Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: (A) Western blot of total HIF-2α and DDX28 protein levels in normoxia (21% O 2 ) and hypoxia (1% O 2 ). GAPDH used as a loading control. (B) Western blot of DDX28 and HIF-2α (arrow, hypoxia-inducible lower band) normoxic and hypoxic protein levels in control (Ctrl) cells stably expressing a non-targeting shRNA or in cells stably expressing one of two shRNAs targeting DDX28 mRNA: Knockdown (KD) 1 and KD2. Actin used as a loading control. (C) GFP co-immunoprecipitation in hypoxic cells stably expressing FLAG-DDX28 and transfected with recombinant GFP-HIF-2α. Cells transfected with no DNA (−) or GFP alone were used as controls. (D) FLAG co-immunoprecipitation of recombinant FLAG-eIF4E2 from hypoxic cells co-transfected with HA-HIF-2α. Cells transfected with empty FLAG vector used as control. (E) FLAG co-immunoprecipitation in hypoxic cells stably expressing FLAG-DDX28 and transfected with recombinant HA-HIF-1α. Cells transfected with empty FLAG vector used as control. SE, short exposure; LE, long exposure. 25 μg of whole cell lysate was used as input. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Immunoprecipitation, Transfection, Recombinant, Plasmid Preparation

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: Western blot and quantification of eIF4E2 capture with m 7 GTP-bound agarose beads in cells stably expressing one of two distinct shRNA sequences targeting DDX28 in 1% O 2 hypoxia (A-B) and 21% O 2 normoxia (C-D). 35 μg of whole cell lysate was used as the input. Ctrl, control cells stably expressing non-targeting shRNA; KD1 and KD2, knockdown cells stably expressing one of two distinct shRNA sequences targeting DDX28; LE, long exposure. Data (n ≥ 3), mean ± s.e.m normalized to input. * represents p < 0.05 using one sample t-test against hypothetical mean (μ = 1). Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control, Knockdown

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: Polysomal distribution of DDX28, eIF4E2 and eIF4E protein measured by western blot in control cells stably expressing non-targeting shRNA in 21% O 2 normoxia (A) and 1% O 2 hypoxia (C) and in Knockdown (KD) cells stably expressing an shRNA targeting DDX28 in normoxia (B) and hypoxia (D). Ribosomal protein L5 (rpL5) used as a marker of protein integrity in each fraction. The eIF4E (E) or eIF4E2 (F) protein associated with polysomes (fractions 4-9) as a percentage of total protein (fractions 1-9) was quantified by densitometry. Data (n = 3), mean ± s.e.m. * represents p < 0.05 using one-way ANOVA and Tukey’s HSD post-hoc test. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Marker

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: ( A) Western blot of HIF-2α protein levels in cytoplasmic and nuclear fractions of control cells expressing a non-targeting shRNA or Knockdown (KD) cells stably expressing an shRNA targeting DDX28 in 1% O 2 hypoxia. Lamin a/c used as nuclear marker and α-tubulin as cytoplasmic marker. (B) The mRNA abundance of HIF-2α gene targets in hypoxia measured via qRT-PCR. Data (n ≥ 3), mean ± s.e.m. represented as log 2 (fold change) in DDX28 KD cells relative to control cells and normalized to endogenous control genes RPLP0 and RPL13A. * represents p < 0.05 using a one-sample t-test against hypothetical mean (μ = 0). CITED2, Cbp/P300-Interacting Transactivator 2; EPO, Erythropoietin; IGFBP3, Insulin Like Growth Factor Binding Protein 3; ITPR1, inositol 1,4,5-trisphosphate receptor type 1; LOXL2, Lysyl Oxidase Like 2; OCT4, octamer-binding transcription factor 4. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Western Blot, Control, Expressing, shRNA, Knockdown, Stable Transfection, Marker, Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: DEAD-box protein family member DDX28 is a negative regulator of HIF-2α and eIF4E2-directed hypoxic translation

doi: 10.1101/632331

Figure Lengend Snippet: Viable cell counts were measured with crystal violet staining after 24 h, 48 h, and 72 h in 21% O 2 normoxia (A) and 1% O 2 hypoxia (B) for control cells expressing a non-targeting shRNA or Knockdown cells stably expressing one of two shRNAs targeting DDX28 (KD1 and KD2). All absolute cell count values were normalized to the number of cells present on day 0 for each individual independent experiment, representing the fold change in the number of cells at each time point relative to day 0. Proliferation was measured as % BrdU-positive control and DDX28 KD cells via immunofluorescence after 24 h in normoxia (C) or hypoxia (D). Data (n ≥ 3), mean ± s.e.m. * represents p < 0.05 using an unpaired two-sample t-test. Experiments performed in U87MG glioblastoma.

