Journal: Medicina (Kaunas, Lithuania)

Article Title: Radiation Induces Bone Microenvironment Disruption by Activating the STING-TBK1 Pathway.

doi: 10.3390/medicina59071316

Figure Lengend Snippet: Figure 3. IR-induced expression changes in the secretion SASP components in a mouse model of bone degenerative injury. (A) The SASP composition in mice serum post-irradiation was detected by Mouse Cytokine microarray membrane analysis. Quantified expression levels of the secretory cytokines, expressed as corrected the pixel density, n = 2. The criteria for defining up- or downregulation was greater or less than 2 SD by the mean value of corrected pixel density in the control group. (B) Heatmap representation of cytokines based on antibody microarray results. Data are expressed as mean ± SD.

Article Snippet: The following reagents were purchased: lipofectamine RNAiMAX (cat. no. 13778100; Thermo Fisher Scientific, Inc., Waltham, MA, USA); opti-MEM (cat. no. 31985062; Gibco, Eggenstein, Germany); STING small interfering RNA (siRNA), negative control (NC)siRNA (GenePharma Co., Ltd., Shanghai, China); STING small-molecule inhibitor, C176 (cat. no. HY-112906; Med-ChemExpress, Princeton, NJ, USA); acid phosphatase, leukocyte (tartrate-resistant acid phosphatase; TRAP) Kit (cat. no. 387A-1KT; SigmaAldrich, St. Louis, MO, USA); mouse cytokine array (cat. no. ARY028; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Irradiation, Microarray, Membrane, Control

Journal: Medicina (Kaunas, Lithuania)

Article Title: Radiation Induces Bone Microenvironment Disruption by Activating the STING-TBK1 Pathway.

doi: 10.3390/medicina59071316

Figure Lengend Snippet: Figure 5. STING-P-TBK1 pathway blocking by siRNA alters inflammatory cytokines secretion and osteoclastogenic activity in an in vitro osteocyte model of irradiation. (A) The knockdown efficiency of STING was verified by western blot analyses. (B) After the STING-siRNA knockdown, the protein expression of P-TBK1 in MLO-Y4 cells was evaluated 3 days post-irradiation by western blot analysis. (C) Effect of STING-targeted siRNA intervention on the expression of bone resorption- related protein RANKL. (D) Effect of STING-targeted siRNA intervention on the mRNA expressions of inflammatory cytokines including IL-1α, IL-6, and NF-κB. (E) STING-targeted siRNA intervention on the paracrine regulation of osteoclastogenesis by IR-induced inflammatory cytokine from OCYs. The RAW264.7 cells were co-cultured with the conditioned medium (CM) of irradiated MLO-Y4 cells following STING-siRNA or NC siRNA intervention. The percentage of TRAP+ OCs was quantified (scale bar, 100 µm; magnification, ×100). Data are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. control), n = 3. NC siRNA: non-STING targeted siRNA interference; STING siRNA: STING targeted siRNA interference. The CM of MLO-Y4 cells was collected at 3 days after 0 Gy or 4 Gy irradiation with STING siRNA or NC siRNA intervention. CM-0 Gy-NC: non- irradiation with NC siRNA intervention; CM-4 Gy-NC: 4 Gy γ-radiation with NC siRNA intervention; CM-0 Gy-SI: non-irradiation with STING-siRNA intervention; CM-4 Gy-SI: 4 Gy γ-radiation with STING-siRNA intervention.

