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 ) Schematic representation of P155 translation. Human MIR155HG spans 13,024 bp and has three exons. P155 is translated by ORF1 (indicated by yellow boxes), which comprises the end of exon 2 and the head of exon 3. The nucleotide and amino acid sequences of ORF1 are highlighted in red and the pre–miR-155 is indicated by a bluish color. ( B ) Schematic representation of P155 EGFP knock-in strategy. The EGFP (without its own ATG) was inserted after the last coding codon (GTT-valine) of P155 by CRISPR/Cas9-mediated homologous recombination in HEK293T cells. The front homologous arm is a 501-bp fragment before the termination codon of P155 sequence and the back homologous arm is a 501-bp fragment starting with the P155 termination codon, E3: exon 3. ( C ) PCR detection of EGFP knock-in efficiency. Target band is indicated by the yellow box. ( D ) Fluorescence imaging of P155-EGFP fusion protein expression. ( E ) Immunoblotting verification of P155-EGFP fusion protein in HEK293T cells. Protein lysate of EGFP plasmid–transfected HEK293T cells served as a negative control. The target band is indicated by black arrowheads, and the EGFP location is visible as a black line. ( F ) Immunoblotting detection of endogenously expressed P155 in human moDCs with P155-specific antibody pre-enrichment. Chemically synthesized P155 served as a positive control, and the target band is indicated by the black arrowheads. ( G ) LC-MS verification of the P155 endogenous expression in OCI-LY-1 cells with P155-specific antibody pre-enrichment. Scale bar, 100 μm. Data (D to F) are representative of three independent experiments. Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: The extracted protein was incubated with a rabbit anti-P155 antibody (40 μg/ml; HuaBio) overnight at 4°C with rotation and then transferred to an Amicon Ultra Centrifugal Filters (Merck) to segregate ingredients ≤10 kDa.

Techniques: Knock-In, CRISPR, Homologous Recombination, Sequencing, Fluorescence, Imaging, Expressing, Western Blot, Plasmid Preparation, Transfection, Negative Control, Synthesized, Positive Control, Liquid Chromatography with Mass Spectroscopy

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: The extracted protein was incubated with a rabbit anti-P155 antibody (40 μg/ml; HuaBio) overnight at 4°C with rotation and then transferred to an Amicon Ultra Centrifugal Filters (Merck) to segregate ingredients ≤10 kDa.

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: The extracted protein was incubated with a rabbit anti-P155 antibody (40 μg/ml; HuaBio) overnight at 4°C with rotation and then transferred to an Amicon Ultra Centrifugal Filters (Merck) to segregate ingredients ≤10 kDa.

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

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 ) Schematic diagram of psoriasis-like mouse model induction. ( B ) Macroscopic views of Scr- or P155-treated IMQ-induced mouse ear (left) and daily measurement of mouse ear thickness (right, n = 4). Scaly plaques are marked by red arrows. ( C ) Representative images of H&E staining from Scr- or P155-treated IMQ-induced mouse ear sections (left) and statistical analysis for skin acanthosis and infiltrating inflammatory cells in the dermis (right, n = 4). a, epidermis; b, dermis. ( D ) Representative immunohistochemical staining of Ki67 (left) and quantitation of Ki67 + epidermal cells (right, a) in Scr- or P155-treated IMQ-induced mouse skin sections ( n = 4). a, epidermis; red arrows indicate the positive expression of Ki67. ( E ) ELISA quantification of IL-17A protein expression in supernatants of Scr- or P155-treated IMQ-induced mouse ear homogenates ( n = 3 to 4). ( F and G ) Representative immunohistochemical staining of IL-17A + cells in Scr- or P155-treated IMQ-induced mouse ear (F, left) and spleen (G, left) sections. a, epidermis; b, dermis. Red arrows indicate the positive staining of IL-17A + cells. Statistical analysis of IL-17A + cell percentage in the epidermis, dermis (F, right), and spleen (G, right, n = 4). The dashed line indicates the border between the epidermis and dermis. Scale bars, 50 μm. All data are representative of three independent experiments. ** P < 0.01; *** P < 0.001; and **** P < 0.0001, two-way ANOVA (B) and one-way ANOVA (mean ± SEM). Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: The extracted protein was incubated with a rabbit anti-P155 antibody (40 μg/ml; HuaBio) overnight at 4°C with rotation and then transferred to an Amicon Ultra Centrifugal Filters (Merck) to segregate ingredients ≤10 kDa.

