Journal: PLoS ONE

Article Title: miR-140 Suppresses Tumor Growth and Metastasis of Non-Small Cell Lung Cancer by Targeting Insulin-Like Growth Factor 1 Receptor

doi: 10.1371/journal.pone.0073604

Figure Lengend Snippet: (A) putative binding sequences of miR-140 in the IGF1R 3’ UTR. Mutation was generated in the IGF1R 3’ UTR by mutating 3 nt that is recognized by miR-140. Either wild-type (WT) or mutant (Mut) IGF1R 3’ UTR was subcloned into the dual-luciferase reporter vector. (B) a statistically inverse correlation between miR-140 and IGF1R mRNA levels in NSCLC tissues by Spearman’s correlation analysis. (C, D) the expression of IGF1R in A549, H157 and H520 cells after infection or transfection was measured by qRT-PCR and Western blotting. (E) Luciferase assay in A549 and H157 cells co-transfected with miR-140 and a luciferase reporter containing the IGF1R 3′-UTR (WT) or a mutant (Mut). Luciferase activities were measured 48 h post-transfection. ** P <0.01.

Article Snippet: The coding sequence of IGF1R was purchased from Origene and cloned into pcDNA3.1 (+) to generate IGF1R expression vectors.

Techniques: Binding Assay, Mutagenesis, Generated, Luciferase, Plasmid Preparation, Expressing, Infection, Transfection, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: miR-140 Suppresses Tumor Growth and Metastasis of Non-Small Cell Lung Cancer by Targeting Insulin-Like Growth Factor 1 Receptor

doi: 10.1371/journal.pone.0073604

Figure Lengend Snippet: A549 cells were infected with specific si IGF1R, or transfected with IGF1R plasmid lacking 3’ UTR along with miR-140. (A) Western blotting analysis. (B) cell viability assay (CCK-8). (C) cell cycle analysis. (D) cell apoptosis assays. (E) Transwell invasion assays. * P <0.05, ** P <0.01.

Article Snippet: The coding sequence of IGF1R was purchased from Origene and cloned into pcDNA3.1 (+) to generate IGF1R expression vectors.

Techniques: Infection, Transfection, Plasmid Preparation, Western Blot, Viability Assay, CCK-8 Assay, Cell Cycle Assay

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: (A) Diagram of in vitro experiments in this study. Monocytes treated with exNef or exCont in the presence or absence of methyl-β-cyclodextrin (MβCD), fluvastatin (Flu), or picropodophyllin (PPP) were analyzed by western blotting (WB) for flotillin 1 (Flot1) or by flow cytometry for lipid rafts (staining with CTB) and IGF1R. After differentiation in the presence of M-CSF, MDMs were characterized by t-SNE for activation markers or by ddPCR for gene expression. Following LPS stimulation, gene expression was characterized by RNA-seq and cytokine production measured by ELISA. See also and . (B) Analysis of TNF-α and IL-6 production by LPS-treated MDMs. The graph shows fold increase of cytokine production by exNef-treated over exCont-treated cells from 8 donors (8 biological replicates, 3 technical replicates for each donor), analyzed by Friedman multiple comparison test with Dunn’s correction. Adjusted p values are shown above bars. (C) Dose response analysis. The graph shows mean ± SEM (3 technical replicates of cells from a single donor) fold increase of TNF-α produced by MDMs exposed to exNef with indicated concentrations of Nef versus MDM exposed to exCont at the same concentration of EVs. (D) Gene set enrichment analysis (GSEA) showing normalized enrichment scores for genes differentially expressed between exNef and exCont and participating in the inflammatory response (left panel) and cytokine pathway (right panel); the gene sets were obtained from BioCarta. (E) Leading edge analysis shows that a number of cytokine genes drive the changes of pathways dysregulated by exNef. See also - .

Article Snippet: IGF1R PCR primers , OriGene , cat#HP200815.

Techniques: In Vitro, Western Blot, Flow Cytometry, Staining, Activation Assay, Gene Expression, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Comparison, Produced, Concentration Assay

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: (A) Analysis of lactate in the supernatant of MDM cultures on day 6 after washing out the EVs. Results are presented as means ± SD of six replicate determinations (technical replicates) using cells from a single donor (1 biological replicate). p value was calculated using unpaired two-tailed parametric t test. See also . (B) Results of ddPCR analysis of monocytes treated with exCont or exNef (24 h after removal of EVs). Bars show fold increase of gene expression in exNef-treated, relative to exCont-treated, cells. Analysis was performed in cells from 4 donors (4 biological replicates, each one is average of 3 technical replicates), and results were analyzed by ANOVA with Dunnett correction for multiple comparisons. Adjusted *p = 0.0464. (C) Incorporation of [ 14 C]acetate into cholesterol after 2 h incubation with MDMs treated with exCont or exNef. Bars show means ± SD of disintegrations per minute (dpm) per mg of total cell protein from quadruplicate determinations (4 technical replicates, 1 biological replicate). p value was calculated by unpaired two-tailed parametric t test. (D) ExNef-induced upregulation of TNF-α production by LPS-treated MDMs was inhibited by Flu and PPP (added with EVs) and by MβCD (added after washout of EVs). Analysis was performed on cells from one donor (1 biological replicate) assayed in 6 replicate wells (6 technical replicates). Results were analyzed by one-way ANOVA, with Holm-Sidak’s correction for multiple comparisons. Adjusted **p = 0.0045. (E) MTT assay was performed on MDMs from a single donor (1 biological replicate) exposed to indicated agents prior to LPS stimulation. Bars show means ± SD of 6 determinations (6 technical replicates). Results were analyzed by ANOVA with Dunnett’s multiple comparison test. *p = 0.0205. (F) IGF1R presentation on MDMs exposed to exNef and exCont. Monocytes were analyzed 48 h after washout of EVs.

Article Snippet: IGF1R PCR primers , OriGene , cat#HP200815.

Techniques: Two Tailed Test, Gene Expression, Incubation, MTT Assay, Comparison

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: IGF1R PCR primers , OriGene , cat#HP200815.

Techniques: Virus, Plasmid Preparation, Recombinant, Modification, Staining, RNA HS Assay, Reverse Transcription, SYBR Green Assay, Clinical Proteomics, Membrane, Isolation, Cell Fractionation, Lysis, Blocking Assay, Enzyme-linked Immunosorbent Assay, Software, Simple Western