Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO, bars 1-4) or 660 nM TSA (TSA, bars 5-8) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of seven independent experiments. Asterisks denote significant differences (***p<0.001). NS, not significant. ## p<0.01 bar 5 vs 1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with increasing TSA doses (0, 6.6, 66, 660, 6600 nM) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of three independent experiments. Asterisks denote significant differences (p<0.05). NS, not significant. # p<0.05 vs 0 nM control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were transfected with non-targeting luciferase siRNA (Luc), or siRNA against SMRT, NCoR, or PPARγ. The levels of mRNA were determined by qPCR. Each point represents the mean ± S.E. of at least three independent experiments. (B) 3T3-L1 adipocytes were transfected with control (Luc), SMRT, NCoR, or PPARγ siRNA. 24 h post transfection, cells were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα) for additional 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01). NS, not significant.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were treated with DMSO, 660 nM TSA, 20 µM SAHA, 10 µM MS275, or 5 µM MC1568 for 24 h. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. Samples were treated in duplicate. Representative immunoblots from three independent experiments are shown. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA, 10 µM MS275, 5 µM MC1568, or combination of MS275 and MC1568 (MS275+MC1568) for 24 h. (C) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA (TSA), 5 or 20 µM SAHA (SAHA) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01; ***p<0.001). NS: not significant. # p<0.05, ## p<0.01 vs the corresponding DMSO Control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A and B) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 660 nM TSA, together with or without 1 µM Rosi (Rosi) for 24h. Cells were then treated with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western analysis with the indicated antibodies. Representative immunoblots and quantification data from five independent experiments are shown in 5A and B, respectively. (C) ERK phosphorylation correlates highly with lipolysis in the treatments of Rosi, TNFα, or both in the presence or absence of TSA, as shown by fitting with linear regression. Individual values were obtained from the experiments described in Figures. 1 and 7B.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 25 µM U0126 for 1 h, followed by treatment with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with DMSO (bars 1-4), 660 nM TSA (bars 5-8), 25 µM U0126 (bars 9-12), or both (bars 13-16) for 24h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (**: p<0.01). NS, not significant. ## p<0.01 vs Treatment No.1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Representative temporal color-coded maps of viral infection time for selected BMDM-fibroblast co-culture conditions (scale bar, 100 μm). The key (far left) demonstrates the color-coding scheme to interpret the remainder of A . Regions infected early during infection are colored darker whereas regions infected later during the experiment are colored lighter. Black regions represent areas without infection. B Quantification of viral spread across all BMDM-fibroblast co-culture conditions. Infection was determined based on fluorescence intensity of tagged viral VP26 in fibroblasts at 48hpi. C Quantification of cells initially infected only from virus (MOI=1) added to the media after being pulsed overnight with different doses of TNF. Infection was determined based on fluorescence intensity of tagged viral VP26 in fibroblasts at 24hpi. D Quantification of cells that stained positive for active, phosphorylated STING (pSTING) after being pulsed overnight with different doses of TNF and infected with MOI 10 of HSV-1. pSTING+ cells were identified based on antibody fluorescence intensity at the specified timepoint post-infection. E Quantification of cells that stained positive for pSTING after being pulsed overnight with different doses of TNF and exposed to dose titrations of the pSTING agonist DMXAA. pSTING+ cells were identified based on antibody fluorescence intensity 1.5 h after DMXAA treatment. Data in B – E are mean ± s.d.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Co-Culture Assay, Fluorescence, Virus, Staining

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Quantification of cell death in NIH 3T3 fibroblasts after treatment with 100 ng/ml TNF or TNF supplemented with 0.25 μg/ml Actinomycin D. Cell death was quantified over time based on Hoechst 33342 fluorescence intensity which increases sharply on nuclear condensation. B Quantification of cell death in fibroblasts 24 h after simultaneous treatment with 0.3 ng/ml TNF and 50 pg/ml LCL-161. Cell death was quantified based on incorporation of a nucleic acid stain (sytox green). C Diagram of experiment for data shown in D . 3T3 fibroblasts were treated for 24 h with 0.3 ng/ml TNF and then washed and rested overnight. The next day, LCL-161 was added at the indicated doses and cell death was quantified after 24 h of LCL-161 exposure. D Quantification of cell death for experiment described in C . Cell death was quantified based on incorporation of sytox green. E Quantification of 3T3 fibroblasts that stained positive for cleaved Caspase-8 during and after treatment with 100 ng/ml TNF. The shaded region indicates the time period before TNF was washed out. Cleaved Caspase-8 was detected by immunostaining and cells were distinguished as positive by setting a threshold on the antibody fluorescence intensity. F Quantification of cell death for 3T3 fibroblasts treated constitutively with 30 ng/ml TNF or washed after overnight treatment with 30 ng/ml TNF. Cell death was quantified based on incorporation of sytox green. G Diagram of experiment for data shown in H . 3T3 fibroblasts were treated with 1 ng/ml TNF for 24 h and then washed. At different times after washing, 0.25 μg/ml Actinomycin D was added to cells for 4 h before quantifying cell death. H Quantification of cell death for experiment described in G . Cell death was quantified based on incorporation of sytox green. B , D , F bars denote the mean of replicates (filled black circles). E , H are mean ± s.d.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Fluorescence, Staining, Immunostaining

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Representative single-cell traces of 3T3 fibroblasts treated with different doses of TNF and infected with MOI 10 of HSV-1. Purple and orange lines represent the fluorescence over time of the virus and nucleic acid stain (sytox green, death marker), respectively. Dotted purple and yellow lines indicate the timing of initial infection and death. The timing of initial infection was determined by setting a threshold on viral VP26 fluorescence intensity. The timing of death was determined by setting a threshold based on incorporation of nucleic acid stain (sytox green) and nuclear condensation identified as a sharp increase in Hoechst 33342 intensity (for details on thresholding, see methods). Shaded regions represent the time interval between infection and death (death decision time, Δt). B Distribution of death decision times obtained by single-cell tracking of 3T3 fibroblasts treated with a single dose of TNF (50 ng/ml) and infected with MOI 10 of HSV-1. Orange line shows an exponential fit of the data. C The same sample that generated B , plotted as the percentage of live 3T3 fibroblasts over time. Orange line shows the same exponential fit plotted in B . D Death rates obtained from exponential fits of 3T3 fibroblasts treated with dose titrations of TNF and infected with MOI 10 of HSV-1. E Quantification of the percentage of uninfected 3T3 fibroblasts killed by TNF (false positives) at 24 h post treatment. Cell death was determined by incorporation of a nucleic acid stain (Sytox green or red), accompanied with nuclear condensation measured as a sharp increase in Hoechst 33342 intensity. F Receiver Operating Characteristic (ROC) curve where false-positive rate represents 3T3 fibroblasts killed by TNF at 24 h post treatment and true positive rate represents infected cells that die during the first viral life cycle (10 h after initial infection is called based on viral VP26 fluorescence intensity). G Speed versus accuracy tradeoff shown by plotting the percentage of false-positive 3T3 fibroblasts (from E ), versus the death rate for infected 3T3 fibroblasts (from D ). D – G are mean ± s.d. F , G colorbar shows TNF concentration.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Fluorescence, Virus, Staining, Marker, Single Cell Tracking, Generated, Concentration Assay

