by high-performance liquid chromatography. U-251 MG ( a , c ) and DBTRG-05MG ( b , d ) were cultured in DMEM ( a , b ) or Plasmax ( c , d ) media prior to analysis. Data are presented as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01 based on two-way ANOVA with Bonferroni test adjustment.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, High Performance Liquid Chromatography, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Cytopathogenic effect of poliovirus-infected GBM cells treated with inhibitors or inducers of polyamine-metabolizing enzymes. U-251 MG or DBTRG-05MG cells maintained in DMEM were pretreated with DFMO ( a ), DENSpm ( b ), MDL72.527 ( c ), or SAM486a ( d ), and infected with PV at different MOI in the presence of these compounds at the same concentrations. Cell viability was measured using resazurin assay. The values were normalized to values of untreated mock-infected cells. The data are presented as mean ± SD.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Resazurin Assay

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Imprint of poliovirus on levels of polar metabolites of GBM cell lines. U-251 MG or DBTRG-05MG cells maintained in DMEM or Plasmax media were infected with PV at MOI 1, and metabolites levels were measured 24 h.p.i. by gas chromatography-mass spectrometry. The graphs represent amino acids ( a ) and other metabolites ( b ). Levels were normalized to levels in mock-infected cells maintained in respective medium and expressed as log2 fold change (Log 2 FC).

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Gas Chromatography, Mass Spectrometry

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Levels of neutral lipids in GBM cells during poliovirus infection. U-251 MG or DBTRG-05MG cells maintained in DMEM or Plasmax were infected with PV at MOI 1 and stained with BODIPY 493/503 (neutral lipids, green fluorescence) and DAPI (nuclei) at various time points. Fluorescence was visualized on ZOE Fluorescent Cell Imager.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Effect of poliovirus on glycolytic and respiratory activities of GBM cell lines. U-251 MG ( a , c ) or DBTRG-05MG ( b , d ) cells maintained in DMEM or Plasmax media were infected with PV at MOI 1, and extracellular acidification (ECAR) reflecting glycolysis ( a , b ) and oxygen consumption rate (OCR) reflecting respiratory activity ( c , d ) were measured by Seahorse technology using GlycoStress and MitoStress assays. Results are presented as mean ± SD.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: 2-deoxyglucose enhances cytopathogenic effect of poliovirus in GBM cells. U-251 MG ( a , c ) or DBTRG-05MG ( b , d ) cells maintained in DMEM ( a , b ) or Plasmax ( c , d ) were pretreated with 2-deoxyglucose (2DG) and infected with PV at different MOI in presence of this drug. Cell viability was measured using resazurin assay. Values were normalized to values of untreated mock-infected cells. Data are presented as mean ± SD.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Resazurin Assay

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Poliovirus infection in U-251 MG cells is not accompanied by increased production of superoxide anion. Superoxide production was assessed using dihydroethidium staining with subsequent measurement of fluorescence of specific 2-hydroxyethidine product by flow cytometry. tBuOOH was used as positive control. Data are presented as mean ± SD. * p ≤ 0.05 by ANOVA with Tukey post hoc test.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Staining, Fluorescence, Flow Cytometry, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Metabolic Imprint of Poliovirus on Glioblastoma Cells and Its Role in Virus Replication and Cytopathic Activity

doi: 10.3390/ijms26157346

Figure Lengend Snippet: Poliovirus infection does induce changes in hydrogen peroxide levels. H 2 O 2 levels in HEK293TΔIFNR1 cells stably expressing the genetically encoded ratiometric HyPer7-Lifeact sensor were measured by confocal microscopy. The cells were infected with poliovirus at MOI = 1 for 14 h; green fluorescence was quantified after excitation at 405 and 488 nm. As a positive control, the cells were treated with hydrogen peroxide 5 min prior to analysis. Ratiometric analysis is presented as the ratio of excitation wavelengths 488/405.

Article Snippet: Primary mouse antibodies to poliovirus (Santa Crus Biotechnologies, Santa-Clara, CA, USA, #sc-80633) were diluted in 2% bovine serum albumin (BSA) in PBS, added to the wells, and incubated for 1 h at 37 °C.

