Journal: eLife

Article Title: Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling

doi: 10.7554/eLife.77340

Figure Lengend Snippet: Predicted targets of miR-306 and miR-79. ( B, C ) Wing disc of indicated genotypes with puc-lacZ background stained with anti-β-galactosidase antibody ( B,C and B’, C’ , 5 or 6 days after egg laying). ( D ) Schematic of the wild-type and mutation-type 3′UTR vector with miRNA binding sites for miR-306 and miR-79, respectively. Red letters shows the mutation sites. Red box shows the seed sequence pairing region. ( E ) RLU/FLU rate from dual-luciferase assay. n = 3, error bars, SD; n.s., p>0.05 (not significant), **p<0.01 by two-tailed Student’s t -test. ( F ) Lysates of adult heads of indicated genotypes were subjected to Western blots using indicated antibodies. ( G ) Quantification of relative levels of RNF146 protein in ( F ) from three independent experiments. Error bars, SD; *p<0.05, **p<0.01 by one-way ANOVA multiple-comparison test. ( H–J ) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (7 days after egg laying). ( K ) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of ( H–J ). Error bars, SD; ****p<0.0001 by two-tailed Student’s t -test. ( L–M ) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (7 days after egg laying). ( N ) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of ( L–M ). Error bars, SD; n.s., p>0.05 (not significant) by two-tailed Student’s t -test. ( O–R ) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes (7 days after egg laying). ( S ) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of ( O–R ). Error bars, SD; *p<0.05, **p<0.01 by one-way ANOVA multiple-comparison test. Figure 5—source data 1. Quantitative data or raw data for . Figure 5—source data 2. Genotypes for and – .

Article Snippet: The cell lysates were then subjected to SDS-PAGE, followed by Western blots using anti-α-tubulin monoclonal antibody (Sigma-Aldrich, Cat #T5168, 1:5000), anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #9251, 1:1000), anti-JNK monoclonal antibody (Santa Cruz Biotechnology, Cat #sc-7345, 1:1000), anti-RNF146 polyclonal antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China, 1:2000), anti-V5 tag monoclonal antibody (Thermo Fisher Scientific, Cat #R960-25, 1:5000), anti-myc tag polyclonal antibody (MBL, Code #562, 1:1000), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7076, 1:5000), or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7074, 1:5000).

Techniques: Staining, Mutagenesis, Plasmid Preparation, Binding Assay, Sequencing, Luciferase, Two Tailed Test, Western Blot, Comparison, Labeling, Clone Assay

Journal: eLife

Article Title: Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling

doi: 10.7554/eLife.77340

Figure Lengend Snippet: ( A ) Lysates of adult heads of indicated genotypes were subjected to Western blots using indicated antibodies. ( B ) Quantification of relative levels of RNF146 protein in ( A ) from three independent experiments. Error bars, SD; *p<0.05 by two-tailed Student’s t -test. Figure 5—figure supplement 3—source data 1. Quantitative data or raw data for .

Article Snippet: The cell lysates were then subjected to SDS-PAGE, followed by Western blots using anti-α-tubulin monoclonal antibody (Sigma-Aldrich, Cat #T5168, 1:5000), anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #9251, 1:1000), anti-JNK monoclonal antibody (Santa Cruz Biotechnology, Cat #sc-7345, 1:1000), anti-RNF146 polyclonal antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China, 1:2000), anti-V5 tag monoclonal antibody (Thermo Fisher Scientific, Cat #R960-25, 1:5000), anti-myc tag polyclonal antibody (MBL, Code #562, 1:1000), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7076, 1:5000), or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7074, 1:5000).