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Diagram of the reactions in our spatial stochastic model. All model parameters were measured in independent experiments shown in Supplementary Fig. , summarized in Supplementary Table . B Ensemble simulation results for different TNF concentrations. Purple area and intensity correspond to the probability of the cell being infected. Black area and intensity correspond to the probability of the cell being dead following infection. Cyan area and intensity correspond to the probability of the cell being alive and healthy. C Quantification of viral spread as a function of TNF concentration, mean ± s.d. D Boxplot summary of simulation results for a dose titration TNF conditions showing the percentages of healthy, infected, dead following infection, and dead but uninfected cells. Central mark denotes the median, boxes denote the upper and lower quartiles, whiskers extend to the most extreme points not considered outliers (≥1.5 interquartile range from top/bottom of box), and outliers are plotted as points. E Quantification of the percentages of healthy, infected, dead following infection, and dead but uninfected 3T3 fibroblasts treated with dose titrations of TNF and infected with MOI 1 of HSV-1. Data are mean ± s.d. F Simulation of the dynamics of actively infected cells for different concentrations of TNF. Lines represent the mean values of 500 simulations. G Effective reproductive rates (Rt) distributions in different conditions represented as violin plots, which were generated with non-negative support and a bandwidth of 0.05. In this context, Rt represents the ratio of the probability that a cell is infected by its infected neighbor to the probability of that neighboring cell dying. The red line denotes a separatrix: Above it, the virus is more likely to spread further and below it, the virus is more likely to be extinguished. Black lines indicate median. For simulations ( B – D , F , G ), each condition was simulated 500 times.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Concentration Assay, Titration, Generated, Virus

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Cartoon diagram with cornea emphasized to show different perspectives of images. B Representative transverse image of R26-H2B-mCherry mouse corneas 36 h after infection with HSV-1 and treated with either 0 or 50 ng/ml TNF. C Enlarged regions within images shown in B with cell death channel included. D Quantification of the total sum of virus pixels for C57Bl/6 corneas treated with 0 or 50 ng/ml TNF, infected with HSV-1, and imaged at 48hpi. E Percentage of total virus pixels also positive for Sytox Green death marker. F Representative epithelial projections of C57Bl/6 corneas treated with 0 or 50 ng/ml TNF and imaged 24 h after treatment. G Quantification of the total Sytox Green intensity for uninfected corneas. H Speed versus accuracy tradeoff shown by plotting the percentage of false-positive primary corneal epithelial cells versus the death rate for infected primary corneal epithelial cells. Colorbar represents TNF concentration. Data are mean ± s.d. D , E , G bars denote the mean of replicate eyes (filled black circles). All scale bars, 100 μm.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Virus, Marker, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: For experiments in Fig. , co-housed 8-week-old female C57BL/6J wild-type mice were administered either 300 μg/kg TNF (R&D Systems Cat#410-MT/CF Lot CS152103 ) in endotoxin-free DPBS or endotoxin-free DPBS alone (Merck Cat# TMS-012-A) via bolus tail vein injection.

Techniques: Derivative Assay, Polymer, Plasmid Preparation, Blocking Assay, Membrane, Staining, RNA HS Assay, dsDNA Assay, Protease Inhibitor, Software, Imaging, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: Bovine leukemia virus reduces anti‐viral cytokine activities and NK cytotoxicity by inducing TGF‐β secretion from regulatory T cells

doi: 10.1002/iid3.93

Figure Lengend Snippet: Reduced anti‐viral cytokine production in BLV‐infected cattle. IFN‐γ (A) and TNF‐α (B) in the supernatants of cultured PBMCs from BLV‐infected aleukemic (AL: n = 9) or persistent lymphocytotic (PL: n = 7) cattle and uninfected cattle ( n = 4) were determined using ELISA. Detection of IFN‐γ producing CD4 + T cells in BLV‐uninfected and BLV‐infected cattle at different disease stages (C: BLV(−): n = 7, AL: n = 14, PL: n = 13). Correlations between IFN‐γ producing CD4 + T cells and lymphocyte numbers (D) and proviral loads (E), TNF‐α producing CD4 + T cells, lymphocyte numbers (F), and proviral loads (G) in BLV‐infected cattle ( n = 27). Lymphocyte numbers in BLV‐infected cattle were counted using Celltac α MEK‐6450 and animals were diagnosed with PL when at least two consecutive lymphocyte counts were >7,500/µL; proviral loads were quantified using real‐time PCR. IFN‐γ‐ and TNF‐α‐producing CD4 + T cells in BLV‐infected cattle were detected using flow cytometry.

