Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Enzyme Replacement Therapy Can Reverse Pathogenic Cascade in Pompe Disease

doi: 10.1016/j.omtm.2020.05.026

Figure Lengend Snippet: AT-GAA Reversed the Levels of the Autophagic Markers and Alleviated the Burden of Accumulated Protein Aggregates and ER-Stress in Muscle from KO Mice Muscle biopsies were collected from age and sex-matched WT, untreated (KO), and AT-GAA-treated KO (KO-ERT) mice. (A) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with the indicated antibodies (n = 4 for each group; n = 5 for western blot with LC3 antibody). (B) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with K63-linked ubiquitin specific antibody (n = 4 for each group). Western blot with anti-GAPDH and Ponceau S staining were used as loading controls. (C) Western blot analysis of muscle lysates from WT, untreated KO (KO), and treated KO (KO-ERT) mice with anti-Grp 78 antibody (ER-stress marker; n = 4 for each group); GAPDH was used as loading control. Each data point represents an individual mouse. Statistical significance was determined by one-way ANOVA. Graphs represent mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: SQSTM1/p62 (#ab56416; mouse monoclonal), galectin 9 (#ab69630; rabbit polyclonal), Ubiquitin K48 (Linkage Specific) (#ab140601; rabbit monoclonal), and glyceraldehyde-3-phosphate dehydrogenase (#ab9485; rabbit polyclonal) were purchased from Abcam.

Techniques: Western Blot, Ubiquitin Proteomics, Staining, Marker, Control

Journal: ImmunoTargets and Therapy

Article Title: Recombinant Antithrombin Alleviated Pulmonary Injury and Inflammation in LPS-Induced ARDS by Inhibiting IL17a/NF-κB Signaling

doi: 10.2147/ITT.S502925

Figure Lengend Snippet: rAT reduced pulmonary inflammation in LPS-induced ARDS model mice. ( A–D ) The levels of inflammatory factors, including IL-6 ( A ), TNF-α ( B ), IL-8 ( C ), and hs-CRP ( D ), were decreased in the serum of the rAT-treated group, as detected by ELISA. ( E ) Immunohistochemical staining for F4/80 revealed that rAT treatment reduced the proportion of macrophages in the lung tissue of LPS-induced ARDS model mice. ( F ) Immunohistochemical staining for MRCI, a marker of M2 macrophages, showed that rAT treatment increased the proportion of M2 macrophages in the lung tissue of LPS-induced ARDS model mice. ( G ) Immunohistochemical staining for Ly6G, a marker of neutrophils, showed that rAT treatment reduced the proportion of neutrophils in the lung tissue of LPS-induced ARDS model mice. All the data are presented as the means ± SDs of three independent experiments. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant; scale bar, 50 μm.

Article Snippet: A mouse CXCL15 ELISA Kit (E-EL-M0269) and a mouse hs-CRP ELISA Kit (E-EL-M0677) were purchased from Elabscience (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Marker

Journal: ImmunoTargets and Therapy

Article Title: Recombinant Antithrombin Alleviated Pulmonary Injury and Inflammation in LPS-Induced ARDS by Inhibiting IL17a/NF-κB Signaling

doi: 10.2147/ITT.S502925

Figure Lengend Snippet: The efficacy of rAT in mitigating lung injury, suppressing the immune response, and inhibiting the activation of the NF-κB signaling pathway in LPS-induced ARDS mice were diminished by the administration of IL-17a. ( A ) ELISA results demonstrated that the administration of IL17a inhibited the ability of rAT to reduce inflammatory factors, including IL-6, TNF-α, and IL-8, in the serum of LPS-induced ARDS mice. ( B ) The analysis of the wet/dry weight ratio of the lung tissue revealed that the administration of IL17a counteracted the ability of rAT to alleviate pulmonary exudation in LPS-induced ARDS mice. ( C ) The administration of IL17a did not significantly affect the ability of rAT to reduce the number of cells in the BALF of LPS-induced ARDS mice. ( D ) The administration of IL17a attenuated the ability of rAT to reduce the concentrations of proteins in the BALF of LPS-induced ARDS mice. ( E ) Real-time PCR results showed that the administration of IL17a blocked the ability of rAT to downregulate the expression of target genes in the IL17a/NF-κB signaling pathway. ( F ) The protein levels of the NF-κB signaling pathway were assessed by Western blotting, and gray intensity analysis of the blots showed that the administration of IL17a in LPS-induced ARDS mice counteracted the ability of rAT to suppress the phosphorylation of IκBα, IKKα/β, and P65. The data are expressed as the means ± SDs (n=3 in each group). One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, and ns not significant.

