pbabe puro c myc t58a  (Addgene inc)

Journal: Cellular and Molecular Life Sciences

Article Title: HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells

doi: 10.1007/s00018-019-03399-5

Figure Lengend Snippet: HAS3-EVs induce c-Myc and EMT by HH signaling. Protein expression of HH and EMT markers were tested in HaCaT ( a , c , e ) and WM115 cells ( b , d , f ). Both HaCaT and WM115 cells were treated with a Gli inhibitor, GANT58 and a constitutively expressing c-Myc T58A plasmid, along with HAS3-EVs. The impact of the treatments on proliferation was also tested in both HaCaT ( g ) and WM115 ( h ) cells. All the statistical significance tests were done in comparison to control. The data represent mean ± S.E. of four independent experiments. * P value < 0.05, ** P value < 0.01, *** P value < 0.001, one-way ANOVA (Tukey’s test). H3 or H3-EVs HAS3-EVs, Clsi and Myci siRNAs for claspin and c-Myc, E-cad E-cadherin, Gli-inhi or Gli Gli inhibitor

Article Snippet: Plasmid transfection was done with Lipofectamine 3000 (Invitrogen) and siRNA transfections were done with Lipofectamine RNAiMAX (Invitrogen), according to the manufacturers’ instructions. c-Myc T58A (Addgene, USA), EGFP-C1 (Clontech, USA), pcDNA, GFP-mHAS3 D216A [ ] and GFP-ΔHAS3 plasmids were used in 12 well, 6 cm and 15 cm plate formats.

Techniques: Expressing, Plasmid Preparation, Comparison, Control

Journal: eLife

Article Title: Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation

doi: 10.7554/eLife.13374

Figure Lengend Snippet: ( A ) Immunoblotting analysis of the representative metabolic genes in glycolysis, tricarboxylic acid cycle (TCA) pathways. 20 µg of protein lysate from NPCs and from neurons differentiated for 3, 7 and 21 days (D3, D7, D21) were loaded. ( B ) Immunostaining analysis of HK2 and LDHA in NPCs and 3-week neurons. ( C ) Effects of HK2 or LDHA knockdown on NPC proliferation. NPCs at early passage (P2) were seeded in 24-well plates one day before infection. The NPC number was determined at 5 days after infection with lenti-shRNA virus against HK2 or LDHA. Two effective shRNA lentiviral vectors targeting different regions of HK2 or LDHA were used. Scramble shRNA vector was used as control. Error bars represent ± SD, n= 3. The knockdown efficiency was confirmed by immunoblotting. ( D , E ) Immunostaining analysis of LDHA in neurons directly converted from fibroblasts. Two protocols were applied: one was to knockdown PTB1, a single RNA-binding protein, and the other was to overexpress proneuronal transcription factors Ngn2 and Ascl1. Tuj1 (ß-III tubulin) and Tau were stained as early and mature neuronal markers, respectively. The above experiments were repeated at least three times. ( F ) ChIP analysis of HK2 and LDHA promoters using anti-cMYC or N-MYC antibodies and rabbit IgG as control. Chromatin were prepared from NPCs. The enrichment values are shown as percentage normalized to input. N.C. stands for non-specific control. Bars are mean ± SD, n= 3. Immunoblotting and real time PCR analysis of HK2 and LDHA expression in NPCs with inducible c-MYC. mRNA expression levels of HK2 and LDHA relative to those from non-induction control were calculated after normalization to β-actin. Bars are mean ± SD, n= 3. ( G ) Immunoblotting analysis of c-MYC, N-MYC and Max in NPCs and 3-week neurons. . DOI: http://dx.doi.org/10.7554/eLife.13374.017 10.7554/eLife.13374.018 Figure 3—source data 1. Knockdown effect on NPC proliferation and Myc control of HK2 and LDHA in NPCs. DOI: http://dx.doi.org/10.7554/eLife.13374.018

Article Snippet: The inducible c-MYC (T58A, a mutation stabilizing c-MYC) lentivirus expression vectors, FUdeltaGW-rtTA (#19780) and FU-tet-o-hc-MYC (#19775) were developed by and obtained from Addgene.

Techniques: Western Blot, Immunostaining, Infection, shRNA, Plasmid Preparation, RNA Binding Assay, Staining, Real-time Polymerase Chain Reaction, Expressing