Journal: Journal of Traditional and Complementary Medicine

Article Title: Betanin combined with virgin coconut oil inhibits neuroinflammation in aluminum chloride-induced toxicity in rats by regulating NLRP3 inflammasome

doi: 10.1016/j.jtcme.2023.11.001

Figure Lengend Snippet: Effect of BET and VCO on molecular markers. (A) NLRP3 (B) IL-1β Data are expressed as mean ± SEM, **p < 0.01, *p < 0.05when compared with normal control, ****p < 0.0001 ***p < 0.001**p < 0.01,*p < 0.05 when compared to treatment group.

Article Snippet: To determine NLRP3, a commercially available Rat Nod-like Receptor Pyrin-3, NLRP3 GENLISA™ ELISA (Krishgen Biosystems, India), was used per the manufacturer's instructions.

Techniques: Control

Journal: Journal of Traditional and Complementary Medicine

Article Title: Betanin combined with virgin coconut oil inhibits neuroinflammation in aluminum chloride-induced toxicity in rats by regulating NLRP3 inflammasome

doi: 10.1016/j.jtcme.2023.11.001

Figure Lengend Snippet: BET combined with VCO prevents neuroinflammation by inhibiting NLRP3 inflammasome activation in microglia.

Article Snippet: To determine NLRP3, a commercially available Rat Nod-like Receptor Pyrin-3, NLRP3 GENLISA™ ELISA (Krishgen Biosystems, India), was used per the manufacturer's instructions.

Techniques: Activation Assay

Journal: Oncotarget

Article Title: Alcohol intake aggravates adipose browning and muscle atrophy in cancer-associated cachexia

doi: 10.18632/oncotarget.22243

Figure Lengend Snippet: (A-C) Pro-caspase 1, activated caspase 1, Pro-caspase 3 and activated caspase 3 protein contents in GA muscle. (D-F) NLRP3 and IL-1β protein contents in GA muscle. ( * p < 0.05, mean ± SEM).

Article Snippet: Antibody against PYD-domains-containing protein 3 (NLRP3) (#PA1665) was purchased from Boster Biological Technology (Fremont, CA).

Techniques:

Journal: PLoS Pathogens

Article Title: Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells

doi: 10.1371/journal.ppat.1008752

Figure Lengend Snippet: (A) Schematic presentation of the domain structure of human PYHIN proteins. Numbers indicate amino acid positions. (B) Expression of human PYHIN proteins. HEK293T cells were transfected with IRES-GFP constructs expressing the indicated HA-tagged PYHIN proteins. GFP and GAPDH were used as controls. Stars indicates bands of the expected size. (C) Levels of indicated viral transcripts in HEK293T cells cotransfected with increasing doses of expression constructs for IFI16, MNDA, PYHIN1 and AIM2 and constant quantities of the proviral HIV-1 NL4-3 construct determined by qRT-PCR in multiplex reactions. Data show mean percentages (±SEM; n = 3) relative to those detected in the presence in the empty control plasmid (100%). * p <0.05, ** p <0.01, *** p <0.001. Primers and probes were designed as previously described . (D) Infectious virus yields in cell supernatants (left) and levels of LTR-dependent eGFP expression (right) in HEK293T cells transfected with vectors coexpressing the indicated PYHIN proteins and eGFP and the proviral HIV-1 NL4-3 IRES-eGFP construct were determined by TZM-bl cell infection assay and flow cytometric analysis at two days post-transfection. (E) HEK293T cells were cotransfected with luciferase reporter constructs under the control of the indicated promoters (MIEP, major immediate early promoter) and expression constructs for IFI16, PYHIN1, MNDA and AIM2 or a vector control. A construct expressing NL4-3 Tat under the control of the CMV IE promoter was used to activate the LTR. Shown are mean values (±SEM) measured in the presence of PYHIN proteins relative to the vector control (100%) obtained in three experiments.

Article Snippet: Proteins were stained using primary antibodies directed against IFI16 (Santa Cruz #sc-8023), HA-tag (Abcam #ab18181 or Cell Signaling #3724), PYHIN1 (ProSci #61–908), MNDA (Santa Cruz #sc-390739), AIM2 (Santa Cruz #sc-515514), GFP/BFP (Abcam #ab290) GAPDH (BioLegend #607902 or Santa Cruz #sc-365062), Sp1 (Abcam #157123 or Abcam #227383) and Infrared Dye labeled secondary antibodies (LI-COR IRDye).