Techniques: Infection, Stable Transfection, Expressing, Confocal Microscopy, Fluorescence, Positive Control

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A Metascape functional enrichment analysis. Common and coherent metabolic DEGs, extracted from GSE24112 and GSE48363, were analyzed for ontology purposes. B PHGDH, PSAT1, PSPH protein levels in CRC cell lines. Cell lysates from HCT-116, HT29, HCT8, CACO2, RKO, and LS174T cells were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. C AA composition of cancer conditioned media (CM). Amino acids content in CM was analyzed by GC-MS on media collected following 48 h of incubation with muscle cells. Data are reported as normalized to serum-free DMEM abundance and are average of three independent experiments. Black label indicates relative value as 1. D Ser media content analyzed by GC-MS on media collected following 48 h of incubation with CRC cells. Data are expressed as relative to serum-free DMEM. One-way ANOVA with Sidak’s post hoc test ( n = 3). Each dot represents a single experiment. E PHGDH protein levels-CM Ser content correlation analysis. Pearson correlation between PHGDH protein levels in CRC cell lines and Ser content from corresponding CM. F Schematic representation of CM-culturing experiments. G Alterations in myotubes width following CM incubation. Relative C2C12 myotubes fibers width following 96 h of incubation with boiled CMs derived from CRC cell lines cultures. CRC cell lines were incubated in serum-free DMEM medium for 48 h before collecting conditioned media (CM) and boiling them for 20 min to remove all protein components. Data are expressed as relative to serum-free DMEM. One-way ANOVA with Sidak’s post hoc test ( n = 3). Each dot represents a single experiment. H Ser media content-myotubes width correlation analysis. Pearson correlation between Ser content in CM from CRC cell lines and C2C12 myotubes width following 96 h of incubation with CM. Data are normalized on values from C2C12 myotubes incubated in normal serum-free DMEM. I PHGDH protein levels-myotubes width correlation analysis. Pearson correlation between PHGDH protein levels in CRC cell lines and C2C12 myotubes width following 96 h of incubation with corresponding CM. J Alterations in myotubes width following incubation with CM from HT29-high and HT29-low cells. Relative C2C12 myotubes fibers width following 96 h of incubation with boiled CM derived from high PHGDH HT29 and low PHGDH HT29-expressing cells. Data are normalized to width values from C2C12 cells incubated in serum-free DMEM. One-way ANOVA with Dunnett’s post hoc test ( n = 3). Each dot represents a single experiment. K Ser supplementation rescues C2C12 myotubes width reduction under CM incubation. Relative C2C12 myotubes fibers width following 96 h of incubation with boiled CM derived from CACO2 cells supplemented or not with exogenous Ser (0.4 mM). Data are normalized to width values from C2C12 cells incubated in serum-free DMEM. One-way ANOVA with Dunnett’s post hoc test ( n = 3). Each dot represents a single experiment. L Representative microscopic pictures of C2C12 myotubes. C2C12 myotubes were incubated with boiled CM derived from CACO2 cells supplemented or not with exogenous Ser (0.4 mM). Pictures were taken following 96 h of incubation. The image is representative of three independent experiments. Data are represented as mean ± standard error of the mean of at least three independent biological replicates. ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Functional Assay, Western Blot, Gas Chromatography-Mass Spectrometry, Incubation, Derivative Assay, Expressing

