Journal: PLoS Pathogens

Article Title: MERS-CoV ORF4b is a virulence factor involved in the inflammatory pathology induced in the lungs of mice

doi: 10.1371/journal.ppat.1010834

Figure Lengend Snippet: Taqman assays.

Article Snippet: , CXCL10 , Interferon gamma-induced protein 10 (IP-10) , Mm00445235-m1.

Techniques: Ubiquitin Proteomics

Journal: Biomedicines

Article Title: Suppression of Bcl3 Disrupts Viability of Breast Cancer Cells through Both p53-Dependent and p53-Independent Mechanisms via Loss of NF-κB Signalling

doi: 10.3390/biomedicines12010143

Figure Lengend Snippet: Bcl3 loss induces p53-independent apoptosis and p53-dependent senescence in breast cancer cells. Bcl3 was suppressed in breast cancer cell lines with the addition of Annexin V reagent to determine apoptosis using IncuCyte analysis software over 6 days. ( A ) MCF-7, ( B ) ZR751, ( C ) T47D and ( D ) BT474 cells each showing a significant increase in Annexin V staining; ( E ) representative images of each cell line at the endpoint. Following 6 days of siRNA, cells were harvested or fixed for further analysis. ( F ) PUMA expression determined by qRT-PCR showing an increase in three of the cell lines following Bcl3 suppression. Immunofluorescence staining for cell cycle markers ( G ) ki67 and ( H ) pH3 showed a reduction in the percentage of positively stained MCF-7 and ZR751 cells. ( I ) Senescence-associated β-gal staining was increased in Bcl3 suppressed MCF-7 and ZR751 cells, ( J ) representative images at endpoint, scale bar = 100 µm. qRT-PCR analysis showed a significant increase in ( K ) p21 expression in MCF-7 and ZR751 cells, while ( L ) p15 expression was upregulated in each cell line following Bcl3 suppression. SASP genes ( M ) IL-6, ( N ) IL-8 and ( O ) CXCL10, were also upregulated in p53 wildtype MCF-7 and ZR751 cells but not in p53-mutant T47D or BT474 cells. Error bars represent ± SEM of n = 3 minimum, with statistical differences determined using a 2-tailed unpaired t test, * p < 0.05. ** p < 0.01. Expanded versions of ( E , J ) are available in , respectively.

Article Snippet: Multiplex qRT-PCR reactions were performed using a QuantStudio 7 Real-Time PCR machine (Applied Biosystems, Altrincham, UK) using predesigned TaqMan primers (ThermoFisher Scientific, Altrincham, UK) designed to target Bcl3 (Hs00180403_m1), CDKN1A (p21) (Hs00355782_m1), CDKN2B (p15) (Hs00793225_m1), IL-6 (Hs00985639_m1), IL-8 (Hs00174103_m1), CXCL10 (Hs01124251_g1), BBC3 (PUMA) (Hs00248075_m1) and ACTB (Hs99999903_m1).

Techniques: Software, Staining, Expressing, Quantitative RT-PCR, Immunofluorescence, Mutagenesis

Journal: Biomedicines

Article Title: Suppression of Bcl3 Disrupts Viability of Breast Cancer Cells through Both p53-Dependent and p53-Independent Mechanisms via Loss of NF-κB Signalling

doi: 10.3390/biomedicines12010143

Figure Lengend Snippet: Loss of p53 rescues senescence in Bcl3-suppressed cells. Bcl3 and p53 were suppressed either alone or together in MCF-7 and ZR751 cells for 6 days. The suppression of Bcl3 and p53 was confirmed via qRT-PCR in ( A ) MCF-7 and ( E ) ZR751 cells. ( B , F ) Senescence-associated β-gal staining was increased following Bcl3 suppression, with no change observed when p53 was suppressed alone or with Bcl3. The percentage of cells positively stained for the cell cycle markers ( C , G ) ki67 and ( D , H ) pH3 was reduced in MCF-7 and ZR751 cells following Bcl3 inhibition, with no change following p53 inhibition alone or with Bcl3. ( I , J ) qRT-PCR analysis of p15 and p21 expression showed that the significant increase in p21 expression following Bcl3 inhibition was mitigated when p53 was also suppressed in both cell lines. ( K ) The expression of SASP genes IL-6, IL8 and CXCL10 was not rescued in MCF-7 cells following the combined suppression of p53 and Bcl3. Cell titre blue analysis demonstrated no further loss in cell viability in ( L ) MCF-7 or ( M ) ZR751 cells following combined Bcl3 and p53 suppression Error bars represent ± SEM of n = 3 with statistical differences determined using a one-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Multiplex qRT-PCR reactions were performed using a QuantStudio 7 Real-Time PCR machine (Applied Biosystems, Altrincham, UK) using predesigned TaqMan primers (ThermoFisher Scientific, Altrincham, UK) designed to target Bcl3 (Hs00180403_m1), CDKN1A (p21) (Hs00355782_m1), CDKN2B (p15) (Hs00793225_m1), IL-6 (Hs00985639_m1), IL-8 (Hs00174103_m1), CXCL10 (Hs01124251_g1), BBC3 (PUMA) (Hs00248075_m1) and ACTB (Hs99999903_m1).