Article Snippet: After 24 h, supernatants were harvested and IFN‐γ (Mabtech, Nacka Strand, Sweden) and TNF‐α (Kingfisher Biotech) production was determined using ELISA kits with a microplate reader MTP‐650FA (Corona Electric, Ibaraki, Japan) at an absorbance of 450 nm.

Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Immunity, Inflammation and Disease

Article Title: Bovine leukemia virus reduces anti‐viral cytokine activities and NK cytotoxicity by inducing TGF‐β secretion from regulatory T cells

doi: 10.1002/iid3.93

Figure Lengend Snippet: Inhibition of IFN‐γ and TNF‐α production from CD4 + T cells by TGF‐β. PBMCs from normal cattle (IFN‐γ: n = 12, TNF‐α: n = 9) and BLV‐infected cattle ( n = 11) were pretreated with TGF‐β for 2 h and were cultivated with IL‐2 alone (A and B) or synthesized peptides from the BLV envelop region (C and D). IFN‐γ‐ or TNF‐α‐producing CD4 + T cells were detected using flow cytometry.

Article Snippet: After 24 h, supernatants were harvested and IFN‐γ (Mabtech, Nacka Strand, Sweden) and TNF‐α (Kingfisher Biotech) production was determined using ELISA kits with a microplate reader MTP‐650FA (Corona Electric, Ibaraki, Japan) at an absorbance of 450 nm.

Techniques: Inhibition, Infection, Synthesized, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Effect of microglial Pd1 on glial scar formation after spinal cord injury in mice

doi: 10.1016/j.jbc.2025.108489

Figure Lengend Snippet: Microglia was involved in the functional recovery after SCI by regulating inflammatory response . A , immunofluorescence staining of Iba1 was conducted on the spinal cords of mice in sham group, control group, SCI + saline group and SCI + minocycline group. The sham group's mice underwent laminectomy without SCI and were sampled concurrently with the other groups. The scale bar represented 100 μm. B , quantification of Iba1 immunostaining intensity in the spinal cords of mice in sham group, control group, SCI + saline group, and SCI + minocycline group. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, as determined by one-way ANOVA. B : one-way ANOVA, F (3, 16) = 8.570, p = 0.0013; sham versus control, p = 0.0112; sham versus SCI + saline, p = 0.0012; SCI + saline versus SCI + minocycline, p = 0.0442; n = 5 mice per group). C , locomotor recovery evaluated by the BMS score acquired at different time points after SCI in mice treated with nothing, saline, or minocycline. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, as determined by two-way ANOVA. C : two-way ANOVA, F (12, 80) = 29.89, p < 0.0001; 7 days, SCI + saline versus SCI + minocycline, p = 0.0017; 14 days, SCI + saline versus SCI + minocycline, p = 0.0099; n = 6 mice per group). D – G , minocycline treatment reduced SCI-induced IL-6 and TNF-α mRNA expression and increased IL-10 mRNA expression in the injured spinal cord. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA. D : one-way ANOVA, F (3, 16) = 13.27, p = 0.0001; sham versus control, p = 0.0078; sham versus SCI + saline, p = 0.0002; SCI + saline versus SCI + minocycline, p = 0.0022; n = 5 mice per group) E : one-way ANOVA, F (3, 16) = 2.660, p = 0.0834; n = 5 mice per group) F : one-way ANOVA, F (3, 15) = 13.48, p = 0.0002; sham versus control, p = 0.0013; sham versus SCI + saline, p = 0.0002; sham versus SCI + minocycline, p = 0.0014; control versus SCI + minocycline, p = 0.0001; n = 4–5 mice per group) G : one-way ANOVA, F (3, 16) = 26.44, p < 0.0001; sham versus control, p < 0.0001; sham versus SCI + saline, p = 0.0001; control versus SCI + minocycline, p < 0.0001; SCI + saline versus SCI + minocycline, p < 0.0001; n = 5 mice per group). H , locomotor recovery evaluated by the BMS score acquired at different time points after SCI in mice treated with nothing, saline, minocycline, minocycline (+TNF-α), minocycline (+IL-6), and minocycline (+IL-10 neutralizing antibody). All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by two-way ANOVA. H : two-way ANOVA, F (18, 134) = 1.336, p = 0.1754; 14 days, SCI + minocycline versus SCI + minocycline (+TNF-a), p = 0.0002; SCI + minocycline versus SCI + minocycline (+IL-6), p = 0.0014; SCI + minocycline versus SCI + minocycline (+IL-10 neutralizing antibody), p = 0.0160; n = 5 to 6 mice per group). BMS, Basso Mouse Scale; IL-6, interleukin-6; SCI, spinal cord injury; TNF-α, tumor necrosis factor-alpha.