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A , B Total tumor mass. Mass quantification of tumors obtained from HT29 low-PHGDH ( A ) and CT26 ( B ) tumor-bearing mice treated as in Fig. . Mass values were calculated based on tumor weight measured at the endpoint of the experiment. Each dot represents a plasma sample derived from a single mouse. Data are represented as mean ± SEM, Student’s t test. C , D Intra-tumor Ser levels. Relative Ser levels quantification in tumor tissue-derived samples from HT29 low-PHGDH ( C ) and CT26 ( D ) tumor-bearing mice treated as in Fig. . Metabolites from tissue samples were extracted as described in the “Materials and methods” section and tissue Ser content was quantified by GC-MS analysis. Each dot represents a plasma sample derived from a single mouse. Data are represented as mean ± SEM, Student’s t test. E PHGDH protein levels in CRC cell lines under Ser starvation. HCT-116, CACO2, and RKO cells were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly for 24 h before protein extraction. Cell lysates were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. F Representative images of western blotting analysis evaluating PHGDH expression in HCT-116, CACO2, and RKO cells incubated in +Ser +Gly or -Ser -Gly medium for 24 h. G PHGDH protein levels in C2C12 myotubes under Ser starvation. C2C12 myotubes were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly for 6, 12, and 24 h before protein extraction. Cell lysates were analyzed by western blotting with the anti-PHGDH antibody. An anti-actin antibody was used to ensure equal protein loading. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. H Representative images of western blotting analysis evaluating PHGDH expression in C2C12 myotubes incubated in +Ser +Gly or -Ser -Gly medium for 24 h. I Ser synthesis pathway (SSP) activity analysis by [U- 13 C]-glucose labeling assay. Relative incorporation of [U- 13 C]-glucose-derived carbons in Ser. C2C12, CACO2, RKO, and HCT116 cells were incubated in a (-Ser -Gly) medium containing [U- 13 C]-Glucose for 3 h. Metabolite abundance and labeling enrichment were evaluated by GC-MS analysis. One-way ANOVA with Dunnett post hoc test ( n = 3). Each dot represents a single experiment. J Competition for Ser availability between tumor and muscle cells. C2C12 myotubes were pre-incubated with a medium containing 13 C 1 -Ser for 24 h before replacing growth medium with standard serum-starved-DMEM and adding tumor cells in the upper compartment of a Boyden chamber (pores diameter 0.4 µm). The fate of C2C12-derived 13 C 1 -Ser was assessed by GC-MS analysis 24 h after tumor cell’s introduction. K Workflow for the 13 C 1 -Ser labeling experiment described in J . One-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. Data are represented as mean ± SEM of n independent experiments, ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Clinical Proteomics, Derivative Assay, Gas Chromatography-Mass Spectrometry, Incubation, Protein Extraction, Western Blot, Expressing, Activity Assay, Labeling

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A Representative microscopic pictures of C2C12 myotubes. C2C12 myotubes were incubated in a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and Gly. Pictures were taken following 96 h of incubation. The image is representative of three independent experiments. B Alterations in myotubes width following Ser/Gly starvation. Relative C2C12 myotubes fibers width following 96 h of incubation with a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and/or Gly. 10 nM TNFα was used as wasting-inducer positive control. Data are expressed as relative to width values of myotubes grown in normal serum-free DMEM. One-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. C Alterations in total protein content in myotubes width following Ser/Gly starvation. C2C12 myotubes fibers were incubated for 96 h of incubation with a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and/or Gly before lysing cells for protein quantification analysis. Data are expressed as relative to protein content of myotubes grown in normal serum-free DMEM. One-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. D Myotubes AAs content following Ser/Gly starvation. C2C12 myotubes were incubated for 48 h with a medium containing (+Ser +Gly) or lacking (-Ser -Gly) Ser and/or Gly before metabolomic analysis. Metabolites were extracted as described in the “Materials and methods” section and intracellular AAs content was quantified by GC-MS. Data are expressed as relative to AAs levels measured in samples from cells incubated in serum-free DMEM. Data are reported as average of three independent experiments. E , F Atrogin-1 and MURF-1 expression levels. mRNA levels of Atrogin-1 ( E ) and MuRF-1 ( F ) were analyzed by quantitative RT-PCR in C2C12 myotubes following 48 h of incubation with +Ser +Gly/-Ser -Gly media. +Ser +Gly condition was used as comparator. Student’s t-test ( n > 3). Each dot represents a single experiment. G Total protein synthesis in C2C12 myotubes under Ser/Gly starvation. Protein synthesis was evaluated on the incorporation of puromycin into newly synthesized proteins. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 24 h, starved in Hank’s balanced salt solution (HBSS) for 1 h, and subsequently reactivated (30 min) in a medium containing puromycin (10 mg/mL). Cell lysates were analyzed by western blotting with the anti-puromycin antibody. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. H Phosphorylation of AKT and mTORC1 targets S6 and S6K1 in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 6, 16, and 24 h before analyzing cell lysates by western blotting. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. I Phosphorylation of AKT and S6K1 in C2C12 myotubes incubated in low Ser containing CM. C2C12 myotubes were incubated with boiled CM derived from CACO2 cells supplemented or not with exogenous Ser (0.4 mM) for 24 h before analyzing cell lysates by western blotting. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. J Proliferation curves of C2C12 cells cultured in presence or absence of Ser/Gly. Proliferation curves of C2C12 cell lines grown in medium with Ser/Gly (+ser+gly, gray dots) or without Serine/Gly (-ser-gly, blue dots). Cell number was assessed every 24 h ( n = 3). K Proliferation curves of C2C12 cells following 24 h of Ser/Gly starvation. C2C12 cells were incubated for 24 h in presence (+ser+gly, gray dots) or absence (-ser-gly, blue dots) of Ser/Gly before assessing cell proliferation in normal medium by counting cell number every 24 h ( n = 3). L Cell Cycle distribution of C2C12 cells cultured in presence or absence of Ser/Gly. Quantitative analysis of relative cell number in G1, S, and G2/M of C2C12 cells incubated in grown in medium with or without Serine/Gly for 24. C2C12 cells were stained with Propidium Iodide (PI) to analyze the cell cycle distribution by flow cytometry. Quantitation was determined by measuring the area corresponding to the indicated phase. M Time-dependent changes in MHC and MYOD protein levels during differentiation under Ser/Gly starvation. C2C12 myotubes were incubated with differentiation media (dialyzed FBS 2%) in presence or not of Ser/Gly for 1, 2, 3, 6 days before analyzing cell lysates by western blotting with the anti-MHC and anti-MYOD antibodies. An anti-Vinculin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. Data are represented as mean ± SEM of n independent experiments, ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Incubation, Positive Control, Gas Chromatography-Mass Spectrometry, Expressing, Quantitative RT-PCR, Synthesized, Western Blot, Phospho-proteomics, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Quantitation Assay