Techniques: Quantitative RT-PCR, Staining, Inhibition, Expressing

Journal: Biomedicines

Article Title: Suppression of Bcl3 Disrupts Viability of Breast Cancer Cells through Both p53-Dependent and p53-Independent Mechanisms via Loss of NF-κB Signalling

doi: 10.3390/biomedicines12010143

Figure Lengend Snippet: Loss of p52 and p50 induces senescence and SASP, respectively. NF-κB family members p50 and p52 were suppressed by siRNA for 6 days in ( A ) MCF-7 and ( B ) ZR751 cells and confirmed via qRT-PCR. ( C , D ) Senescence-associated β-gal staining was significantly increased following p52 suppression compared to that of scRNA controls in both cell lines in a similar response to that seen following Bcl3 knockdown. Analysis via qRT-PCR identified a significant increase in ( E ) p15 and ( F ) p21 expression in MCF-7 cells following p52 inhibition, with a similar trend in p15 expression also observed in ZR751 cells. The percentage of ki67- ( G , H ) and pH3- ( I , J ) positive cells was reduced in both cell lines treated with p52 siRNA in a similar manner to that of Bcl3 suppression, with no change observed following the suppression of p50. The expression of SASP genes ( K ) IL-6, ( L ) IL-8 and ( M ) CXCL10 was increased following p50 suppression and not following p52 knockdown. ( N ) Graphical illustration of how Bcl3 suppression in p53 wildtype breast cancer cells results in the induction of senescence and SASP through inhibiting p52 and p50 signalling, respectively. Error bars represent ± SEM of n = 3 with statistical differences determined using a one-way ANOVA; * p < 0.05, ** p < 0.01, and *** p < 0.01.

Article Snippet: Multiplex qRT-PCR reactions were performed using a QuantStudio 7 Real-Time PCR machine (Applied Biosystems, Altrincham, UK) using predesigned TaqMan primers (ThermoFisher Scientific, Altrincham, UK) designed to target Bcl3 (Hs00180403_m1), CDKN1A (p21) (Hs00355782_m1), CDKN2B (p15) (Hs00793225_m1), IL-6 (Hs00985639_m1), IL-8 (Hs00174103_m1), CXCL10 (Hs01124251_g1), BBC3 (PUMA) (Hs00248075_m1) and ACTB (Hs99999903_m1).

Techniques: Quantitative RT-PCR, Staining, Knockdown, Expressing, Inhibition

Journal: Journal of Neuroimmunology

Article Title: N-acylethanolamine regulation of TLR3-induced hyperthermia and neuroinflammatory gene expression: A role for PPARα

doi: 10.1016/j.jneuroim.2021.577654

Figure Lengend Snippet: The effect of meth-AEA (i.c.v.) on poly I:C induced (a) hyperthermia and increases in (b) IP-10 , (c) TNF-α and (d) IL-1β expression in the hypothalamus, 4 h post poly I:C administration. Data expressed as mean ± SEM ( n = 5–9 per group). * p < 0.05; ** p < 0.01 vs vehicle-saline-treated counterparts.

Article Snippet: Assay IDs for the genes were as follows: IP-10 (Rn00594648_m1), IRF7 (Rn01450778_g1), TNFα (Rn99999017_m1), IL-1β (Rn00580432_m1), IL-10 (Rn00563409_m1), iNOS (NOS2) (Rn00561646_m1), COX-2 (Rn01483828_m1), m-PGE-s (Rn00572047_m1), SOCS1 (Rn00595838_s1) and SOCS3 (Rn00585674_s1) . β-actin was used as an endogenous control to normalise gene expression data.

