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

Journal: The Journal of biological chemistry

Article Title: Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration.

doi: 10.1016/j.jbc.2023.105232

Figure Lengend Snippet: Figure 6. Nogo-A amino-terminal fragment interacts with HSPA8. A, cortical neurons transfected with GFP or FLAG-NogoA 1 to 213 were without or with axotomy for 16 h and then the lysate was immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were resolved by SDS-PAGE and visualized by silver staining. Red arrow band was excised and analyzed by LC/MS to determine its identity. Black arrow shows immunoprecipitated NogoA 1 to 213 protein band. B, cortical culture neurons of nogoAtrap/trap or nogoAatg/atg without or with axotomized for 16 h were lysed and immunoprecipitated with anti-NogoA-N antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-HSPA8, anti-NogoA-N, and anti-actin antibody. C, quantification of HSPA8 protein levels in the immunoprecipitates normalized to total cell lysate. Mean ± SEM, n = 5 biological replicates. ***p < 0.005, Student’s two-tailed t test. D, HEK293T cells were transfected with FLAG-tagged NogoA WT or FLAG-tagged NogoA G214A-N215A mutant. At 36 h after transfection, cells were lysed and immunoprecipitated with anti-FLAG antibody. Then, the immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-HSPA8 and anti-FLAG antibodies. E, quantification of HSPA8 protein levels in the immunoprecipitates normalized to total cell lysate. Mean ± SEM, n = 3 biological replicates. *p < 0.05, one-way ANOVA followed by Tukey’s test (F = 9.66, DF = 8). HEK, human embryonic kidney.

Article Snippet: Briefly, recombinant HSPA8 protein (400 ng) (catalog #NBP130278; Novus) was incubated with GST or NogoA-213 (400 ng) at 37 C for 15 min. Firefly luciferase (2 × 108 U) (catalog #SRE0045; Merck) in refolding buffer (25 mM Tris– HCl pH7.6, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, 2.5 mM ATP) was heat denatured at 45 C for 7 min. Denatured luciferase was mixed with HSPA8/GST or HSPA8/ NogoA-213 and incubated on ice for 10 min, then the mixture was incubated at 30 C for the indicated time points.

Techniques: Transfection, Immunoprecipitation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Mutagenesis

Journal: The Journal of biological chemistry

Article Title: Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration.

doi: 10.1016/j.jbc.2023.105232

Figure Lengend Snippet: Figure 7. HSPA8 regulates axonal regeneration in vitro. A, representative images of regenerating axons in shNC, shHSPA8 #1, and #2 transfected neurons 3 days after axotomy. The microphotographs show ßIII-tubulin (in axons; green) and phalloidin (to stain F-actin; magenta). The scale bar represents 200 μm. B, quantification of axonal regeneration relative to shNC. Mean ± SEM, n = 3 biological replicates. ***p < 0.005, one-way ANOVA followed by Dunnett’s multiple comparisons test (F = 77.83, DF = 8). C, representative images of regenerating axons in GFP or HSPA8 transfected neurons 3 days after axotomy. The scale bar represents 200 μm. D, quantification of axonal regeneration relative to GFP. Mean ± SEM, n = 3 biological replicates. ***p < 0.005, Student’s two-tailed t test. E, cortical neurons taken from wt mouse were transfected with FLAG-HSPA8 and scraped at DIV 8 and regenerated for 3 days. Low magnification image of FLAG-HSPA8 (anti-FLAG antibody; green), axons (anti-ßIII-tubulin antibody; blue), and growth cones (rhodamine-phalloidin; red) are taken. The scale bar represents 200 μm in both panels. F, cortical neurons taken from wt mouse were transfected with FLAG-HSPA8 and scraped at DIV 8 and regenerated for 3 days. Confocal microscope images of FLAG-HSPA8 (anti-FLAG antibody; green), and growth cones (rhodamine-phalloidin; red) in scraped area are taken. The scale bar represents 20 μm. G, cortical neurons taken from nogoAtrap/trap mouse were transfected with FLAG-HSPA8 and scraped at DIV 8 and regenerated for 3 days. Confocal microscope images of FLAG-HSPA8 (anti-FLAG antibody; green), Nogo-A amino-terminal fragment (anti- NogoA-N antibody; blue), and growth cones (rhodamine-phalloidin; red) in scraped area are taken. The scale bar represents 20 μm.

