Journal: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: Amelioration of LPS-Induced AKI in MBD2-KO Mice The MBD2-KO and -WT littermate mice were injected with 10 mg/kg LPS for days 0 to 7 or saline as control. (A) Representative of survival cure. (B and C) Blood samples were collected for the measurement of serum nitrogen (BUN) (B) and creatinine (C) concentration at 24 h. (D and E) Representative of TNF-α (Δ) and IL-1β (E). (F) The kidney sections were stained with hematoxylin and eosin (H&E). (G and H) Tubular damage scores of kidney cortex (G) and OSOM (H). Scale bar, 100 μM. For the survival, the animal number is 12; for other experiments, the data are expressed as mean ± SD (n = 6). #p < 0.05 versus the saline group; ∗p < 0.05 versus the MBD2-WT with LPS group. Original magnification, ×200.

Article Snippet: TNF-α, IL-1β, and ICAM1 are from Proteintech North America (Rosemont, IL, USA). p38MAPK activator dehydrocorydaline chloride and pERK1/2 activator honokiol were purchased from MedChemExpress USA (Deer Park, NJ, USA).

Techniques: Injection, Saline, Control, Concentration Assay, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: MBD2 Mediates Septic AKI through Activation of PKCη/p38MAPK and the ERK1/2 Axis

doi: 10.1016/j.omtn.2020.09.028

Figure Lengend Snippet: MBD2-Mediated LPS Induced the Expression of TNF-α, IL-1β, and ICAM1 in BUMPT Cells BUMPT cells were transfected with MBD2 plasmid or MBD2 siRNA or primary macrophages from bone marrow of MBD2 and MBD2-KO mice, followed by treatment with 300 μg/mL LPS for 24 h. (A) Real-time PCR analysis of TNF-α, IL-1β, and ICAM1 expression after LPS treatment with or without transfection of MBD2. (B) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH after LPS treatment with or without transfection of MBD2. (C)The protein signal was quantified by densitometry and normalized to internal control of GAPDH after LPS treatment with or without transfection of MBD2. (D) Real-time PCR analysis of TNF-α, IL-1β, and ICAM1 expression after LPS treatment with or without transfection of MBD2 siRNA. (E) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH after LPS treatment with or without transfection of MBD2 siRNA . (F)The protein signal was quantified by densitometry and normalized to internal control of GAPDH after LPS treatment with or without transfection of MBD2 siRNA. (G) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH in MBD2 KO primary tubular cells with LPS treatment. (H) The protein signal was quantified by densitometry and normalized to internal control of GAPDH in MBD2 KO primary tubular cells with LPS treatment. (I) Immunoblot analysis of TNF-α, IL-1β, ICAM1, or MBD2, and GAPDH in MBD2 KO primary macrophage cells with LPS treatment. (J) The protein signal was quantified by densitometry and normalized to internal control of GAPDH in MBD2 KO primary macrophage cells with LPS treatment. Data are expressed as mean ± SD (n = 6). #p < 0.05 versus the saline group; ∗p < 0.05 versus the MBD2 plasmid or MBD2 siRNA with LPS group; Δp > 0.05 versus the LPS group.

Article Snippet: TNF-α, IL-1β, and ICAM1 are from Proteintech North America (Rosemont, IL, USA). p38MAPK activator dehydrocorydaline chloride and pERK1/2 activator honokiol were purchased from MedChemExpress USA (Deer Park, NJ, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, Saline

Journal: Nature immunology

Article Title: Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry

doi: 10.1038/s41590-019-0378-1

Figure Lengend Snippet: High-dimensional transcriptomic scRNA-seq clustering reveals distinct cell type subpopulations. a . 18 clusters across 5,265 cells from all cell types on a tSNE visualization. b . Cluster abundances across donors. c . Fibroblasts: three types of THY1 + sublining fibroblasts (SC-F1, SC-F2, and SC-F3) and CD55 + lining fibroblasts (SC-F4). d . Monocytes: two activated cell states of IL1B + pro-inflammatory (SC-M1) and IFN-activated (SC-M4) monocytes. e . T cells: CD4 + subsets: SC-T1, SC-T2, SC-T3, and CD8 + subsets: SC-T4, SC-T5, and SC-T6. f . B cells: HLA + (SC-B1, SC-B2, and SC-B3) and plasmablasts (SC-B4). The cluster colors in c-f are consistent with ( a ).