Techniques: Expressing, Saline

Journal: Journal of Neuroimmunology

Article Title: N-acylethanolamine regulation of TLR3-induced hyperthermia and neuroinflammatory gene expression: A role for PPARα

doi: 10.1016/j.jneuroim.2021.577654

Figure Lengend Snippet: The effect of OEA or PEA on poly I:C induced (a) hyperthermia and increases in inflammatory gene expression of (b) IP-10 , (c) IRF7, (d) TNF-α, (e) IL-1β, (f) IL-10, (g) iNOS, (h) COX2, (i) MPGES, (j) SOCS1 and (k) SOCS3 in the hypothalamus, 4 h post poly I:C administration. Data expressed as mean ± SEM ( n = 6–8 per group). *** p < 0.001; ** p < 0.01; * p < 0.05 vs vehicle-saline-treated counterparts. ++ p < 0.01; + p < 0.05 vs vehicle-poly I:C-treated counterparts.

Article Snippet: Assay IDs for the genes were as follows: IP-10 (Rn00594648_m1), IRF7 (Rn01450778_g1), TNFα (Rn99999017_m1), IL-1β (Rn00580432_m1), IL-10 (Rn00563409_m1), iNOS (NOS2) (Rn00561646_m1), COX-2 (Rn01483828_m1), m-PGE-s (Rn00572047_m1), SOCS1 (Rn00595838_s1) and SOCS3 (Rn00585674_s1) . β-actin was used as an endogenous control to normalise gene expression data.

Techniques: Gene Expression, Saline

Journal: Journal of Neuroimmunology

Article Title: N-acylethanolamine regulation of TLR3-induced hyperthermia and neuroinflammatory gene expression: A role for PPARα

doi: 10.1016/j.jneuroim.2021.577654

Figure Lengend Snippet: The effect of OEA or PEA on poly I:C induced increases in inflammatory gene expression of (a) IP-10 , (b) IRF7, (c) TNF-α and (d) IL-1β in the spleen, 4 h post poly I:C administration. Data expressed as mean ± SEM (n = 6–8 per group). *** p < 0.001 vs vehicle-saline-treated counterparts.

Article Snippet: Assay IDs for the genes were as follows: IP-10 (Rn00594648_m1), IRF7 (Rn01450778_g1), TNFα (Rn99999017_m1), IL-1β (Rn00580432_m1), IL-10 (Rn00563409_m1), iNOS (NOS2) (Rn00561646_m1), COX-2 (Rn01483828_m1), m-PGE-s (Rn00572047_m1), SOCS1 (Rn00595838_s1) and SOCS3 (Rn00585674_s1) . β-actin was used as an endogenous control to normalise gene expression data.

Techniques: Gene Expression, Saline

Journal: Journal of Neuroimmunology

Article Title: N-acylethanolamine regulation of TLR3-induced hyperthermia and neuroinflammatory gene expression: A role for PPARα

doi: 10.1016/j.jneuroim.2021.577654

Figure Lengend Snippet: The effect of GW6471 on OEA-induced changes in (a) temperature and f (b) IP-10 , (c) IRF7, (d) TNF-α and (e) IL-1β expression in the hypothalamus. Data expressed as mean ± SEM ( n = 7–8 per group). * p < 0.05; ** p < 0.01 vs Veh-Veh-Saline. ++ p < 0.01 vs Veh-Veh-poly I:C. $ p < 0.05 vs Veh-OEA-poly I:C.

Article Snippet: Assay IDs for the genes were as follows: IP-10 (Rn00594648_m1), IRF7 (Rn01450778_g1), TNFα (Rn99999017_m1), IL-1β (Rn00580432_m1), IL-10 (Rn00563409_m1), iNOS (NOS2) (Rn00561646_m1), COX-2 (Rn01483828_m1), m-PGE-s (Rn00572047_m1), SOCS1 (Rn00595838_s1) and SOCS3 (Rn00585674_s1) . β-actin was used as an endogenous control to normalise gene expression data.