Article Snippet: The following reagents were purchased: lipofectamine RNAiMAX (cat. no. 13778100; Thermo Fisher Scientific, Inc., Waltham, MA, USA); opti-MEM (cat. no. 31985062; Gibco, Eggenstein, Germany); STING small interfering RNA (siRNA), negative control (NC)siRNA (GenePharma Co., Ltd., Shanghai, China); STING small-molecule inhibitor, C176 (cat. no. HY-112906; Med-ChemExpress, Princeton, NJ, USA); acid phosphatase, leukocyte (tartrate-resistant acid phosphatase; TRAP) Kit (cat. no. 387A-1KT; SigmaAldrich, St. Louis, MO, USA); mouse cytokine array (cat. no. ARY028; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Blocking Assay, Activity Assay, In Vitro, Irradiation, Knockdown, Western Blot, Expressing, Cell Culture, Control

Journal: Nature Communications

Article Title: High-fat diet-induced upregulation of exosomal phosphatidylcholine contributes to insulin resistance

doi: 10.1038/s41467-020-20500-w

Figure Lengend Snippet: a Plasma and WAT cytokine array of mice that received CD63 + A33 + exosomes (L-Exo or H-Exo) for 14 days. b Fold change in H-Exo vs. L-Exo-induced plasma cytokine expression for all cytokines showing greater than two-fold change. Red bars show cytokines/factors known to be involved in insulin resistance. c TNF-α (left) and IL-6 (right) upregulation following treatment with H-Exo were confirmed by ELISA in plasma. Filled circle—PBS, filled triangle—L-Exo, and filled rectangle—H-Exo. d ITT performed on C57BL/6 mice that received exosomes via adoptive transfer for 14 days followed with or without macrophage depletion. Filled rectangle—macrophage-depleted mice treated with H-Exo; filled diamond—mice without macrophage depletion treated with PBS and circle—mice without macrophage depletion treated with H-Exo. e Glucose uptake assay performed on hepatocytes cultured with different concentrations of H-Exo (as indicated in the figure). f Glucose uptake assay performed on mouse hepatocytes supplemented with supernatant derived from macrophages cultured with nanoparticles derived from H-Exo total lipids (H-Exo Nano) and PC (34:2). g Supernatants from H-Exo-treated macrophages (monocytes+ 5 × 10 6 ) were preneutralized with anti-TNF-α and/or anti-IL-6 antibodies. Glucose uptake by hepatocytes cultured in the presence of preneutralized supernatant was estimated. Data are represented as the mean ± SD. One-way ANOVA with a Tukey post hoc test. * < 0.05; **** < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Cytokines were analyzed with a Proteome Profiler Mouse XL Cytokine Array Kit (R&D Systems, ARY028) as per the manufacturer’s instructions.

Techniques: Clinical Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Cell Culture, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Pten loss in the bone marrow leads to G-CSF–mediated HSC mobilization

doi: 10.1084/jem.20122768

Figure Lengend Snippet: Increased level of mobilizing cytokines in pten ΔMx and pten ΔScl mice. (A and B) FACS plot (A) and histogram showing GCSF expression in BM CD11b + Gr-1 + cells of control and Pten ΔMx mice. Two independent experiments were done for controls ( n = 7) and mutants ( n = 8; *** indicates statistically significant change, P = 0.0008). (C) Cytokine array screen showing serum levels of indicated cytokines 15 d ( Pten ΔMx ) or 28 d ( Pten ΔScl ) after deletion. This screen has been performed once with n = 2 per group. (D) MCP-5 and IL-9 levels in splenic Gr-1 high /CD115 − and CD4 + CD8 + cells or BM CD11b + Gr-1 + and CD4 + CD8 + cells of Pten ΔScl mice and control counterparts. Data are shown as fold change as compared with control levels. P = 0.01, MCP-5 in Gr-1 high /CD115 − cells. P = 0.007, MCP-5 in CD4 + CD8 + cells. P = 0.03, IL-9 in BM CD4 + CD8 + cells. P = 0.04, IL-9 in BM CD11b + Gr-1 + cells. Three independent experiments were performed, n = 11 controls and n = 6 mutants. (E) CXCL-12 levels observed in osteoblasts (CD45 − CD31 − Ter119 − CD51 + Sca-1 − cells) and endothelial cells (CD45 − Ter119 − CD31 + ) of Pten ΔMx mice and controls. Data are shown as fold change as compared with control levels. Two independent experiments (5 controls and 10 mutants) were performed. P < 0.01, osteoblasts, P < 0.05 endothelial cells. Data are provided as mean ± SE. Unless otherwise indicated, * indicates statistically significant change with P < 0.05.