Article Snippet: Briefly, recombinant HSPA8 protein (400 ng) (catalog #NBP130278; Novus) was incubated with GST or NogoA-213 (400 ng) at 37 C for 15 min. Firefly luciferase (2 × 108 U) (catalog #SRE0045; Merck) in refolding buffer (25 mM Tris– HCl pH7.6, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, 2.5 mM ATP) was heat denatured at 45 C for 7 min. Denatured luciferase was mixed with HSPA8/GST or HSPA8/ NogoA-213 and incubated on ice for 10 min, then the mixture was incubated at 30 C for the indicated time points.

Techniques: In Vitro, Transfection, Staining, Two Tailed Test, Microscopy

Journal: The Journal of biological chemistry

Article Title: Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration.

doi: 10.1016/j.jbc.2023.105232

Figure Lengend Snippet: Figure 8. NogoA N-terminal fragment enhances HSPA8 refolding activity. A, quantification of axonal regeneration of GFP, NogoA-213, HSPA8, and NogoA-213+HSPA8 transfected neurons relative to GFP. Mean ± SEM, n = 4 biological replicates. n.s., not significant (p > 0.05), **p < 0.01, ****p < 0.001, one-way ANOVA followed by Tukey’s test (F = 17.23, DF = 19). B, recombinant HSPA8 protein was incubated with GST or NogoA-213 at 37 C for 15 min. Denatured luciferase mixed with HSPA8/GST or HSPA8/NogoA-213 was incubated at 30 C for the indicated time points. To determine the luciferase activity, samples were added to luciferin and the luminescence was measured. The graph sows fold change. Mean ± SEM, n = 3 replicates. *p < 0.05, ***p < 0.005, one-way ANOVA followed by Dunnett’s test (30 min, F = 5.44, DF = 8, 60 min, F = 26.89, DF = 8). HSP, heat shock protein.

Article Snippet: Briefly, recombinant HSPA8 protein (400 ng) (catalog #NBP130278; Novus) was incubated with GST or NogoA-213 (400 ng) at 37 C for 15 min. Firefly luciferase (2 × 108 U) (catalog #SRE0045; Merck) in refolding buffer (25 mM Tris– HCl pH7.6, 50 mM KCl, 10 mM MgCl2, 5 mM DTT, 2.5 mM ATP) was heat denatured at 45 C for 7 min. Denatured luciferase was mixed with HSPA8/GST or HSPA8/ NogoA-213 and incubated on ice for 10 min, then the mixture was incubated at 30 C for the indicated time points.

Techniques: Activity Assay, Transfection, Recombinant, Incubation, Luciferase

Journal: American Journal of Translational Research

Article Title: CircRNAs in exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells

doi:

Figure Lengend Snippet: Isolation and identification of exosomes from glomerular endothelial cells treated with high glucose and low glucose. A: Exosomal size analysis of HG-GEC-EXO and NG-GEC-EXO by NTA. B: The number of exosomes analyzed in the two different groups by NTA. C: Western blot analysis of HspA8 and Alix in the exosomes of the two different groups. ****P-value < 0.001. HG-GEC-EXO: exosomes from glomerular endothelial cells treated with high glucose. NG-GEC-EXO: exosomes from glomerular endothelial cells treated with normal (low) glucose; NTA: Nanoparticle tracer analysis.

Article Snippet: After blocking for 1 h at room temperature, the membrane was incubated with rabbit polyclonal anti-mouse HspA8 (1:1000) and Alix (1:1500) antibody (BOSTER, USA) for 12 hours.

Techniques: Isolation, Western Blot