Journal: Cell Death Discovery

Article Title: Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia

doi: 10.1038/s41420-024-02271-1

Figure Lengend Snippet: A Total ROS levels in C2C12 myotubes under Ser/Gly starvation. Intracellular ROS were quantified in C2C12 myotubes incubated with +Ser +Gly/-Ser -Gly media for 24 h by staining with DCFDA probe and analyzing them by FACS. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. B Mitochondrial ROS levels in C2C12 myotubes under Ser/Gly starvation. Mitochondrial ROS were quantified in C2C12 myotubes incubated with +Ser +Gly/-Ser -Gly media for 24 h by staining with MitoSOX probe and analyzing by FACS. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. C Intracellular ATP levels in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 24 h before analyzing ATP content. Data are expressed as relative to +Ser+Gly condition. Student’s t-test ( n = 3). Each dot represents a single experiment. D Phosphorylation of AMPK in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media 24 h before analyzing cell lysates by western blotting with the anti-phospho-AMPK and anti-AMPK antibody. AMPK phosphorylation levels are reported as relative to total AMPK levels. An anti-actin antibody was used to ensure equal protein loading. Student’s t-test ( n = 3). Each dot represents a single experiment. E – G Oxidative metabolism in myotubes under Ser/Gly starvation. Oxygen consumption rate (OCR) was measured in C2C12 myotubes previously incubated with +Ser +Gly/-Ser -Gly media for 24 and 48 h. E Basal OCR was calculated as the OCR values before Oligomycin injection. F ATP-linked respiration was calculated by subtracting OCR values obtained following Oligomycin injection to basal OCR. G Non-mitochondrial respiration was calculated as the OCR values after Rotenone/Antimycin A injection. Data are normalized to those from C2C12 cells incubated with +Ser +Gly medium. One-way ANOVA with Dunnett’s post hoc test ( n = 3). Each dot represents a single experiment. H Mitochondrial respiration complexes in C2C12 myotubes under Ser/Gly starvation. C2C12 myotubes were incubated with +Ser +Gly/-Ser -Gly media for 24 h before analyzing cell lysates by western blotting with the anti-OXPHOS complexes antibody. An anti-actin antibody was used to ensure equal protein loading. The image is representative of three independent experiments. I Mitochondrial ROS accumulation in Ser starved C2C12 myotubes upon mitoTEMPO treatment. C2C12 cells were incubated with +Ser +Gly/-Ser -Gly media 24 h in presence or not of 2.5 µM mitoTEMPO before quantifying mitochondrial ROS by staining them with MitoSOX probe and analyzing by FACS. Data are normalized to those from C2C12 cells incubated with +Ser +Gly medium. Two-way ANOVA with Tukey’s post hoc test ( n = 3). Each dot represents a single experiment. J Quantification of Ser starved C2C12 myotubes upon MitoTEMPO treatment. C2C12 cells were incubated with +Ser +Gly/-Ser -Gly media 72 h in presence or not of 2.5 µM mitoTEMPO before quantifying fiber width. Data are normalized to those from C2C12 cells incubated with +Ser +Gly medium. Two-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± SEM of n independent experiments, ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse C2C12 myoblasts were obtained from ATCC and cultured in DMEM with 10% fetal bovine serum (FBS).