Article Snippet: To identify IL1B + monocytes, we used a mixture of goat anti human CD14 (119–13402, RayBiotech) biotinylated rabbit anti-human IL1b (OABF00305-Biotin, Aviva Systems Biology) and mouse anti-human CD16 (clone DJ130c, LifeSpan Biosciences).

Techniques:

Journal: Nature immunology

Article Title: Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry

doi: 10.1038/s41590-019-0378-1

Figure Lengend Snippet: Connection between cell populations determined by mass cytometry and scRNA-seq clusters and disease associations. Bold mass cytometry clusters are significantly enriched in leukocyte-rich RA (one-sided Benjamini-Hochberg FDR q value < 0.05). Two significant digits are given to the one-sided F-tests conducted on nested models with MASC. 95% confidence interval (CI) for the odds ratio (OR) is given for each mass cytometry cluster. Where possible, we have identified the most similar scRNA-seq clusters for each cluster found by mass cytometry. The mass cytometry analysis is performed on downsampled datasets of 25,161 fibroblasts from 23 patients, 15,298 monocytes from 26 patients, 19,985 T cells from 26 patients, and 8,179 B cells from 23 patients.

Article Snippet: To identify IL1B + monocytes, we used a mixture of goat anti human CD14 (119–13402, RayBiotech) biotinylated rabbit anti-human IL1b (OABF00305-Biotin, Aviva Systems Biology) and mouse anti-human CD16 (clone DJ130c, LifeSpan Biosciences).

Techniques: Mass Cytometry

Journal: Nature immunology

Article Title: Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry

doi: 10.1038/s41590-019-0378-1

Figure Lengend Snippet: Unique activation states define synovial monocytes heterogeneity. a . scRNA-seq analysis identified four subsets: IL1B + pro-inflammatory monocytes (SC-M1), NUPR1 + monocytes (SC-M2) with a mixture of leukocyte-poor RA and OA cells, C1QA + (SC-M3), and IFN-activated monocytes (SC-M4). Differential analysis by bulk RNA-seq on leukocyte-rich RA samples ( n = 17) and OA samples ( n = 13) revealed upregulation/downregulation of cluster marker genes. Effect sizes with 95% CI are given. b . By querying the bulk RNA-seq, we found scRNA-seq cluster IL1B + pro-inflammatory monocytes (two-sided Student’s t-test P =6×10 −5 , t-value=4.56, df =26.33) and IFN-activated monocytes (two-sided Student’s t-test P =6×10 −3 , t-value=3.28, df =23.68) are upregulated in leukocyte-rich RA ( n = 17) compared to OA ( n = 13), while SC-M2 is depleted (two-sided Student’s t-test P =2×10 −5 , t-value=−5.62, df=26.81) in leukocyte-rich RA. Error bars indicate mean and 95% CI. c . Pathway enrichment analysis indicates the potential pathways for each subset. Two-sided Kolmogorov-Smirnov test with 10 5 times permutation was performed; Benjamini-Hochberg was used to control the FDR of multiple tests. The standard names for the immunological gene sets from up to bottom are: Genes down-regulated in neutrophils versus monocytes (GSE22886); Genes down-regulated in healthy myeloid cells versus SLE myeloid cells (GSE10325); Genes down-regulated in control microglia cells versus those 24 h after stimulation with IFNG (GSE1432); Genes down-regulated in unstimulated macrophage cells versus macrophage cells stimulated with LPS (GSE14769); Genes up-regulated monocytes treated with LPS versus monocytes treated with control IgG (GSE9988); Genes up-regulated in monocytes versus myeloid dendritic cells (mDC) (GSE29618); Genes up-regulated in monocytes versus plasmacytoid dendritic cells (pDC) (GSE29618). d . Detection of pro-inflammatory IL-1β in inflamed synovium by multicolor immunofluorescent staining with antibodies CD14 (red), IL-1β (green), and counterstained with DAPI (blue) identified CD14 + IL-1β + cells (white arrow). The experiment was repeated > 5 times with staining of 6 independent leukocyte-rich RA samples with similar results. Image was acquired at 200 magnification. Scale bar is 50 μm. e–f . Identified subpopulations from monocytes ( n = 15,298) and disease status from 6 leukocyte-rich RA, 9 leukocyte-poor RA, and 11 OA by mass cytometry on the same gating with scRNA-seq. g-h . Normalized intensity of distinct protein markers by tSNE visualization and averaged for each cluster in heatmap. i . Integration of identified mass cytometry clusters with bulk RNA-seq reveals genes that are associated with CD11c + CD38 + and CD11c + CCR2 + , like IFITM3, CD38, HBEGF, ATF3, and HLA+ genes. j . Integration of mass cytometry clusters and scRNA-seq clusters revealed that CD11c+CD38+ by mass cytometry are significantly associated with IL1B + pro-inflammatory (SC-M1) monocytes.