Article Snippet: A mouse CXCL15 ELISA Kit (E-EL-M0269) and a mouse hs-CRP ELISA Kit (E-EL-M0677) were purchased from Elabscience (Wuhan, China).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: L5-LDL from ST-elevation myocardial infarction patients induces IL-1β production via LOX-1 and NLRP3 inflammasome activation in macrophages.

doi: 10.1152/ajpheart.00509.2016

Figure Lengend Snippet: Fig. 4. Knockdown of lectin-type oxidized LDL receptor (LOX-1) reduced NF-B activation and L5-LDL-induced IL-1 and cleaved caspase-1. A: effects of copper oxidation on L5-LDL-induced IL-1 production in THP-1 macrophages. L5-LDL was oxidized with copper for 0, 6, and 16 h in vitro and then the copper-oxidized L5-LDL was used to treat THP-1 macrophages for 24 h. The levels of IL-1 in the medium were determined by ELISA. *P 0.05, compared with the 0 h. B: THP-1 cells were infected with lentivirus carrying specific shRNA for LOX-1 and CD36, and knockdown efficiency was evaluated by RT-qPCR. *P 0.05, compared with the shLuc control. C: macrophages derived from shLOX-1, shCD36, or shLuc knockdown cells were incubated with L5-LDL (20 g/ml) for 24 h. D: macrophages derived from shLOX-1 or shLuc knockdown cells were incubated with LPS (100 pg/ml) for 24 h. Levels of IL-1 in the medium were determined by ELISA. LPS was used as a positive control. *P 0.05, compared with the L5-LDL-treated shLuc cells. E: levels of cleaved caspase-1 in the medium of PBS or L5-LDL-treated shLuc and shLOX-1 knockdown cells were detected by Western blotting. Coomassie blue staining was used as loading control. Values are the means SD for 3 independent experiments.

Article Snippet: A Quantikine Human IL-1 Enzyme-Linked Immunosorbent Assay (ELISA) kit and the Quantikine High Sensitivity Human IL-1 ELISA were obtained from R&D Systems (Minneapolis, MN).

Techniques: Knockdown, Activation Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Infection, shRNA, Quantitative RT-PCR, Control, Derivative Assay, Incubation, Positive Control, Western Blot, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Paeonol inhibits the translocation of HMGB1 from the nucleus to the cytoplasm. A, Total protein (left), nuclear protein (middle) and cytoplasmic protein (right) were extracted. The level of HMGB1 in each fraction was tested by using Western blotting. B, HMGB1 was stained with Cy3 (red), and nuclei were stained with DAPI (blue). HMGB1 was distributed in the cytoplasm (indicated by the white arrows). C, The relative mRNA expression level of HMGB1 was tested by using RT‐qPCR. The bar graphs are representative of three independent experiments. * P < .05, ** P < .01 and *** P < .001

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques: Translocation Assay, Western Blot, Staining, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Construction of HMGB1 + RAW264.7 and HMGB1 m RAW264.7 cells. A, Diagram of HMGB1 + and HMGB1 m RAW264.7 cell construction. B, Endogenous HMGB1 was stained red, exogenous HMGB1 (Flag tagged, expressed from a plasmid) was stained green, and nuclei were stained blue. HMGB1 in the cytoplasm is indicated by white arrows. C, The intensity of cytoplasmic HMGB1 staining was measured with ImageJ software. *** P < .001

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques: Staining, Plasmid Preparation, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Influence of paeonol on the inflammatory response in HMGB1 + RAW264.7 and HMGB1 m RAW264.7 cells. A, The mRNA levels of IL‐1β, TNF‐α, iNOS, TGF‐β1 and P65 in HMGB1 + RAW264.7 cells were measured by using RT‐qPCR. B, The protein levels of IL‐1β, TNF‐α, iNOS, TGF‐β1 and P65 in HMGB1 + RAW264.7 cells were measured by using Western blotting. C, The relative mRNA expression levels of IL‐1β, TNF‐α, iNOS, TGF‐β1 and P65 in HMGB1m RAW264.7 cells were measured by using RT‐qPCR. D, The protein levels of IL‐1β, TNF‐α, iNOS, TGF‐β1 and P65 in HMGB1 m RAW264.7 cells were measured by using Western blotting. E, HMGB1 was stained red, and nuclei were stained blue. HMGB1 in the cytoplasm is indicated with white arrows. The bar graphs are representative of three independent experiments. * P < .05, ** P < .01 and *** P < .001