Techniques: Expressing, Saline

Journal: Journal of Neuroimmunology

Article Title: N-acylethanolamine regulation of TLR3-induced hyperthermia and neuroinflammatory gene expression: A role for PPARα

doi: 10.1016/j.jneuroim.2021.577654

Figure Lengend Snippet: The effect of systemic administration of WY14643 on poly I:C induced (a) hyperthermia and (b) increases in (b) IP-10 and (c) TNF-α expression in the hypothalamus. Data expressed as mean ± SEM (n = 6–8 per group). *** p < 0.001 ** p < 0.01 * p < 0.05 vs vehicle-saline.

Article Snippet: Assay IDs for the genes were as follows: IP-10 (Rn00594648_m1), IRF7 (Rn01450778_g1), TNFα (Rn99999017_m1), IL-1β (Rn00580432_m1), IL-10 (Rn00563409_m1), iNOS (NOS2) (Rn00561646_m1), COX-2 (Rn01483828_m1), m-PGE-s (Rn00572047_m1), SOCS1 (Rn00595838_s1) and SOCS3 (Rn00585674_s1) . β-actin was used as an endogenous control to normalise gene expression data.

Techniques: Expressing, Saline

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 1. Circulating CXCL10 in leprosy patients and healthy con- trols. Each point represents one sample from a different individual. The groups were healthy controls, borderline tuberculoid without re- action (BT), borderline lepromatous without reaction (BL), polar lep- romatous without reaction (LL), BT with reaction (BT plus RR), BL with reaction (BL plus RR), and LL patients with T2R (erythema nodosum leprosum). For patients with reaction (BT plus RR, BL plus RR, and T2R), the sample was taken at the time of reaction, prior to treatment. For LL, n 6; for all other groups, n 10. The horizontal lines indicate median values. The median level of CXCL10 in LL patients with type 2 reaction was not significantly elevated (P 0.9).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques:

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 2. Circulating CXCL10 in repeated samples from leprosy patients who developed T1R. Each graph depicts the measurements made from serum samples obtained at 1 month intervals in borderline tuberculoid (A, no. 1 to 10) and borderline lepromatous (B, no. 11 to 20) patients. All patients with clinical T1R received standard multidrug treatment starting at the first visit. The arrows indicate the visits at which T1R was diagnosed. The number above each time point indicates the dose of prednisone started on that day, tapered monthly as indicated; in some cases, corticosteroids were not used initially but were started 1 to 2 months after the initial clinical diagnosis of T1R. Each serum specimen was obtained before corticosteroid treatment was initiated. CXCL10 was significantly elevated during T1R in the 10 BT patients (A) (P 0.0006) and the 10 BL patients (B) (P 0.0001). In patients 12, 16, and 17 (B), T1R was diagnosed by histopathological criteria only; when they were excluded from the analysis, the association of T1R with CXCL10 remained significant (P 0.05).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Biomarker Discovery

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 3. Expression levels of CXCL10 and IFN- genes in skin biopsy specimens from leprosy patients. (A to H) Real-time quantitative RT-PCR was performed on cDNA obtained from sequential skin biopsy specimens from individual leprosy patients who were placed on MDT, none of whom had T1R at the time of the first biopsy. Five patients (A to E) had clinical symptoms of T1R at the time of the second biopsy; two of these (C and D) had a third biopsy after the reaction had resolved. Three patients (F to H) had no clinical symptoms of T1R at the time of the initial or second biopsy. Data were obtained using the standard-curve method and were normalized using 18S rRNA values, repeated three times for all determinations. The results are presented as the mean standard deviation.

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Cell death and differentiation

Article Title: RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8(+) T cells.

doi: 10.1038/cdd.2014.96

Figure Lengend Snippet: Figure 1 RLH activation induces secretion of proinflammatory cytokines and induction of apoptosis in murine pancreatic cancer cells. (a) Panc02 cells were stimulated with indicated amounts of ppp-RNA, poly(I:C) or left untreated. OH-RNA served as transfection control. IFN-b levels were analyzed with qRT-PCR relative to HPRT and secretion of CXCL10 or IL-6 was measured with ELISA; (b) Panc02 cells were stimulated with RNA (24 h for poly(I:C) and 48 h for ppp-RNA) and viability was assessed by FACS analysis using annexin V/PI staining; (c) Panc02 cells were incubated with siRNA specific for RIG-I or MDA5 for 24 h and subsequently stimulated with ppp-RNA or poly(I:C). Induction of apoptosis was measured by annexin V/PI staining. Silencing efficacy, as assessed by western blot, is shown; (d) activated caspase-9 (green) was visualized using green FLICA caspase-9 assay kit. Cell membranes were costained with cholera toxin B subunit (red) and nuclei with DAPI (blue); (e and f) Panc02 cells were treated as indicated for 48 h. Full length PARP-1 (116 kDa) and the cleaved large fragment of PARP-1 (89 kDa) (e) as well as the autophagy markers LC3B-I and LC3B-II (f) were analyzed by western blot. Results are representative of at least three independent experiments