Techniques: Incubation, Staining, Phospho-proteomics, Western Blot, Injection

Journal: Cell host & microbe

Article Title: Defined human Clostridia consortia reverse colitis via dual effects of tryptophan metabolites on microbiota and immunity

doi: 10.1016/j.chom.2026.08.003

Figure Lengend Snippet: (A and B) Germ-free (GF) Il10 -eGFP reporter mice were colonized with vehicle (Eggerth-Gagnon media), the 11- or 17-mix Clostridium strain consortia (2 × 10 7 CFU in 200 μL) for 2 weeks. cLP GFP (IL-10) + , FOXP3 + CD4 + T, and FOXP3 neg CD4 + T cells were analyzed by flow cytometry. N = 5−7/group. (B) Representative plots. (C) cLP RORγt+FOXP3+CD4+ T cells in GF WT mice 2 weeks after vehicle, 11-mix, or 17-mix. N = 4−5/group. (D) Cecal luminal metabolites from GF Il10 -eGFP reporter mice treated with vehicle, 11-mix, or 17-mix were analyzed by GC/MS and LC-MS/MS. Heat map shows log2-transformed, Z-scored metabolite abundance with FDR-corrected ANOVA q < 0.05 and Tukey post-hoc comparisons. Colored boxes indicate p < 0.05 red (Dn: decreased); green (Up: increased) boxes or p ≥ 0.05 white denotes no significant change (N.S.) compared with vehicle. N = 5−7/group. Arrows indicate AhR agonists/activators. (E) Selected metabolites. Data are mean ± SE; each point represents one mouse. One-way ANOVA with Tukey test, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Article Snippet: Mouse Naïve CD4+ T Cell Isolation Kit , Miltenyi Biotec , Cat# 130–104-453.

Techniques: Flow Cytometry, Gas Chromatography-Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay

Journal: Cell host & microbe

Article Title: Defined human Clostridia consortia reverse colitis via dual effects of tryptophan metabolites on microbiota and immunity

doi: 10.1016/j.chom.2026.08.003

Figure Lengend Snippet: (A) Germ-free Rag2 −/− mice were colonized with donor-A human stool, received naïve CD4 + T cells 2 weeks later, and were treated with vehicle, 11-mix, or 17-mix (2 × 10 7 CFU in 200 μL) beginning 4 weeks after transfer. N = 7−8/group. (B) Stool images and scores. (C) Serial f-Lcn. The y-axis break retains an outlier while showing lower-range data. (D) Representative photomicrographs and histological score of 3 combined segments (cecum, proximal colon (Pro-colon), and rectum). Yellow arrowheads indicate infiltrating cells. Bar = 100 μm. (E) Colonic mRNA by qPCR relative to vehicle. N = 4/group. (F) Significant family- and genus-level stool microbiota changes by 16S rRNA sequencing. N = 4/group. Data are mean ± SE; each point represents one mouse. One-way ANOVA with Tukey test for B, D-F; Kruskal-Wallis with Dunn’s test for C. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Mouse Naïve CD4+ T Cell Isolation Kit , Miltenyi Biotec , Cat# 130–104-453.

Techniques: Adoptive Transfer Assay, Sequencing