vhl dna  (Addgene inc)

Journal: Oncotarget

Article Title: Dicer suppresses the malignant phenotype in VHL-deficient clear cell renal cell carcinoma by inhibiting HIF-2α

doi: 10.18632/oncotarget.7807

Figure Lengend Snippet: ( A ) Volcano plot of 61 mature miRNAs for which there were significant differences in expression between 8 pairs of VHL− and VHL+ ccRCC tissue samples as assessed by microarray analysis. ( B and C ) qRT-PCR analysis of expression levels of representative downregulated mature miRNAs and corresponding upregulated precursor miRNAs in VHL− ccRCCs ( n = 36) compared to VHL+ ccRCCs ( n = 36). ( D ) mRNA levels of miRNA biogenesis components in VHL− ccRCCs ( n = 36) compared to VHL+ ccRCCs ( n = 36). ( E ) Representative immunoblots and quantification of protein levels of miRNA biogenesis components in VHL− ccRCCs ( n = 12) compared with VHL+ ccRCCs ( n = 12). ( F ) Dicer mRNA levels in VHL− ccRCCs ( n = 36) and VHL+ ccRCCs ( n = 36) compared to their corresponding adjacent normal tissues ( n = 36 for both groups). ( G ) Representative immunoblots and quantification of Dicer protein levels in VHL-deficient and pVHL-restored 786-O cells. ( H ) Dicer mRNA levels in VHL-deficient and pVHL-restored 786-O cells. ( I ) Representative immunoblots and quantification of Dicer protein levels in wild-type VHL and VHL-knockdown Caki-1 cells. ( J ) Dicer mRNA levels in wild-type VHL and VHL-knockdown Caki-1 cells. VHL+ denotes wild-type VHL, and VHL− denotes VHL-deficient. Data are presented as mean ± SEM ( n ≥ 3). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student's t -test).

Article Snippet: For knockdown of VHL, annealed DNA oligonucleotides targeting VHL were cloned into a pLVTHM lentiviral vector (Addgene).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Knockdown

Journal: Oncotarget

Article Title: Dicer suppresses the malignant phenotype in VHL-deficient clear cell renal cell carcinoma by inhibiting HIF-2α

doi: 10.18632/oncotarget.7807

Figure Lengend Snippet: ( A ) Schematic representation of the potential binding site of miR-182-5p in the HIF-2α 3′-UTR and its wild type (WT) or mutated (MUT) sequence within the luciferase reporter vector. ( B and C ) Levels of mature miR-182-5p and precursor miR-182-5p after Dicer ectopic expression (Dicer ORF) in VHL wild-type and VHL knockdown Caki-1 cells. ( D ) Association between HIF-2α mRNA levels and miR-182-5p levels in ccRCCs ( n = 48). ( E ) Representative immunoblots and quantification of HIF-2α protein levels after miR-182-5p ectopic expression (mimic miR-182-5p) or miR-182-5p inhibition (inhibitor miR-182-5p) in OS-RC-2 cells. ( F and G ) HIF-2α mRNA levels after miR-182-5p ectopic expression (mimic miR-182-5p) or miR-182-5p inhibition (inhibitor miR-182-5p) in OS-RC-2 cells. ( H ) Representative immunoblots and quantification of HIF-2α protein levels after miR-182-5p ectopic expression (mimic miR-182-5p) or miR-182-5p inhibition (inhibitor miR-182-5p) in 786-O cells. ( I and J ) HIF-2α mRNA levels after miR-182-5p ectopic expression (mimic miR-182-5p) or miR-182-5p inhibition (inhibitor miR-182-5p) in 786-O cells. ( K ) Luciferase activity in OS-RC-2 and 786-O cells transfected with HIF-2α 3′-UTR luciferase reporter vectors with intact or mutated seed sequences in the presence of miR-182-5p mimics or NC, respectively. ( L ) Luciferase activity in OS-RC-2 and 786-O cells transfected with HIF-2α 3′-UTR luciferase reporter vectors with intact or mutated seed sequences in the presence of Dicer ORF or Empty vector, respectively. ORF = open reading frame. Data are presented as mean ± SEM ( n ≥ 3). * p < 0.05; ** p < 0.01; *** p < 0.001 (Student's t -test).

Article Snippet: For knockdown of VHL, annealed DNA oligonucleotides targeting VHL were cloned into a pLVTHM lentiviral vector (Addgene).

Techniques: Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Expressing, Knockdown, Western Blot, Inhibition, Activity Assay, Transfection