Techniques: Expressing, Transfection, Construct, Quantitative RT-PCR, Multiplex Assay, Control, Plasmid Preparation, Virus, Infection, Luciferase

Journal: PLoS Pathogens

Article Title: Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells

doi: 10.1371/journal.ppat.1008752

Figure Lengend Snippet: (A) Expression of PYHIN proteins in primary HIV-1 target cells. CD4+ T lymphocytes were stimulated with IL-2 alone, IL-2 + IFNα (500 U/ml), IL-2 + IFNγ (200 U/ml) or IL-2 + anti-CD3/CD28 beads. Macrophages (MDM) were stimulated with either IFNα (500 U/ml) or IFNγ (200 U/ml). 72 hours post stimulation, cells were harvested and lysed for Western Blot analysis. (B). Knock-down efficiencies in human MDM. Cells were treated with PYHIN-specific or control siRNA. Each point represents protein expression levels compared to cells treated with control siRNA (100%) obtained from one donor. (C) Expression levels of the indicated PYHIN proteins in HIV-1 infected compared to uninfected MDM cultures (100%). **, p<0.01. (D) Infectious virus production at 3 or 6 days post-infection (dpi) from MDM treated with the indicated siRNAs. In the nuclear PYHIN panel, siRNAs targeting IFI16, PYHIN1 and MNDA were combined. Numbers give average n-fold enhancement of infectious virus yield compared to cells treated with the control siRNA.

Article Snippet: Proteins were stained using primary antibodies directed against IFI16 (Santa Cruz #sc-8023), HA-tag (Abcam #ab18181 or Cell Signaling #3724), PYHIN1 (ProSci #61–908), MNDA (Santa Cruz #sc-390739), AIM2 (Santa Cruz #sc-515514), GFP/BFP (Abcam #ab290) GAPDH (BioLegend #607902 or Santa Cruz #sc-365062), Sp1 (Abcam #157123 or Abcam #227383) and Infrared Dye labeled secondary antibodies (LI-COR IRDye).

Techniques: Expressing, Western Blot, Knockdown, Control, Infection, Virus

Journal: PLoS Pathogens

Article Title: Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells

doi: 10.1371/journal.ppat.1008752

Figure Lengend Snippet: (A) HEK293T cells were transfected with constructs coexpressing the indicated full-length (wt) form of IFI16, PYHIN1, MNDA and AIM2 or just the N-terminal PYD or PYD and linker region and BFP or a vector control and expression was analyzed by Western blot. BFP and GAPDH are used as transfection and loading control, respectively. (B) Effect of mutant PYHIN proteins on infectious virus yield (black) and levels of LTR-dependent eGFP expression (green). HEK293T cells were cotransfected with constructs coexpressing the indicated wt and mutant PYHIN proteins and proviral HIV-1 NL4-3 IRES-eGFP constructs. At 40 hours post-transfection, BFP and eGFP expression was analyzed by flow cytometry. Shown are mean values (±SEM) derived from seven experiments relative to those obtained in the presence of the empty control vector (100%). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Proteins were stained using primary antibodies directed against IFI16 (Santa Cruz #sc-8023), HA-tag (Abcam #ab18181 or Cell Signaling #3724), PYHIN1 (ProSci #61–908), MNDA (Santa Cruz #sc-390739), AIM2 (Santa Cruz #sc-515514), GFP/BFP (Abcam #ab290) GAPDH (BioLegend #607902 or Santa Cruz #sc-365062), Sp1 (Abcam #157123 or Abcam #227383) and Infrared Dye labeled secondary antibodies (LI-COR IRDye).

Techniques: Transfection, Construct, Plasmid Preparation, Control, Expressing, Western Blot, Mutagenesis, Virus, Flow Cytometry, Derivative Assay