Techniques: Staining, Immunohistochemical staining, Quantitation Assay, Expressing, Enzyme-linked Immunosorbent Assay

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 ) Schematic diagram of MOG-induced EAE. ( B ) Clinical score of Scr- or P155-treated EAE mice ( n = 5). ( C and D ) Representative images of H&E, LFB, MBP staining, and statistical analysis for Scr- or P155-treated EAE mouse brain (C, left and right) and spinal cord sections (D, left and right). n = 3 to 4, red arrows indicate the demyelination. ( E to G ) Flow cytometric analysis of CD4 + T cell (E), T H 17 cell (F), and T reg cell (G) percentages gated on CD4 + T cells from Scr- or P155-treated EAE mouse brain on day 15 after immunization ( n = 4). ( H ) Pathway enrichment analysis of down-regulated DEGs in Scr- or P155-treated EAE mouse spinal cord on day 15 after immunization ( n = 2). Color key represents the value of −log P ; the size of the dot corresponds to the gene counts. ( I ) Heat map of selected genes based on RNA-seq data from the spinal cord of Scr- or P155-treated EAE mice on day 15 after immunization ( P < 0.05, Log2FC < −1, n = 2). Color key represents the normalized expression of genes. Scale bars, 200 μm (C) or 100 μm (D). Data (C to G) are representative of three independent experiments. ** P < 0.01; *** P < 0.001; and **** P < 0.0001, two-way ANOVA (B) and one-way ANOVA (mean ± SEM). Photo credit: Liman Niu (Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine).

Article Snippet: The extracted protein was incubated with a rabbit anti-P155 antibody (40 μg/ml; HuaBio) overnight at 4°C with rotation and then transferred to an Amicon Ultra Centrifugal Filters (Merck) to segregate ingredients ≤10 kDa.

Techniques: Staining, RNA Sequencing, Expressing

Journal: Scientific Reports

Article Title: Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa

doi: 10.1038/srep25543

Figure Lengend Snippet: Changes in colistin minimum inhibitory concentration (MIC) during evolution of colistin resistance ( A ) and its loss ( B ) in P. aeruginosa P5 and P155 lineages. The points at which whole-genome sequencing WT strains and mutants of P5 and P155 lineages was conducted are indicated by the black and white triangles (▴ and ▵), respectively. Intermediate-stage mutants derived from P5 and P155 and subjected to sequence analysis for genes with alterations are indicated by arrows (↑ and ↓). ( C ) Competitive index (CI) as determined by competition assays. The CI represents the ratio of colony-forming units (CFUs) of a colistin-susceptible strain to the CFUs of a colistin-resistant strain (P5:P5R, P5R-rev:P5R, P155:P155R, and P155R-rev:P155R).

Article Snippet: Whole-genome sequencing of P5, P155, P5R, P155R, P5R-rev, and P155R-rev was conducted at Macrogen Inc. (Seoul, Korea) with an Illumina HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA).

Techniques: Concentration Assay, Sequencing, Derivative Assay

Journal: Scientific Reports

Article Title: Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa

doi: 10.1038/srep25543

Figure Lengend Snippet: Non-synonymous mutations identified in P155R and P155R-rev.

Article Snippet: Whole-genome sequencing of P5, P155, P5R, P155R, P5R-rev, and P155R-rev was conducted at Macrogen Inc. (Seoul, Korea) with an Illumina HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa

doi: 10.1038/srep25543

Figure Lengend Snippet: Amino acids substitution during the evolution and loss of colistin resistance in P. aeruginosa P5 (A) and P155 (B). The sequences of P5, P5R, P5R-rev, P155, P155R, and P155R-rev were determined by genome sequencing, while the sequences of all other strains were determined by the Sanger sequencing method.

Article Snippet: Whole-genome sequencing of P5, P155, P5R, P155R, P5R-rev, and P155R-rev was conducted at Macrogen Inc. (Seoul, Korea) with an Illumina HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA).

Techniques: Sequencing