Article Snippet: To identify IL1B + monocytes, we used a mixture of goat anti human CD14 (119–13402, RayBiotech) biotinylated rabbit anti-human IL1b (OABF00305-Biotin, Aviva Systems Biology) and mouse anti-human CD16 (clone DJ130c, LifeSpan Biosciences).

Techniques: Activation Assay, RNA Sequencing, Marker, Control, Staining, Mass Cytometry

Journal: Oncotarget

Article Title: Upregulation of calprotectin in mild IgE-mediated ovalbumin hypersensitivity

doi: 10.18632/oncotarget.16954

Figure Lengend Snippet: Body weight ( A ) and length ( B ) were monitored weekly from the beginning of the study to the end. There was no significant difference between two groups from days 7 to days 42 (day 7 to 35, n = 5/group; day 42, n = 15/group, P all > 0.05).

Article Snippet: Immunohistochemistry was performed using standard protocols with the following antibodies: IL-1β, (1:100, both from Origene, Inc., Rockville, MD, USA); TNF-α, S100A8, S100A9 (1:200, Abcam, Inc., Cambridge, MA, USA); Toll-like receptor 4 (TLR4), S100A8/A9 (1:200, NOVUSbio, Inc., Littleton, CO, USA); nuclear factor kappa B (NF-κB) (1:200, Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques:

Journal: Oncotarget

Article Title: Upregulation of calprotectin in mild IgE-mediated ovalbumin hypersensitivity

doi: 10.18632/oncotarget.16954

Figure Lengend Snippet: PCR was used to detect the mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, and IL-6. ( A , B , C , D , and E ) show the expression of TLR4, NF-κB, TNF-α, IL-1β, and IL-6 in jejunum; ( F , G , H , I and J ) show the expression of TLR4, NF-κB, TNF-α, IL-1β, and IL-6 mRNA in liver. * P < 0.05 ; # P < 0.01. ( K ) shows the expression of TLR4, NF-κB, TNF-α, and IL-1β. from left to right are control group, hypersensitivity groups from day 7 to day 42; and from top to bottom, expression of TLR4, NF-κB, TNF-α, and IL-1β in jejunum.

Article Snippet: Immunohistochemistry was performed using standard protocols with the following antibodies: IL-1β, (1:100, both from Origene, Inc., Rockville, MD, USA); TNF-α, S100A8, S100A9 (1:200, Abcam, Inc., Cambridge, MA, USA); Toll-like receptor 4 (TLR4), S100A8/A9 (1:200, NOVUSbio, Inc., Littleton, CO, USA); nuclear factor kappa B (NF-κB) (1:200, Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Control