Journal: Nature Communications
Article Title: JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma
doi: 10.1038/s41467-019-11132-w
Figure Lengend Snippet: THZ1 and panobinostat synergistically induce tumor regression by reducing JMJD6. a , b CHP134 and SK-N-AS cells were treated with vehicle control, THZ1, panobinostat, or their combination for 72 h, followed by Alamar blue assays ( a ). Error bars represent normalized standard errors from three independent experiments. Isobolograms showed the actual concentrations of THZ1 and panobinostat required to achieve inhibition of cell viability by 85% (IC 85 , solid line), as compared with additivity [dotted line, combination index (CI) = 1] ( b ). c CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by Alamar blue assays. Error bars represent normalized standard errors from four independent experiments (* p < 0.05, *** p < 0.001, two-tailed unpaired Student’s t test). d CHP134 and SK-N-AS cells were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by staining with propidium iodide and flow cytometry analysis of the cell cycle. Error bars represent standard errors from four independent experiments (* p < 0.05, two-tailed unpaired Student’s t test). e Seventy-two nude mice were xenografted with SK-N-AS cells stably transfected with an empty vector or JMJD6 ORF expression construct. When tumors reached 0.05 mm 3 , the mice were divided into four subgroups of nice mice each and treated intraperitoneally with control solvent, THZ1 (10 mg/kg body weight/twice a day), panobinostat (7.5 mg/kg body weight/3 days), or THZ1 plus panobinostat for 21 days. Tumor mass was measured every other day. Error bars represent standard errors (* p < 0.05, *** p < 0.001, two-way ANOVA). f Protein was extracted from the tumor tissues, and subjected to immunoblot analysis of JMJD6 and E2F2 protein expression. Source data are provided as a Source Data file
Article Snippet: The membranes were blocked of nonspecific antibody binding with 10% skim milk powder in phosphate-buffered saline, and probed with the following primary antibodies: mouse anti-E2F2 antibody (1:2000) (sc-633×), mouse anti-JMJD6 antibody (1:500) (sc-28348), mouse anti-N-Myc antibody (1:1000) (sc-53993), or rabbit anti-c-Myc antibody (1:500) (sc-764) (all from Santa Cruz Biotechnology).
Techniques: Control, Inhibition, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Construct, Two Tailed Test, Staining, Flow Cytometry, Solvent, Western Blot
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![THZ1 and panobinostat synergistically induce tumor regression by reducing JMJD6. a , b CHP134 and SK-N-AS cells were treated with vehicle control, THZ1, panobinostat, or their combination for 72 h, followed by Alamar blue assays ( a ). Error bars represent normalized standard errors from three independent experiments. Isobolograms showed the actual concentrations of THZ1 and panobinostat required to achieve inhibition of cell viability by 85% (IC 85 , solid line), as compared with additivity [dotted line, combination index (CI) = 1] ( b ). c CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by Alamar blue assays. Error bars represent normalized standard errors from four independent experiments (* p < 0.05, *** p < 0.001, two-tailed unpaired Student’s t test). d CHP134 and SK-N-AS cells were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by staining with propidium iodide and flow cytometry analysis of the cell cycle. Error bars represent standard errors from four independent experiments (* p < 0.05, two-tailed unpaired Student’s t test). e Seventy-two nude mice were xenografted with SK-N-AS cells stably transfected with an empty vector or JMJD6 ORF expression construct. When tumors reached 0.05 mm 3 , the mice were divided into four subgroups of nice mice each and treated intraperitoneally with control solvent, THZ1 (10 mg/kg body weight/twice a day), panobinostat (7.5 mg/kg body weight/3 days), or THZ1 plus panobinostat for 21 days. Tumor mass was measured every other day. Error bars represent standard errors (* p < 0.05, *** p < 0.001, two-way ANOVA). f Protein was extracted from the tumor tissues, and subjected to immunoblot analysis of JMJD6 and E2F2 protein expression. Source data are provided as a Source Data file](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8504/pmc06658504/pmc06658504__41467_2019_11132_Fig7_HTML.jpg)