Techniques: Western Blot, Two Tailed Test

Journal: eLife

Article Title: Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling

doi: 10.7554/eLife.77340

Figure Lengend Snippet: ( A ) Drosophila S2 cells were transfected with plasmids expressing indicated proteins. Cell lysates were subjected to Western blots using indicated antibodies. ( B ) Quantification of relative Tnks-myc levels in ( A ) from three independent experiments. Error bars, SD; ***p<0.001 by two-tailed Student’s t -test. ( C ) Quantification of relative p-JNK levels in ( A ) from three independent experiments. Error bars, SD; *p<0.05 by one-way ANOVA multiple-comparison test. ( D ) Drosophila S2 cells were transfected with plasmid expressing indicated protein and dsRNA targeting indicated gene. ( E, F ) Quantification of relative Tnks-myc levels ( E ) and p-JNK ( F ) levels in ( D ) from three independent experiments. Error bars, SD; *p<0.05, ***p<0.001 by one-way ANOVA multiple-comparison test. ( G–J ) Eye-antennal disc bearing GFP-labeled clones of indicated genotypes ( G, H , 5 days after egg laying, I, J , 7 days after egg laying). ( K ) Quantification of clone size (% of total clone area per disc area in eye-antennal disc) of ( G–J ). Error bars, SD; ****p<0.0001 by one-way ANOVA multiple-comparison test. ( L ) Adult eye phenotype of flies with indicated genotypes. ( M ) Eclosion rate of flies with indicated genotypes. Data from three independent experiment, n > 30 for each group in one experiment; error bars, SD. ( N, O ) Drosophila S2 cells were transfected with plasmid expressing indicated protein and dsRNA targeting indicated gene. After 36 hr, cells were treated with 50 μg/ml cycloheximide (CHX) for the indicated periods. Cell lysates were subjected to Western blots using indicated antibodies. ( P ) Quantification of relative Tnks-myc levels in ( N, O ) from three independent experiments. Error bars, SD. ( Q ) A model for tumor elimination by miR-306/79. Tumor cell with elevated canonical JNK signaling via Eiger/TNF, dTAK1/JNKKK, and Hep/JNKK grows in a Bsk/JNK-dependent manner. Overexpression of miR-306 or miR-79 in JNK-activated tumor cell results in overactivation of JNK signaling to the lethal level via RNF146-Tnks-mediated noncanonical JNK-activating signaling. Overexpression of miR-306 or miR-79 in normal cells has no significant effect on JNK signaling. Figure 6—source data 1. Quantitative data or raw data for (part 1). Figure 6—source data 2. Quantitative data or raw data for (part 2). Figure 6—source data 3. Genotypes for .

Article Snippet: The cell lysates were then subjected to SDS-PAGE, followed by Western blots using anti-α-tubulin monoclonal antibody (Sigma-Aldrich, Cat #T5168, 1:5000), anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #9251, 1:1000), anti-JNK monoclonal antibody (Santa Cruz Biotechnology, Cat #sc-7345, 1:1000), anti-RNF146 polyclonal antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China, 1:2000), anti-V5 tag monoclonal antibody (Thermo Fisher Scientific, Cat #R960-25, 1:5000), anti-myc tag polyclonal antibody (MBL, Code #562, 1:1000), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7076, 1:5000), or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7074, 1:5000).

Techniques: Transfection, Expressing, Western Blot, Two Tailed Test, Comparison, Plasmid Preparation, Labeling, Clone Assay, Over Expression

Journal: eLife

Article Title: Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling

doi: 10.7554/eLife.77340

Figure Lengend Snippet:

Article Snippet: The cell lysates were then subjected to SDS-PAGE, followed by Western blots using anti-α-tubulin monoclonal antibody (Sigma-Aldrich, Cat #T5168, 1:5000), anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #9251, 1:1000), anti-JNK monoclonal antibody (Santa Cruz Biotechnology, Cat #sc-7345, 1:1000), anti-RNF146 polyclonal antibody (raised in rabbits against the peptide HSGGGSGEDPAVGSC, GenScript antibody service, Nanjing, China, 1:2000), anti-V5 tag monoclonal antibody (Thermo Fisher Scientific, Cat #R960-25, 1:5000), anti-myc tag polyclonal antibody (MBL, Code #562, 1:1000), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7076, 1:5000), or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Cat #7074, 1:5000).

Techniques: Transfection