Journal: PLoS Pathogens

Article Title: Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells

doi: 10.1371/journal.ppat.1008752

Figure Lengend Snippet: (A) Inhibition of different primary HIV-1 IMCs by human PYHIN proteins. HEK293T cells were cotransfected with the indicated proviral HIV-1 constructs and a control plasmid or vectors expressing IFI16, PYHIN1 or MNDA. Infectious virus yields in the presence of one PYHIN protein relative to the vector control (100%) was determined by infection of TZM-bl cells. Each symbol represents the average values obtained in three independent experiments. (B) The PYD plus linker region of IFI16, MNDA, PYHIN1 and AIM2 is sufficient for antiretroviral activity. HEK293T cells were transfected and analyzed for infectious virus yield as described in panel A. Shown are average values obtained from four experiments (± SD). * p< 0.05, ** p <0.01, *** p <0.001. (C-E) PYHIN proteins compete with DNA for Sp1 binding. HEK293T cells were transfected with empty vector or expression constructs for the C-terminally HA-tagged PYHIN proteins. 40 hours post transfection, cells were lysed in Co-IP buffer and left untreated or treated with Benzonase before immunoprecipitating the PYHIN proteins using an anti-HA mouse antibody and magnetic beads. (C) Prior to immunoprecipitation, lysates were run on a 1% agarose gel to verify DNA degradation. (D) Proteins were blotted onto PVDF membranes and stained using anti-Sp1 goat, anti-HA rabbit and anti-GAPDH rat antibodies. (E) Mean Sp1 signal intensities (±SD) minus background control measured in three independent experiments. (F, G) HEK293T cells were transfected with expression constructs for the indicated PYHIN protein or their N-terminal PYD and linker regions. 40 hours later, cells were harvested and normalized quantities of nuclear extracts were used for the TransAM Sp1 binding assay (ActiveMotif). Data represent mean values (± SEM) obtained from three experiments.

Article Snippet: Proteins were stained using primary antibodies directed against IFI16 (Santa Cruz #sc-8023), HA-tag (Abcam #ab18181 or Cell Signaling #3724), PYHIN1 (ProSci #61–908), MNDA (Santa Cruz #sc-390739), AIM2 (Santa Cruz #sc-515514), GFP/BFP (Abcam #ab290) GAPDH (BioLegend #607902 or Santa Cruz #sc-365062), Sp1 (Abcam #157123 or Abcam #227383) and Infrared Dye labeled secondary antibodies (LI-COR IRDye).

Techniques: Inhibition, Construct, Control, Plasmid Preparation, Expressing, Virus, Infection, Activity Assay, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Magnetic Beads, Immunoprecipitation, Agarose Gel Electrophoresis, Staining

Journal: PLoS Pathogens

Article Title: Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells

doi: 10.1371/journal.ppat.1008752

Figure Lengend Snippet: (A, B) Impact of IFI16, PYHIN1, MNDA and AIM2 on (A) SV40 and (B) HBV infection. An empty vector was used as negative control. (C, D) Determinants of the ability of IFI16 or other human PYHIN proteins to inhibit (C) SV40 and (D) HBV). CV-1 and HepG2 cells were transfected with either an empty vector or expression constructs for full length or the indicated mutants of IFI16 and PYHIN proteins. At 24 h post transfection, cells were infected with SV40 or HBV. SV40 infection was evaluated via intracellular staining of the Large T antigen while HBV replication was assessed by quantification of the HBeAg released into the supernatant. Shown are the results from two to three independent experiments ± SD (E, F) HEK293T cells were cotransfected with (C) LINE-1 or (D) CMV promoter constructs driving luciferase gene expression and expression constructs for IFI16 or a control vector. Luciferase activities (relative light units, RLU) in cell lysates were determined two days later (n = 3 ± SEM). (G) HEK293T cells were cotransfected with a retrotransposition-competent (L1) or -defective (L1 neg) LINE 1-GFP reporter plasmid and a vector control or expression constructs for PYHIN proteins or SAMHD1. Five days post-transfection, GFP+ cells were quantified by flow cytometry (n = 3 ± SEM).

Article Snippet: Proteins were stained using primary antibodies directed against IFI16 (Santa Cruz #sc-8023), HA-tag (Abcam #ab18181 or Cell Signaling #3724), PYHIN1 (ProSci #61–908), MNDA (Santa Cruz #sc-390739), AIM2 (Santa Cruz #sc-515514), GFP/BFP (Abcam #ab290) GAPDH (BioLegend #607902 or Santa Cruz #sc-365062), Sp1 (Abcam #157123 or Abcam #227383) and Infrared Dye labeled secondary antibodies (LI-COR IRDye).