Article Snippet: Cytokine levels in the serum were quantified using the RayBio Mouse Cytokine Array III kit (Hoelzel Biotech) according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Journal of Neuroinflammation

Article Title: Inflammatory monocytes and microglia play independent roles in inflammatory ictogenesis

doi: 10.1186/s12974-022-02394-1

Figure Lengend Snippet: Viral load in the brain is not reduced in the low inoculum group, but inflammatory chemokine and cytokine production are reduced by more than 90% at 1 dpi. A Viral load was assessed at 1, 3, and 7 dpi in mice inoculated with 200,000 PFU or 12,500 PFU of TMEV. Despite a 16-fold reduction in the amount of virus delivered to the brain, the groups exhibit similar viral loads at each timepoint. F (5,70) = 0.1534, P = 0.6964 between 200,000 PFU and 12,500 PFU by two-way ANOVA on ranks (inoculum x day); Shapiro–Wilk P = 0.0033; Tukey HSD pairwise analysis on ranks: 200,000 PFU vs 12,500 PFU @ 1 dpi: P = 0.6938; @ 3 dpi: P = 0.9900; @ 7 dpi: P = 0.9989; results from 5 separate experiments. B , C Representative images of TMEV immunostaining (green) in CA1 pyramidal neurons at 3 dpi in a mouse inoculated with 200,000 PFU ( B ) or 12,500 PFU ( C ) of virus; DAPI is shown in blue. Boxes indicate higher magnification insets “ i ” and “ ii ”. The amount of CCL2 ( D ), TNFα ( E ), and IL6 ( F ) in hippocampal homogenates was measured at 24 and 72 hpi. All of the factors were profoundly reduced at 24 hpi in the mice inoculated with 12,500 PFU of TMEV relative to mice inoculated with 200,000 PFU, with TNFα and IL6 indistinguishable from vehicle control mice. D CCL2, F (2,12) = 318.3, P < 0.0001 by two-way ANOVA; Shapiro–Wilk P = 0.8826 (24 hpi); Tukey’s pairwise analysis: 24 hpi: 200,000 PFU vs 12,500 PFU: P < 0.0001; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P = 0.5123; 72 hpi: 200,000 PFU vs 12,500 PFU: P = 0.4512; 200,000 PFU vs vehicle: P = 0.9999; 12,500 PFU vs vehicle: P = 0.5252; d s @ 24 hpi = 11.8; results from 3 separate experiments. E TNFα, F (2,28) = 15.92, P < 0.0001 by two-way ANOVA; Shapiro–Wilk P = 0.7674; Tukey’s pairwise analysis: 24 hpi: 200,000 PFU vs 12,500 PFU: P < 0.0001; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P > 0.9999; 72 hpi: 200,000 PFU vs 12,500 PFU: P = 0.1562; 200,000 PFU vs vehicle: P = 0.3342; 12,500 PFU vs vehicle: P > 0.9999; d s @ 24 hpi = 4.6; results from 3 separate experiments. F IL6, F (2,28) = 28.975, P < 0.0001 by two-way ANOVA; Shapiro–Wilk P = 0.5052; Tukey’s pairwise analysis: 24 hpi: 200,000 PFU vs 12,500 PFU: P < 0.0001; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P = 0.9993; 72 hpi: 200,000 PFU vs 12,500 PFU: P = 0.9975; 200,000 PFU vs vehicle: P = 0.9914; 12,500 PFU vs vehicle: P > 0.9999; d s @ 24 hpi = 4.15; results from 3 separate experiments. Scale bar in C is 200 μm and refers to B ; scale bar in i is 100 μm and refers to i

Article Snippet: Levels of CCL2, TNFα, and IL6 were measured using a mouse inflammatory cytokine cytometric bead array multiplex panel (#552364, BD Biosciences).

Techniques: Immunostaining