Journal: Frontiers in Immunology

Article Title: Tribbles-1 Expression and Its Function to Control Inflammatory Cytokines, Including Interleukin-8 Levels are Regulated by miRNAs in Macrophages and Prostate Cancer Cells

doi: 10.3389/fimmu.2020.574046

Figure Lengend Snippet: TRIB1 3’ UTR encodes for evolutionarily conserved miRNA binding sites. (A) Schematic representation of TRIB1 mRNA, showing the length of the untranslated regions and the protein coding sequence. (B) Schematic overview of TRIB1 3’UTR multi-species alignment, generated with the software DNASTAR Lasergene (v16). (C) TRIB1 3’UTR alignment tree, generated with the software DNASTAR Lasergene (v16) using the ClustalW algorithm. (D) Relative luciferase activity measured in HEK293T cell line after transfection of renilla luciferase reporter (control, 95 ng/well) and TRIB1 3’UTR renilla luciferase reporter (TRIB1 3’UTR, 95 ng/well) along with a firefly luciferase reporter plasmid (5 ng/well), used for data normalization (n = 12, biological replicates, unpaired t-test, ***p < 0.001). (E) Overview of miRNAs-target prediction analysis: seven different tools (miRanda, TargetScan, StarBase, miRDB, miRwalk, TarBase, and MicroT-CDS) were used to predict miRNAs targeting the 3’UTR of TRIB1; the list of distinct miRNAs was filtered selecting only “high-confidence” miRNAs (according to miRbase v.22) and miRNAs predicted at least by three different tools.

Article Snippet: The substrates of Firefly luciferase (LAR II, Promega) and Renilla luciferase (Stop & Glo, Promega) were sequentially added to the cell lysates (1:1 ratio).

Techniques: Binding Assay, Sequencing, Generated, Software, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Tribbles-1 Expression and Its Function to Control Inflammatory Cytokines, Including Interleukin-8 Levels are Regulated by miRNAs in Macrophages and Prostate Cancer Cells

doi: 10.3389/fimmu.2020.574046

Figure Lengend Snippet: miR-101-3p has a functional binding site in the 3’UTR of TRIB1. (A) Schematic alignment of miR-101-3p with the 3’UTR of TRIB1 and its “binding site” mutant. (B) Relative luciferase activity measured in HEK293T cell line after co-transfection of the TRIB1 3’UTR renilla reporter (95 ng/well) with miR-101-3p mimic and a negative control (+firefly luciferase reporter, 5 ng/well), (n = 9, biological replicates, unpaired t-test, **p < 0.01). (C) Relative luciferase activity measured in HEK293T cell line after co-transfection of the TRIB1 3’UTR renilla reporter “mutant” (95 ng/well) with miR-101-3p mimic and a negative control (+ firefly luciferase reporter, 5 ng/well), (n = 7, biological replicates, unpaired t-test, no statistical significance p > 0.05). (D) Relative luciferase activity measured in HEK293T cell line after co-transfection of the renilla luciferase reporter (control, 95 ng/well) and TRIB1 3’UTR renilla reporter (TRIB1 3’UTR, 95 ng/well) with miR-101-3p inhibitor and a negative control (+ firefly luciferase reporter, 5 ng/well), (n = 6, biological replicates, ordinary one-way ANOVA with Sidak’s post-test, **p < 0.01, no statistical significance p > 0.05). (E, F) Trib1 protein levels relative to the housekeeping α-tubulin in MDMs transiently transfected with miR-101-3p mimic/negative control and representative immunoblot (n = 5, biological replicates, paired t-test, *p < 0.05). (G) Schematic representation of TSB/endogenous miRNA competition for the target site. (H) Relative TRIB1 mRNA expression normalized to the housekeeping GAPDH in MDMs transiently transfected with miR-101/TRIB1 TSB and a negative control (n = 7, biological replicates, paired t-test, **p < 0.01). (I, J) Trib1 protein levels relative to the housekeeping Hsp90 in MDMs transiently transfected with miR-101/TRIB1 TSB/negative control and representative immunoblot (n = 4, biological replicates, paired t-test, no statistical significance p > 0.05). Data are presented as mean ± SEM (scatter dot plot where each dot represents an individual donor of MDMs or different passages of HEK293T cells). Each biological replicate represents the average of 3 technical replicates. Transient transfection of both DNAs and RNAs was carried out for 24 h. Mimic/control, inhibitor/control and TSB/control were used at 50, 25, and 50 nM, respectively. RT-qPCR data were analyzed using the 2(−Δct) method.

Article Snippet: The substrates of Firefly luciferase (LAR II, Promega) and Renilla luciferase (Stop & Glo, Promega) were sequentially added to the cell lysates (1:1 ratio).

Techniques: Functional Assay, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Negative Control, Control, Transfection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Tribbles-1 Expression and Its Function to Control Inflammatory Cytokines, Including Interleukin-8 Levels are Regulated by miRNAs in Macrophages and Prostate Cancer Cells

doi: 10.3389/fimmu.2020.574046

Figure Lengend Snippet: TRIB1 is a target of miRNAs downregulated in prostate cancer. (A) Relative mRNA expression of TRIB1 normalized to the housekeeping GAPDH in prostate cancer cell lines (n = 4–6, biological replicates, ordinary one-way ANOVA with Sidak’s post-test, ****p < 0.0001, ***p < 0.001, **p < 0.01, no statistical significance p > 0.05). (B) Flowchart for the detection of miRNAs downregulated/silenced in prostate cancer and potentially targeting the 3’UTR of TRIB1. (C–E) Relative expression of miR-132-3p, miR-224-5p and miR-101-3p normalized to the housekeeping U6 in a panel of prostate cancer and control cell lines (n = 4–6, biological replicates, ordinary one-way ANOVA with Sidak’s post-test, ****p < 0.0001, **p < 0.01, ND not detected, ns p > 0.05). (F) Schematic representation of miR-132-3p binding sites in two different positions of TRIB1 3’UTR. (G, H) Relative luciferase activity measured in HEK293T cell line after co-transfection of co-transfection of the TRIB1 3’UTR renilla reporter (95 ng/well) and miR-132-3p mimic/control (left) and miR-132-3p inhibitor/control (right) (+ firefly luciferase reporter, 5 ng/well) (n = 3–5, biological replicates, unpaired t-test, *p < 0.05, no statistical significance p > 0.05). Data are presented as mean ± SEM (scatter dot plot where each dot represents different passages of cell lines). Each biological replicate represents the average of 3 technical replicates. Transfection of plasmids and RNAs was carried out for 24 h. Mimic/control and inhibitor/control were used at 50 and 25 nM, respectively. RT-qPCR data were analyzed using the 2(−Δct) method.

Article Snippet: The substrates of Firefly luciferase (LAR II, Promega) and Renilla luciferase (Stop & Glo, Promega) were sequentially added to the cell lysates (1:1 ratio).

Techniques: Expressing, Control, Binding Assay, Luciferase, Activity Assay, Cotransfection, Transfection, Quantitative RT-PCR