Journal: Molecular Medicine Reports

Article Title: Differentially expressed lncRNAs, miRNAs and mRNAs with associated ceRNA networks in a mouse model of myocardial ischemia/reperfusion injury

doi: 10.3892/mmr.2020.11300

Figure Lengend Snippet: Validation of microarray data using reverse transcription-quantitative PCR. Relative expression levels of (A) one lncRNA, (B) one miRNA and (C) one mRNA were assessed in the Sham and I/R mice hearts. Data are presented as the mean ± SEM of four samples in each group. *P<0.05 vs. Sham group. lncRNAs, long non-coding RNAs; miRNAs, microRNAs; I/R, ischemia/reperfusion; Olfml1, olfactomedin like 1.

Article Snippet: The Arraystar Mouse lncRNA array v3.0 (8×60K; Arraystar Inc.) was designed to profile lncRNAs and mRNAs in the mouse genome.

Techniques: Biomarker Discovery, Microarray, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: lncRNA STAT4-AS1 expression was downregulated accompanied by up-regulation of IL-17 expression in asthmatic patients. (a) The release of IL-17A in peripheral T cells of asthmatic patients was detected using ELISA. (b–d) The mRNA expression of lncRNA STAT4-AS1, IL-17A, and ROR γ t in peripheral blood T cells of patients with asthma was detected using Q-PCR. Three independent experiments were performed, and data are presented as the mean ± SD. ∗∗ p < 0.01, and ∗∗∗∗ p < 0.001.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: lncRNA STAT4-AS1 expression was downregulated accompanied by upregulation of IL-17 expression in PBMCs. (a) The proportion of TH17 cells on day 0 and day 3 of induced differentiation. Flow cytometry figures (left) and statistical analysis (right) of TH17 cells. (b) The upper chart of this panel shows genome organization of the stat4 gene at locus 2q32.2-q32.3. The chart shows the start and end positions of these genes as indicated on the UCSC site. Black arrows indicate transcription direction, green blocks are the exons of STAT4, and purple blocks are the exons of STAT4-AS1. The lower chart of this panel shows that the exons of STAT4 and STAT4-AS1 have an overlapping region (OL). Blocks with colors (orange, blue, and red) represent exons. Red block in STAT4 represents the 23 exon and also OL region. The genomic organization of the human STAT4-AS1 locus. “+” at both sides of strand represents the positive strand; “-” represents the negative strand. (c) The levels of IL-17A in cell supernatant were detected using ELISA. (d) The expression of IL-17A, lncRNA STAT4-AS1, STAT4, and ROR γ t genes was detected using qPCR, respectively. (e) The expression of intracellular ROR γ t protein was detected using immunofluorescence. Three independent experiments were performed, and data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: Expressing, Flow Cytometry, Blocking Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: Downregulation of lncRNA STAT4-AS1 promoted the differentiation of TH17 cells. (a) The proportion of TH17 cells on day 2 and day 4 of induced differentiation. Flow cytometry figures (left) and statistical analysis (right) of TH17 cells. (b) The mRNA level of STAT4-AS1, STAT4, IL-17A, ROR γ t, IL-17F, and IL-21 on days 2 and 4 of induction. (c) The levels of IL-17A in cell supernatant were detected using ELISA. (d) Immunofluorescence was used to determine the protein expression of ROR γ t. Three independent experiments were performed, and data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: Upregulation of lncRNA STAT4-AS1 inhibited the differentiation of TH17 cells. (a) The mRNA levels of lncRNA STAT4-AS1, STAT4, IL-17A, ROR γ t, IL-17F, and IL-21 on day 0, day 2, and day 4 after induction. (b) The level of IL-17A in the supernatant. (c) The proportion of TH17 cells after lncRNA STAT4-AS1 overexpression. Flow cytometry figures (left) and statistical analysis (right) of TH17 cells. Three independent experiments were performed, and data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: Over Expression, Flow Cytometry

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: lncRNA STAT4-AS1 interacted with ROR γ t and inhibited IL-17 expression. (a) Bioinformatics analysis predicted results of the interaction between lncRNA STAT4-AS1 and ROR γ t protein. Left: the interaction between lncRNA STAT4-AS1 and ROR γ t protein. Right: the interaction between lncRNA STAT4-AS1 and ROR γ t protein isoform (ROR γ ). (b) ROR γ t protein was detected in the extracts of the RNA pull-down using Western blot. lncRNA-Con was used as negative control. (c) RT-qPCR detection of lncRNA STAT4-AS1 mRNA in the immunoprecipitation by IgG, AGO2 or anti-ROR γ t specific antibody from PBMCs treated with TH17 polarization. IgG served as a negative control. (d) Dual luciferase reporter assay was used to confirm the inhibition of lncRNA STAT4-AS1 during ROR γ t-induced activation of the IL-17 promoter. Three independent experiments were performed, and data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: Expressing, Western Blot, Negative Control, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Reporter Assay, Inhibition, Activation Assay

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: Subcellular localization of lncRNA STAT4-AS1 ( n = 3). (a) The localization of lncRNA STAT4-AS1 in PBMCs cells before and after induction was determined by FISH. (b) Nuclear-cytoplasmic separation assay. (c) The colocalization of lncRNA STAT4-AS1 and ROR γ t protein in TH17 before and after overexpression of lncRNA STAT4-AS1 was determined by FISH and immunofluorescence. Three independent experiments were performed, and data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: Over Expression, Immunofluorescence

Journal: Journal of Immunology Research

Article Title: lncRNA STAT4-AS1 Inhibited TH17 Cell Differentiation by Targeting ROR γ t Protein

doi: 10.1155/2022/8307280

Figure Lengend Snippet: The mechanism of lncRNA STAT4-AS1 on TH17 differentiation.

Article Snippet: Cell extracts were incubated with biotin-labeled lncRNA STAT4-AS1 at 4°C for 1 h. Then, streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human ROR γ t primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody (Boster, Wuhan, China).

Techniques: