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: Scientific Reports

Article Title: Loco-regional treatment with temozolomide-loaded thermogels prevents glioblastoma recurrences in orthotopic human xenograft models

doi: 10.1038/s41598-023-31811-5

Figure Lengend Snippet: IC 50 for TMZ on cell viability in U87 MG-Red-FLuc human glioblastoma cells by cell counts. The concentration-dependent inhibitory dose-curve data was plotted as log percentage inhibition normalized to controls with applied curve non-fit calculated using GraphPad Prism. Results are shown as the mean of at least three independent experiments ± SEM.

Article Snippet: To track tumor growth in an orthotopic mouse model for glioblastoma, we decided to use commercially available human cancer cells expressing a very bright red-shifted luciferase (Red-FLuc), U87-MG-Red-FLuc cells ( https://www.perkinelmer.com/product/ivisbrite-u87mg-red-f-luc-bw124577 ).

Techniques: Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: Loco-regional treatment with temozolomide-loaded thermogels prevents glioblastoma recurrences in orthotopic human xenograft models

doi: 10.1038/s41598-023-31811-5

Figure Lengend Snippet: In vitro effect of the isolated components and of assembled new gel formulations on Glioblastoma cells. ( a ) U87-MG-Red-FLuc cells were treated with cell medium (CTRL), mesoporous SiO 2 nanoparticles (SiO 2 ) and polycaprolactone microparticles (PCL) for 24 h, causing no significant reduction in viable cells number. ( b ) U87 MG-Red-FLuc cells were treated with cell medium (CTRL), the empty thermogel (THG), and the thermogels containing mesoporous SiO 2 nanoparticles (THG@SiO 2 ) and polycaprolactone microparticles (THG@PCL) for 24 h, causing no significant reduction in viable cells number. ( c ) TMZ-containing SiO 2 nanoparticles and PCL microparticles efficiently release active drug into the cell medium to induce cell death after 24 h treatment in U87 MG-Red-FLuc cells (*p < 0.05). ( d ) THG@SiO 2 -TMZ and THG@PCL-TMZ efficiently release active TMZ into the cell medium to induce cell death after 24 h treatment in U87-MG-Red-FLuc cells (* p < 0.05; ** p < 0.005).

Article Snippet: To track tumor growth in an orthotopic mouse model for glioblastoma, we decided to use commercially available human cancer cells expressing a very bright red-shifted luciferase (Red-FLuc), U87-MG-Red-FLuc cells ( https://www.perkinelmer.com/product/ivisbrite-u87mg-red-f-luc-bw124577 ).

Techniques: In Vitro, Isolation

Journal: Scientific Reports

Article Title: Loco-regional treatment with temozolomide-loaded thermogels prevents glioblastoma recurrences in orthotopic human xenograft models

doi: 10.1038/s41598-023-31811-5

Figure Lengend Snippet: In vivo efficacy of TMZ-based therapeutic thermogels using a GBM resection and recurrence mouse model. ( a ) Experimental timeline for studying the in vivo anticancer activities of gel-based local glioblastoma treatments. Two independent experiments comparing empty THG with THG@SIO 2 -TMZ and THG@PCL-TMZ, respectively, were performed (n = 5). Results were next merged resulting in n = 8 for THG, n = 3 for THG@SIO 2 -TMZ and n = 4 for THG@PCL-TMZ, considering drop-outs due to the severity of the procedure. ( b ) Representative BLI images of mice bearing U87-MG-Red-FLuc implantation in the subcortical striatal region. After primary tumor resection, THG@SIO 2 -TMZ, THG@PCL-TMZ or empty THG were applied. ( c ) Tumor growth was directly related to the increment of luminescence signal normalized to baseline (day 2 after resection, corresponding to Time 0 in panels A and B) and recorded at days 7 (Time 1), 14 (Time 2) and 21 (Time 3) after resection, in THG, THG@SiO 2 -TMZ and THG@PCL-TMZ groups. Tumor growth was expressed as fold increase [Lum(tx)−Lum (t0)/Lum (t0)]. THG@SIO 2 -TMZ or THG@PCL-TMZ reduced tumor recurrences when compared to empty THG treatment (*p < 0.05; two-ways ANOVA p < 0.05). ( d ). Luminescence signals from control (empty THG) animals on day 21 are significantly higher (*p < 0.05; Kruskal–Wallis test p < 0.004). ( e ) Site of the resection in relation to the Paxinos representation of brain coronal section at the injection coordinate (− 0.5 mm). Representative images of H&E-stained brain sections collected 21 days after tumor resection and gel application from mice receiving either THG@SIO 2 -TMZ, THG@PCL-TMZ or empty THG (control animals) show the growth of the tumor mass to the areas adjacent to the cancer cells injection site.

Article Snippet: To track tumor growth in an orthotopic mouse model for glioblastoma, we decided to use commercially available human cancer cells expressing a very bright red-shifted luciferase (Red-FLuc), U87-MG-Red-FLuc cells ( https://www.perkinelmer.com/product/ivisbrite-u87mg-red-f-luc-bw124577 ).

Techniques: In Vivo, Injection, Staining

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