Techniques: Infection, Plasmid Preparation, Negative Control, Transfection, Expressing, Construct, Staining, Luciferase, Gene Expression, Control, Flow Cytometry

Journal: Cellular and Molecular Immunology

Article Title: Carbon monoxide decreases interleukin-1β levels in the lung through the induction of pyrin

doi: 10.1038/cmi.2015.79

Figure Lengend Snippet: ( a ) THP-1 cells were stimulated with LPS (1 μg mL −1 ) in the absence or presence of CORM-2 (20 μM) at indicated time, and then ATP (5 mM) was added for the last 30 min. Secretion of IL-1β in the supernatants was quantified by ELISA. ( b and c ) THP-1 cells were pretreated with CORM-2 at indicated concentrations for 30 min, and then treated with LPS (1 μg mL −1 ) for 12 h and ATP (5 mM) was added for the last 30 min. ( b ) Secreted IL-1β in the supernatants was measured by ELISA. ( c ) The pro-caspase-1, cleaved caspase-1 (p10), pro-IL-1β, and IL-1β p17 in the supernatants (Sup) and the cell lysates (Lys) were analyzed by western blot analysis. ( d and e ) The expression of IL-10 mRNA was measured by quantitative real-time PCR in the various concentrations of CORM-2 or RuCl 2 for 12 h ( d ) and CORM-2 (20 μM) for increasing time intervals ( e ). ( f ) IL-10 protein was determined by western blot. ( g and h ) THP-1 cells were inhaled with different times of CO gas (250 ppm). Expression of IL-10 mRNA ( g ) and protein ( h ) was assayed by quantitative real-time PCR and western blot. ( i ) THP-1 cells were stimulated with LPS (1 μg mL −1 ) in the absence or presence of CORM-2 (20 μM) at indicated time, and then ATP (5 mM) was added for the last 30 min. Secreted IL-10 was detected by ELISA in the supernatants. ( j – l ) THP-1 cells were transfected with IL-10 siRNA or control siRNA (con), pre-treated with or without 20 μM CORM-2 for 30 min, and then treated with LPS (1 μg mL −1 ) for 12 h and ATP (5 mM) was added for the last 30 min. ( j ) The effectiveness of pyrin knockdown was confirmed by quantitative real-time PCR. ( k ) Western blot of the cellular extracts probed for the indicated protein. ( l ) ELISA for IL-1β in the supernatants. GAPDH and β-actin served as a loading control for quantitative real-time PCR and western blot analysis, respectively. The relative band density on western blots was normalized to the β-actin loading control and was quantitated. Representative data are shown. Data are expressed as mean ± SEM ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.005.

Article Snippet: The siRNA duplexes corresponding to human pyrin and IL-10 were purchased from Santa Cruz Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Knockdown

Journal: Cellular and Molecular Immunology

Article Title: Carbon monoxide decreases interleukin-1β levels in the lung through the induction of pyrin

doi: 10.1038/cmi.2015.79

Figure Lengend Snippet: ( a – d ) THP-1 cells were transfected with pyrin siRNA or control siRNA (con), pre-treated with or without 20 μM CORM-2 for 30 min, and then treated with LPS (1 μg mL −1 ) for 12 h and ATP (5 mM) was added for the last 30 min. ( a ) The knockdown of pyrin by siRNA was confirmed by quantitative real-time PCR. ( b ) The pro-caspase-1, cleaved caspase-1 (p10), pro-IL-1β, and IL-1β p17 in the supernatants (Sup) and the cell lysates (Lys) were analyzed by western blot analysis. Secreted IL-1β ( c ) and TNF-α ( d ) in the supernatants were measured by ELISA. ( e ) THP-1 cells were exposed with LPS (1 μg mL −1 ) + ATP (5 mM) in the absence or presence of CORM-2 (20 μM). The interaction of pyrin and pro-caspase-1 was analyzed by immunoprecipitation (IP) of anti-Pyrin followed by western blot (WB) for anti-pro-caspase-1. To evaluate the binding ASC and NLRP3, cell lysates were performed IP with anti-ASC and then followed by WB for anti-NLRP3. Total cell lysates were used as input controls. ( f and g ) THP-1 cells were transfected with pyrin siRNA or control siRNA (con), cells were exposed with 20 μM of CORM-2 for 12 h. The levels of pyrin mRNA ( f ) and protein ( g ) were measured. GAPDH and β-actin were used as a loading control for quantitative real-time PCR or western blot analysis, respectively. The relative band density on western blots was normalized to the β-actin loading control and was quantitated. All experiments were performed in triplicate, and representative data are shown. Data are expressed as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.005.

Article Snippet: The siRNA duplexes corresponding to human pyrin and IL-10 were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Binding Assay