Article Snippet: TNF-α (Novus, NBP2-35185), IL-6 (MCE, HY-P7063), and IL-10 neutralizing antibody (MCE, HY-P990001) were applied at doses of 10 ng, 10 ng, and 2 μg, respectively, dissolved in 2 μl of minocycline solution.

Techniques: Functional Assay, Immunofluorescence, Staining, Control, Saline, Immunostaining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Effect of microglial Pd1 on glial scar formation after spinal cord injury in mice

doi: 10.1016/j.jbc.2025.108489

Figure Lengend Snippet: Cytokine array analysis revealed Pd1-dependent cytokines and chemokines in LPS-treated microglia . A , qPCR showing the decreased Pd1 mRNA in the microglia after Pd1 siRNA (20 nM and 50 nM) treatment for 48 h. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by one-way ANOVA. A : ANOVA, F (2, 12) = 15.32, p = 0.0005; NC siRNA versus Pd1 siRNA 20 nM, p = 0.0022; NC siRNA versus . Pd1 siRNA 50 nM, p = 0.0004; n = 5 wells per group). B , array membranes of protein expression among NC siRNA, NC siRNA + LPS (5 μg/ml), Pd1 siRNA (50 nM), and Pd1 siRNA (50 nM) + LPS (5 μg/ml) groups in culture medium for microglia. C , the expression of one colony-stimulating factor (G-CSF), one intercellular cell adhesion molecule (CD54), two inflammatory factors (IL-6 and TNF-α), and four chemokines (CXCL10, CCL12, CXCL9, and CCL5) significantly increased following LPS treatment, while the expression of one chemokine (CCL4) decreased. A significant increase in the expression of one inflammatory factor (TNF-α) and one chemokine (CXCL9) after treatment with Pd1 siRNA + LPS. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by two-way ANOVA. (Figure 6C: two-way ANOVA; G-CSF, NC siRNA versus Pd1 siRNA + LPS, p = 0.0016, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0021; CD54, NC siRNA versus Pd1 siRNA, p = 0.0245, NC siRNA versus Pd1 siRNA + LPS, p = 0.0185; IL-6, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001; Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; CXCL10, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0481; CCL12, NC siRNA versus NC siRNA + LPS, p = 0.0012, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017, NC siRNA + LPS versus Pd1 siRNA, p = 0.0012; Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0017; CXCL9, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0156, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0145; CCL4, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p = 0.0062, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0143; CXCL2, NC siRNA versus NC siRNA + LPS, p = 0.0121, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017; CCL5, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; TNF-α, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0442, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; n = 3–4 separate experiments per group). G-CSF, granulocyte colony-stimulating factor; IL-6, interleukin-6; LPS, lipopolysaccharide; NC, negative control; Pd1, programmed cell death protein 1; qPCR, quantitative real-time PCR; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-alpha.

Article Snippet: TNF-α (Novus, NBP2-35185), IL-6 (MCE, HY-P7063), and IL-10 neutralizing antibody (MCE, HY-P990001) were applied at doses of 10 ng, 10 ng, and 2 μg, respectively, dissolved in 2 μl of minocycline solution.

Techniques: Expressing, Negative Control, Real-time Polymerase Chain Reaction, Small Interfering RNA