Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Modulation of RASD2 by miRNA ‐485‐5p Drives Thyroid Cancer Progression and Metastasis

doi: 10.1002/kjm2.70028

Figure Lengend Snippet: miR‐485‐5p regulation of RASD2 in thyroid cancer. (A) Venn diagram showing overlapping miRNA predictions from three databases (TargetScan, miRDB, StarBase). (B) Sequence alignment between miR‐485‐5p and RASD2 3′‐UTR showing predicted binding site. (C) Dual‐luciferase reporter assay results in IHH4 and TPC‐1 cells transfected with wild‐type or mutant RASD2 3′‐UTR constructs and miR‐485‐5p mimic or negative control. (D) Western blot analysis of RASD2 protein levels in cells transfected with miR‐485‐5p mimic or negative control. (E) TCGA database analysis of miR‐485‐5p expression in thyroid cancer vs. normal tissues. (F) miR‐485‐5p expression in paired tumor‐normal tissues by qRT‐PCR ( n = 96 pairs). (G) Correlation analysis between miR‐485‐5p and RASD2 expression levels in thyroid cancer tissues. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After 48 h, luciferase activities were measured using the Dual‐Luciferase Reporter Assay System (HY‐K1013, MedChemExpress).

Techniques: Sequencing, Binding Assay, Luciferase, Reporter Assay, Transfection, Mutagenesis, Construct, Negative Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection

doi: 10.1073/pnas.2412725121

Figure Lengend Snippet: IRF2 promotes OAS3 expression in unstressed cells. ( A and B ) The levels of OAS3, IRF2, RNase L, and GAPDH were analyzed in the indicated U2OS ( A ) or A549 ( B ) cell lines by western blot. ( C ) The OAS3 mRNA levels were monitored by RT-qPCR in indicated U2OS ( Left ) or A549 ( Right ) cell lines. Mean values ± SD (n = 3). **** P < 0.0001 (two-tailed t test). ( D ) The levels of OAS3 were analyzed by western blot using indicated antibodies in A549 IRF2 KO cells expressing either wild-type IRF2 or DNA binding mutant IRF2K78R. ( E and F ) Total RNAs were isolated from A549 WT or IRF2 KO cells expressing wild-type IRF2, IRF2K78R, or OAS3 and monitored for integrity by a bioanalyzer after transfection with poly(I:C) (10 ng/mL, 4 h). The red arrows indicated ribosomal RNA cleavage products. ( G ) IgG and IRF2 ChIP-sequencing in A549 cells. Analysis of ChIP-sequencing data focuses on the promoter region of OAS3. (H) IRF2 ChIP was performed in A549 WT and IRF2 KO cells. IRF2 binding on the OAS3 promoter was determined by qPCR. Mean values ± SD. *** P < 0.001 (two-tailed t test). ( I ) Schematic of pGL3-OAS3-luciferase constructs harboring OAS3 WT promotor region (−1 to −900 bp upstream of transcription start site [TSS]) and IRF2 binding motif region deleted (Δ−1 to −200 bp). ( J ) The relative luciferase activity (ratio of Firefly:Renilla) was measured in U2OS cells transiently transfected with the empty pGL3 and pGL3 vector harboring either WT and Δ200 bp OAS3 promoter region. ( K ) The relative luciferase activity was monitored U2OS WT or IRF2 KO cells 24 h following transfection with the pGL3-OAS3 vector.

Article Snippet: The OAS3 luciferase reporter plasmid was generated by cloning the OAS3 promoter region (TSS −1 to −900) into pGL3 basic luciferase reporter (Addgene #128046).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Mutagenesis, Isolation, Transfection, ChIP-sequencing, Luciferase, Construct, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies – as well as position of the associated uStop codon at position c.125, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR, 5’ Untranslated Region; CDS, CoDing Sequence; WT, wild-type; uoORF, overlapping upstream Open Reading Frame; eCDS, elongated CDS. (B) Schematic presentation of the pcDNA3.1-L-ENG constructs prepared and used for the evaluation of ENG steady-state levels in HeLa cells. Arrows indicate specific primers targeting the cDNA of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Position of the Myc-His tag is represented by the black square on the plasmid. CMV, cytomegalovirus promoter; WT, wild-type; Var, Variant; 5’UTR, 5’ UnTranslated Region; L, Long. (C and D) Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs or from transduced HUVEC cells with 20 MOI of lentiviruses containing ENG, respectively. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers . Anti-Myc and anti-ENG correspond to the used antibodies for the target protein from HeLa and HUVECs, respectively, and anti-βactin corresponds to the antibody used against the reference protein. kDa, kilodalton; M, protein ladder; WT, wild-type, C-, negative control corresponding to pcDNA3.1-empty vector. Shown results are representative of 5 independent experiments. (E) Decrease of luciferase activity observed with uAUG-creating variants in ENG . Schematic presentation of the pGL3b-(ENG)-luciferase prepared and used in this assay is shown in the upper panel. Arrows indicate specific primers targeting the promoter of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Luc, luciferase. In the lower panel, shown results correspond to Firefly/Renilla ratios normalized to the wild-type (WT) in 5 independent experiments. ***, p-value < 10 −3 (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Sequencing, Construct, Cloning, Expressing, Plasmid Preparation, Variant Assay, Western Blot, Transfection, Negative Control, Luciferase, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Novel uAUG creating variants in the 5’UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.12.18.520932

Figure Lengend Snippet: (A) Schematic presentation of the co-transfected constructs in this assay. BRE, BMP-response element; CMV, cytomegalovirus promoter; WT, wild-type; Var, variant; 5’UTR, 5’ UnTranslated Region; L, Long. (B) Decrease of BRE activity observed with all the analyzed variants in co-transfected NIH3T3 cells stimulated with BMP9 (5 pg/ml). Shown results correspond to the quantification of Firefly/Renilla and normalized to the wild-type (WT) (n = 4). ***, p-value < 10 −3 ; *, p-value < 5.10 −2 , ns, non-significant (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Article Snippet: Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer).

Techniques: Transfection, Construct, Variant Assay, Activity Assay, Comparison

Journal: Organic & biomolecular chemistry

Article Title: New bioluminescent coelenterazine derivatives with various C-6 substitutions.

doi: 10.1039/c7ob01554b

Figure Lengend Snippet: Figure 2. (A) Bioluminescence imaging of coelenterazine derivatives with Renilla luciferase. (B) The comparison of bioluminescence intensities of new derivatives and DeepBlueCTM and

Article Snippet: Recombinant Renilla reniformis luciferase was purchased from RayBiotech (Norcross, GA, USA).

Techniques: Imaging, Luciferase, Comparison

Journal: Organic & biomolecular chemistry

Article Title: New bioluminescent coelenterazine derivatives with various C-6 substitutions.

doi: 10.1039/c7ob01554b

Figure Lengend Snippet: Figure 3. (A)Bioluminescence imaging of parts of coelenterazine derivatives in cellulo; (B) the comparison of bioluminescence intensities of new derivatives and DeepBlueCTM and coelenterazine in ES-2 cells expressing Renilla luciferase (Rluc) at various concentrations; (C) parts of compounds that showed bioluminescence in cellulo.

Article Snippet: Recombinant Renilla reniformis luciferase was purchased from RayBiotech (Norcross, GA, USA).

Techniques: Imaging, Comparison, Expressing, Luciferase