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Paeonol attenuates the inflammatory response in vivo. A, A diagram of the animal experimental process is shown. B, The protein levels of iNOS, P65, TGF‐β1 and TNF‐α in peritoneal macrophages were tested by using Western blotting, n = 6. C, The protein levels of iNOS, P65, TGF‐β1 and TNF‐α in lung tissue were tested by using Western blotting, n = 6. D, HMGB1 in peritoneal macrophages and lung tissue was stained red. Nuclei were stained blue. HMGB1 in the cytoplasm is indicated by white arrows. D, HE staining was performed to observe the morphology of spleen, liver and lung tissues. * P < .05, ** P < .01 and *** P < .001

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques: In Vivo, Western Blot, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Paeonol has no effect on the inflammatory response in HMGB1 conditional knockout mice. A, The protein levels of iNOS, P65, TGF‐β1 and TNF‐α in peritoneal macrophages were tested by using Western blotting, n = 6. B, The protein levels of iNOS, P65, TGF‐β1 and TNF‐α in lung tissue were tested by using Western blotting, n = 6. C, HE staining was performed to observe the morphology of spleen, liver and lung tissues. * P < .05, ** P < .01 and *** P < .001

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques: Knock-Out, Western Blot, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Paeonol promotes the colocalization of HMGB1 and P65. A, Pathway analysis of the transcriptome is shown. B, The binding rates among HMGB1/P65/P50 were analysed by using mammalian cell hybrid technology. C, HMGB1 was stained red, P65 was stained green, and nuclei were stained blue. P65 was found in the cytoplasm in the control group. In the LPS group, P65 was found in the nucleus, and HMGB1 was found in the cytoplasm. The complex of P65 and HMGB1 was identified in the PL, PM and PH groups. The bar graphs are representative of three independent experiments. * P < .05, ** P < .01 and *** P < .001

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques: Binding Assay, Staining, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Paeonol attenuates inflammation by confining HMGB1 to the nucleus

doi: 10.1111/jcmm.16319

Figure Lengend Snippet: Combined conformation of paeonol and HMGB1. The yellow molecule is paeonol, and the cyan molecule is HMGB1

Article Snippet: HMGB1 conditional knockout (Lyz‐2 cre, macrophage specific) mice were constructed by Cyagen Company.

Techniques:

Journal: Vision research

Article Title: A Mouse M-opsin Monochromat: Retinal Cone Photoreceptors have increased M-Opsin Expression when S-Opsin is knocked out

doi: 10.1016/j.visres.2010.12.017

Figure Lengend Snippet: a. Schematic of Opn1sw targeting strategy. The targeting vector comprised a Neomycin resistance cassette (Neo) flanked by FRT recombination sites (arrowheads) and homologous sequences (4.5 Kb 3’ and 816 bp 5’) to the Opn1sw gene locus (the calumenin gene, which abuts the 5’ end of the Opn1sw gene and is transcribed in the reverse direction on the complementary strand). The asterisk indicates the site of a targeted point mutation. Southern blotting and PCR confirmed successful targeting.

Article Snippet: Taq-Man ® real time qRT-PCR was performed on serial dilutions of cDNA from Opn1sw Neo/Neo and WT littermate eyes with a TaqMan 7500 Real-Time PCR System (Applied Biosystems/Life Technologies Corporation, Carlsbad, California 92008) using probes for mouse visual opsins and β-actin (exon boundary and catalogue number): rhodopsin (1–2) Mm00520345-m1; M-opsin (1–2) Mm01193546-m1; S-opsin (1–2) Mm01135619_g1; S-opsin (4–5) Mm00432058_m1; β-actin, Mm 00607939-s1.

Techniques: Plasmid Preparation, Mutagenesis, Southern Blot