e2f4 shrna  (OriGene)

Journal: Oncogenesis

Article Title: Heterogeneity of subsets in glioblastoma mediated by Smad3 palmitoylation

doi: 10.1038/s41389-021-00361-8

Figure Lengend Snippet: A IDH1 knockdown elevates HIF-1α levels in the proneural GSC subtype, PN12. IDH1 and HIF-1α protein levels were determined by western blotting from PN12 GSCs transduced with control or siRNA targeting IDH1. The relative intensity (HIF-1α/β-actin) was quantified and indicated below. B A cell-permeable α-KG derivative blocks induction of HIF-1α in cells expressing IDH1 R132H. PN12 GSCs were transfected with IDH1 R132H, and different concentrations of octyl-α-KG ester were added to each transfected cell for 4 h. HIF-1α protein levels were assayed by western blotting. The relative intensity (HIF-1α/β-actin) was quantified and is indicated below. C Analysis of reverse-phase protein array expression data for proneural GBM from The Cancer Genome Atlas containing data for 64 tumor samples. HIF-1α protein levels were upregulated in IDH1 R132H mutant GBM ( n = 16) when compared with wild-type IDH1 GBM ( n = 48). Differences between groups were examined for statistical significance by the Student’s t -test using GraphPad Prism software, and the log-rank p -value was used to determine statistical significance, without multiple testing correction. D IDH1 knockdown or mutant IDH1 increases HIF-1α mRNA. After PN12 GSCs were transfected with control or siRNA targeting IDH1 or with the IDH1 R132H mutant, expression of HIF-1α mRNA was measured by quantitative polymerase chain reaction. E IDH1 knockdown reduces methylation of HIF-1α. Methylation-specific PCR (MSP) measurement of DNA methylation levels of the HIF-1α gene in PN12 GSCs. M and U represent amplification of methylated and unmethylated portions, respectively. Quantification of MSP band density for methylated alleles (closed bars) and unmethylated alleles (open bars) is shown. F Overexpression of HIF-1α reduced TGF-β1 levels in proneural GSCs, PN12. HIF-1α and TGF-β1 protein levels were determined by western blotting from PN12 GSCs transduced with control or Flag-tagged HIF-1α. The relative intensity (TGF-β1/β-actin) was quantified and is indicated below. G HIF-1α reduces the stability of TGF-β1 protein. PN12 GSCs were transfected with Flag-tagged HIF-1α for 24 h and then incubated with cyclohexidine 200 μg/ml, after which the cells were harvested at the indicated times for Western blot analysis. Relative TGF-β1 protein levels normalized to β-actin are presented relative to the level (set as 1.0) at 0 h post-CHX treatment (upper panel). H HIF-1α decreases expression of TGF-β1 mRNA. After PN12 was transfected with Flag-tagged HIF-1α or treated with CoCl 2 (100 μmol/l) for 24 h, expression of TGF-β1 mRNA was measured by quantitative polymerase chain reaction. Analysis of variance coupled with Dunnett’s test (two-sided) was used for post hoc comparisons. * p < 0.05 versus the control group. GBM glioblastoma, GSCs glioblastoma stem cells, HIF-1α hypoxia-inducible factor 1-alpha, IDH1 isocitrate dehydrogenase 1.

Article Snippet: Cells were also transduced with pGFP-C-shLenti virus specks specifically for control shRNA or E2F4 shRNA (#TL313319; Origene, Rockville, MD, USA), HIF-1α shRNA (#TL320380, Origene), ZDHHC19 shRNA (#TL317857, Origene), or with pLenti-C-Myc-DDK-P2A-Purovirus species specifically for ZDHHC19 (#RC221741L3V, Origene).

Techniques: Knockdown, Western Blot, Transduction, Control, Expressing, Transfection, Protein Array, Mutagenesis, Software, Real-time Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Amplification, Over Expression, Incubation

Journal: Oncogenesis

Article Title: Heterogeneity of subsets in glioblastoma mediated by Smad3 palmitoylation

doi: 10.1038/s41389-021-00361-8

Figure Lengend Snippet: A Heatmap showing molecular subtype marker expression in proneural GSCs (PN12, PN16, and PN19), mesenchymal GSCs (ME23, ME27, and ME29), and proneural (or mesenchymal) GSCs infected with the indicated plasmids or shRNA or treated with the indicated inhibitors. HIF-1α inhibitor, GN44028, 12.5 μM. Palmitoylation inhibitor, 2-BP, 100 μM. Smad3 inhibitor, (E)-SIS3, 20 μM. EP300 inhibitor, C646, 15 μM. Z-scores were calculated from the △Ct values obtained in the quantitative polymerase chain reaction analysis. B Viability of proneural PN12 GSCs transfected with the indicated plasmids or shRNA or treated with GN44028, C646, 2-BP, or (E)-SIS3 for 48 h. C A serial dilution assay was used to determine the self-renewal capacity of PN12 GSCs transfected with the indicated plasmids or shRNA or treated with GN44028, C646, 2-BP, or (E)-SIS3. D Viability of mesenchymal ME23 GSCs transfected with the indicated plasmids or shRNA or treated with GN44028, C646, 2-BP, or (E)-SIS3 for 48 h. E A serial dilution assay was used to determine the self-renewal capacity of ME23 GSCs transfected with the indicated plasmids or shRNA or treated with GN44028, C646, 2-BP, or (E)-SIS3. GSCs glioblastoma stem cells, HIF-1α hypoxia-inducible factor 1-alpha.

Article Snippet: Cells were also transduced with pGFP-C-shLenti virus specks specifically for control shRNA or E2F4 shRNA (#TL313319; Origene, Rockville, MD, USA), HIF-1α shRNA (#TL320380, Origene), ZDHHC19 shRNA (#TL317857, Origene), or with pLenti-C-Myc-DDK-P2A-Purovirus species specifically for ZDHHC19 (#RC221741L3V, Origene).

Techniques: Marker, Expressing, Infection, shRNA, Real-time Polymerase Chain Reaction, Transfection, Serial Dilution Assay