Journal: Science Advances

Article Title: A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation

doi: 10.1126/sciadv.aaz2059

Figure Lengend Snippet: ( A ) Two-dimensional visualization of the single immune cell (CD45 + cells) transcriptome in the dermis of healthy donors ( n = 3) and patients with psoriasis ( n = 3). Immune cell compartments are encircled, and feature plots of MIR155HG expression in different subsets are presented. ( B ) Silver staining of P155 interactive protein in the immunoprecipitants pulled down by streptavidin-agarose from human moDCs pretreated with R848 (1 μg/ml) and biotin-Scr/P155 (25 μM). The black box represents target protein. ( C ) Scatterplot of representative data for intensity of proteins detected with MS in human moDCs treated with R848 (1 μg/ml) and Biotin-Scr/P155 (25 μM). The dots represent the intensities (log10-transformed) of all proteins identified in the P155 group ( y axis) and the Scr group ( x axis), and the purple dot represents the protein of interest. ( D ) Immunoblotting verification of the interaction between HSC70 and P155. The black arrowhead indicates the specific band. ( E ) Immunoblotting detection of the P155-specific binding domain in the immunoprecipitants pulled down by streptavidin-agarose from biotin-Scr/P155–pretreated HEK293T cells overexpressing Myc-Tag–labeled HSC70 subdomain plasmids. Anti–Myc-Tag antibody was used and the black box indicates the specific banding. IB, immunoblot; PD, pull-down assay. ( F ) Confocal visualization of PLA signals (red/pink dots) in HEK293T cells overexpressing Myc-Tag–labeled HSC70 or HSC70 subdomain plasmids together with the endogenously expressed P155. Myc-Tag–labeled HSC70 protein served as a positive control, and the red arrows indicate the specific signals. ( G ) ATPase activity of HSC70 in the presence of P155 or Scr ( n = 3). ( H ) GO pathway enrichment analysis of down-regulated differentially expressed genes (DEGs) of RNA-seq data from THP-1–derived DCs treated with R848 (1 μg/ml) and Scr/P155 (25 μM) compared to Scr-treated controls. Dot color represents the value of −log P ; dot size corresponds to the gene counts, and the red words indicate the pathways we are focusing on. Scale bar, 10 μm. Data (B to E and G) are representative of three independent experiments. ** P < 0.01, two-tailed Student’s t test (mean ± SEM). Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: To determine the HSC70 ATPase activity, the recombinant protein of human heat shock 70-kDa protein 8 (HSPA8) transcript variant 1 (OriGene) was prepared in assay buffer at a final concentration of 0.2 μM with P155 or Scr (25 μM) added to the solution.

Techniques: Expressing, Silver Staining, Transformation Assay, Western Blot, Binding Assay, Labeling, Pull Down Assay, Positive Control, Activity Assay, RNA Sequencing, Derivative Assay, Two Tailed Test

Journal: Science Advances

Article Title: A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation

doi: 10.1126/sciadv.aaz2059

Figure Lengend Snippet: ( A ) Silver staining of P155 interactive protein in the immunoprecipitants pulled down by streptavidin-agarose from murine BMDCs pretreated with R848 (1 μg/ml) and biotin-Scr/P155 (25 μM). The black box represents target protein. ( B ) Scatterplot of representative data for intensity of proteins detected with MS in murine BMDCs treated with R848 (1 μg/ml) and Biotin-Scr/P155 (25 μM). The dots represent the intensities (log10-transformed) of all proteins identified in the P155 group ( y axis) and the Scr group ( x axis), and the purple dot represents the protein of interest. ( C ) Immunoblotting verification of the interaction between HSC70 and P155. The black arrowhead indicates the target band. ( D ) Confocal microscopic images of murine BMDCs treated with FITC-OVA with the cells costained with LAMP2A (red); nuclei were stained with DAPI (blue). The white arrows indicate the colocalization of OVA and LAMP2A (yellow), and the white dashed line is the position indicator for the fluorescence colocation analysis. ( E ) Fluorescence intensity analysis of colocalization of FITC-OVA and LAMP2A in murine BMDCs treated with Scr or P155 ( n = 6). ImageJ software was used to analyze the fluorescence colocalization. The white dashed indicator line of statistical analysis centers on the colocation of FITC-OVA and LAMP2A. ( F ) Immunoblotting detection of HSP90 and LAMP2A expression in the immunoprecipitants pulled down by anti-HSC70 from R848- and Scr/P155-pretreated BMDCs, with immunoprecipitants pulled down by rat IgG from R848- and Scr/P155-pretreated BMDCs serving as the negative controls. The black arrows indicate the target protein. IP, immunoprecipitation; IB, immunoblot. The fold (FD) value represents the protein intensity of HSP90 or LAMP2A and is measured with ImageJ software. ( G ) Confocal microscopic images of the distribution of MHC class II (green) in R848- and Scr/P155-pretreated murine BMDCs. Nuclei were stained with DAPI (blue). ( H ) Representative flow cytometry chart (left) and statistics analysis of OT-II T cell proliferation rates (right, indicated by CD4 + CD44 + Violet − cell percentages) in coculture with BMDCs pretreated with soluble OVA ( n = 3 to 4). ( I ) Schematic diagram of MIR155HG encoding P155 function in regulating DC presentation. Scale bars, 10 μm (D) or 25 μm/5 μm (G). All data are representative of three independent experiments. n.s., not significant; ** P < 0.01 and **** P < 0.0001, one-way ANOVA (mean ± SEM). Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: To determine the HSC70 ATPase activity, the recombinant protein of human heat shock 70-kDa protein 8 (HSPA8) transcript variant 1 (OriGene) was prepared in assay buffer at a final concentration of 0.2 μM with P155 or Scr (25 μM) added to the solution.

Techniques: Silver Staining, Transformation Assay, Western Blot, Staining, Fluorescence, Software, Expressing, Immunoprecipitation, Flow Cytometry