Article Snippet: U87MG cells stably expressing C-terminal 3x FLAG tagged DDX28 were generated by transfecting cells with the OmicsLinkTM pEZ-M14 EX-A3144-M14 expression vector encoding the human DDX28 coding sequence (Genecopoeia).

Techniques: Staining, Control, Expressing, shRNA, Knockdown, Stable Transfection, Cell Counting, Positive Control, Immunofluorescence

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: Hypoxia regulated miRNAs. Hierarchical clustering of hypoxia-induced and down-regulated miRNAs (>1.5-fold) in response to hypoxia (0.2% O 2 ) in cell line U87MG (a) . List of hypoxia-regulated miRNA clusters in U87MG cells (b) . A table showing correlation of microRNAs altered in hypoxia or in GBM tumor tissues (c) .

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques:

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: Quantitative RT-PCR data showing miRNA levels in response to hypoxia or HIF1A. Graph showing miRNAs that are upregulated (a) or downregulated (b) in response to hypoxia. (c) U87MG cells were transfected with pCDNA3.1or a HIF1A over-expressing plasmid (pCDNA3.1-HIF1A), and miRNA levels were determined. U87MG cells were transfected with pLK0.1-shGFP or a shHIF1A over-expressing plasmid (pLK0.1-shHIF1A) and levels of HIF1A (d) and miRNAs (e) in response to hypoxia were determined. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: MiR-210-3p induces HIF transcriptional activity. U87MG cells were transfected with a HRE luciferase vector, along with either miR-210-3p overexpression - [(pBABE-miR210) or control (pBABE)] or inhibition - [(miR-210 inhibitor) or (control)] vectors, and HRE transcriptional activity was assayed (a) . U87MG cells were transiently transfected with either a miR-210-3p over-expression vector (pBABE-miR-210) or the empty pBABE-puro parent vector (b) or with either a miR-210-3p inhibitor or control oligos (c) , and VEGF/CA9 levels were determined by qRT-PCR. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Over Expression, Control, Inhibition, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: MiR-210-3p functions. Graphs showing MTT assay results of cell survival on the 3 rd day in U87MG, U251MG and A172 cells in response to either miR-210 overexpression - [miR-210 polyclonals (pBABE-miR-210) or control (pBABE)] or inhibition - [(miR-210 inhibitor) or (control)] under (a) 0.2% hypoxia (b) or serum starvation or (c) chemo drug temozolomide treatment. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: MTT Assay, Over Expression, Control, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: EGFR-Targeted Pentacyclic Triterpene Analogues for Glioma Therapy

doi: 10.3390/ijms222010945

Figure Lengend Snippet: The cytotoxic effects of the compounds on U251, T98G, U87, and Jurkat cells, PBMCs.

Article Snippet: U251, T98G (Japanese Collection of Research Bioresources Cell Bank), and U87 human glioblastoma cell lines were incubated in Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12 (Wako Pure Chemical Industries, Osaka, Japan), Eagle’s Minimum Essential Medium (EMEM; Wako Pure Chemical Industries, Osaka, Japan), and DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) respectively, and supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, MO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: EGFR-Targeted Pentacyclic Triterpene Analogues for Glioma Therapy

doi: 10.3390/ijms222010945

Figure Lengend Snippet: The anticancer effects of compounds 4, 10, 13, and cisplatin at varying concentrations on U251 cells ( a ), T98G cells ( b ), U87 cells ( c ), Jurkat cells ( d ), and PBMCs ( e ). All descriptive data were expressed as the mean ± standard deviation (SD). All experiments were repeated three times.

Article Snippet: U251, T98G (Japanese Collection of Research Bioresources Cell Bank), and U87 human glioblastoma cell lines were incubated in Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12 (Wako Pure Chemical Industries, Osaka, Japan), Eagle’s Minimum Essential Medium (EMEM; Wako Pure Chemical Industries, Osaka, Japan), and DMEM (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) respectively, and supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich, MO, USA).

Techniques: Standard Deviation