Article Snippet: After fixation with paraformaldehyde and permeabilization with saponin (both Sigma), DCs were stained for IL-6 (PE, clone MP5-20F3; BioLegend) or goat anti-mouse CXCL10 (R&D Systems).

Techniques: Activation Assay, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood–Spinal Cord Barrier

doi: 10.3389/fimmu.2020.00477

Figure Lengend Snippet: CCI induces CXCL10/CXCR3 signaling activation. (A) Upregulated CXCL10 expression is observed in the ipsilateral spinal cord after CCI injury (** p < 0.01; independent t -test, n = 3). (B) Immunohistochemistry reveals the CCI-mediated induction of CXCR3 activation. (C) The spinal CXCR3 protein level increased during days 3–7 after CCI as shown by western blotting (** p < 0.01; *** p < 0.001; ANOVA, n = 3). The data are presented as means ± standard errors. GAPDH was used as a loading control. ANOVA, analysis of variance; CCI, chronic constriction injury; CXCL10, C-X-C motif chemokine 10; Contra, contralateral to CCI.

Article Snippet: A 20-μL volume of normal saline or a solution containing an anti-CXCL10 antibody (200 ng/rat, Proteintech) was injected into the subarachnoid space between the L5 and L6 vertebrae with a 30-gauge needle.

Techniques: Activation Assay, Expressing, Immunohistochemistry, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood–Spinal Cord Barrier

doi: 10.3389/fimmu.2020.00477

Figure Lengend Snippet: Neutralizing CXCL10 alleviated hyperalgesia and reduced CCI-induced BSCB disruption. (A,B) Hindpaw PWTs in CCI + anti-CXCL10 antibodies group and CCI + saline group rats on various experimental days (* p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA, n = 5–6). (C) Lumbar spinal cord NaFlu concentrations in the CCI + anti-CXCL10 antibodies group, CCI + saline group, and sham surgery group rats on experimental day 3 (* p < 0.01, CCI + NS group vs. sham surgery group; Mann–Whitney U- test, n = 4–6; ## p < 0.01, CCI + anti-CXCL10 antibodies group vs. CCI + NS group; independent t- test, n = 6). The data are shown as means ± standard errors. ANOVA, analysis of variance; BSCB, blood-spinal cord barrier; CCI, chronic constriction injury; CXCL10, C-X-C motif chemokine 10; NaFlu, sodium fluorescein; PWT, paw withdrawal threshold; NS, normal saline.

Article Snippet: A 20-μL volume of normal saline or a solution containing an anti-CXCL10 antibody (200 ng/rat, Proteintech) was injected into the subarachnoid space between the L5 and L6 vertebrae with a 30-gauge needle.

Techniques: Disruption, Saline, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood–Spinal Cord Barrier

doi: 10.3389/fimmu.2020.00477

Figure Lengend Snippet: Neutralizing CXCL10 reduced CCI-induced T cell infiltration of the dorsal horn. (A,B) CD3-positive T cell levels as percentages of CCI-ipsilateral dorsal horn mononuclear cell populations in the sham surgery group and CCI + NS group rats (** p < 0.01; independent t- test, n = 3–4). (B,C) CD3-positive T cell levels as percentages of CCI-ipsilateral dorsal horn mononuclear cell populations in CCI + NS group and CCI + anti-CXCL10 antibodies group rats ( # p < 0.05; independent t- test, n = 3). (D) Bar chart indicating the statistical analysis. The data are shown as means ± standard errors. CCI, chronic constriction injury; CXCL10, C-X-C motif chemokine 10; NS, normal saline.

Article Snippet: A 20-μL volume of normal saline or a solution containing an anti-CXCL10 antibody (200 ng/rat, Proteintech) was injected into the subarachnoid space between the L5 and L6 vertebrae with a 30-gauge needle.

Techniques: Saline