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: The Journal of Biological Chemistry

Article Title: A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line

doi: 10.1074/jbc.M116.771188

Figure Lengend Snippet: Flow cytometric analysis of U87MG-EGFRvIII (A), A431 (B), and CNE (C) cell lines. Cells were stained with an irrelevant isotype control (black), mAb806 (green), cetuximab (red), and cetuximab/mAb806 mixture (blue). A, panels 1–4, U87MG-EGFRvIII cells were probed with individually produced mAb806 (green) or cetuximab (red) in four different concentrations (0.1, 1, 10, or 100 μg ml−1), or their mixture (blue) was produced in a single cell line, also in four different concentrations (0.2, 2, 20, or 200 μg ml−1). B and C, either A431 or CNE cells were probed with individually produced cetuximab or mAb806 in a saturated concentration (100 μg ml−1) or with the cetuximab/mAb806 mixture produced in a single cell line in a saturated concentration (200 μg ml−1).

Article Snippet: Each mouse was injected s.c. into the right flank with 2 × 10 6 U87MG-EGFRvIII cells (U87MG-EGFRvIII cell line constantly expressing human EGFRvIII protein) in 50% culture medium and 50% Matrigel (BD Biosciences).

Techniques: Staining, Produced, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line

doi: 10.1074/jbc.M116.771188

Figure Lengend Snippet: Flow cytometric analysis of U87MG-EGFRvIII, A431 and CNE cell lines

Article Snippet: Each mouse was injected s.c. into the right flank with 2 × 10 6 U87MG-EGFRvIII cells (U87MG-EGFRvIII cell line constantly expressing human EGFRvIII protein) in 50% culture medium and 50% Matrigel (BD Biosciences).

Techniques: Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line

doi: 10.1074/jbc.M116.771188

Figure Lengend Snippet: Treatment of established U87MG-EGFRvIII xenografts with mAb806, cetuximab, and cetuximab/mAb806 mixture. Mice (n = 6) bearing U87MG-EGFRvIII xenografts were injected i.p. with PBS (■), 0.5 mg/kg mAb806 (▴), 0.5 mg/kg cetuximab (▾), or 1 mg/kg cetuximab/mAb806 mixture (●), three times weekly for 2 weeks.

Article Snippet: Each mouse was injected s.c. into the right flank with 2 × 10 6 U87MG-EGFRvIII cells (U87MG-EGFRvIII cell line constantly expressing human EGFRvIII protein) in 50% culture medium and 50% Matrigel (BD Biosciences).

Techniques: Injection

Journal: Cancers

Article Title: Targeting Glutamine Addiction in Gliomas

doi: 10.3390/cancers12020310

Figure Lengend Snippet: Therapeutic approaches targeting glutamine addiction in gliomas.

Article Snippet: , SAS , D54MG, U87MG, U251MG, STTG1 human GBM cell lines; patient-derived GBM cells; mouse intracranial xenograft (D54MG cells) , decreased cell proliferation and migration; decreased tumor growth and invasion , [ , ] .

Techniques: Inhibition, In Vitro, shRNA, Migration, Over Expression, Activity Assay, In Vivo