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: Oncology Letters

Article Title: ERK-mediated negative feedback regulation of oncogenic EGFRvIII in glioblastoma cells

doi: 10.3892/ol.2020.11760

Figure Lengend Snippet: ERK-mediated Thr-669 phosphorylation suppresses the constitutive tyrosine phosphorylation of EGFR-vIII. (A and B) 293 cells transiently transfected with the expression plasmid for EGFR-vIII were stimulated with 100 ng/ml TPA for the indicated periods. (C) 293 cells transfected with expression plasmids for the WT or T669A mutant of EGFRvIII stimulated with TPA for 10 min. (D) 293 cells co-transfected with EGFRwt and EGFRvIII were pretreated with 0.03 µM Tram for 30 min, and then stimulated with TPA for another 10 min. Whole cell lysates were analyzed by (A) Zn2+ Phos-tag-immunoblotting or (B) normal IB with phospho-EGFR (Thr-669 and Tyr-1068), EGFR, phospho-ERK and actin antibodies. EGFRvIII, epidermal growth factor receptor variant III; TPA, 12-O-tetradecanoylphorbol-13-acetate; wt, wild-type; Tram, trametinib; IB, immunoblotting.

Article Snippet: Cell lines and culture conditions Human U87MG glioblastoma cells that overexpress EGFRwt and EGFRvIII were provided by Professors Webster K. Cavenee (University of California San Diego) and Motoo Nagane (Kyorin University) ( 18 , 19 ).

Techniques: Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Variant Assay

Journal: Oncology Letters

Article Title: ERK-mediated negative feedback regulation of oncogenic EGFRvIII in glioblastoma cells

doi: 10.3892/ol.2020.11760

Figure Lengend Snippet: ERK-mediated feedback inhibition of EGFRvIII in glioma cells. (A) U87MG-vIII cells were stimulated with 100 ng/ml TPA for the indicated periods. Whole cell lysates were separated by Zn2+ Phos-tag SDS-PAGE and probed with primary antibody against EGFR. (B) Cells stimulated with 100 ng/ml TPA for the indicated periods, then whole cell lysates were analyzed by normal immunoblotting. (C) Cells were treated with 0.03 µM trametinib (Tram) for the indicated times, furthermore separated by normal immunoblotting. (D) U87MG-vIII cells were pretreated with 0.03 µM trametinib (Tram) 30 min, and then stimulated with TPA for another 10 min. Whole cell lysates were separated by Zn2+ Phos-tag SDS-PAGE and immunoblotted with antibody against EGFR. (E) Cells were pretreated with 0.03 µM trametinib (Tram) or 0.5 µM SCH772984 (SCH) for 30 min, and then stimulated with TPA for another 10 min. All whole cell lysates from normal SDS-PAGE were immnunoblotted with phospho-EGFR (Thr-669 and Tyr-1068). EGFR, phospho-ERK and actin antibodies. EGFRvIII, epidermal growth factor receptor variant III; TPA, 12-O-tetradecanoylphorbol-13-acetate; Tram, trametinib; IB, immunoblotting.

Article Snippet: Cell lines and culture conditions Human U87MG glioblastoma cells that overexpress EGFRwt and EGFRvIII were provided by Professors Webster K. Cavenee (University of California San Diego) and Motoo Nagane (Kyorin University) ( 18 , 19 ).

Techniques: Inhibition, SDS Page, Western Blot, Variant Assay

Journal: Oncology Letters

Article Title: ERK-mediated negative feedback regulation of oncogenic EGFRvIII in glioblastoma cells

doi: 10.3892/ol.2020.11760

Figure Lengend Snippet: Schematic diagram of negative feedback regulation of EGFRvIII in glioblastoma cells. TPA stimulation leads to down-regulation of the constitutive tyrosine phosphorylation of EGFRvIII via ERK-mediated Thr-669 phosphorylation. Exogenous EGF causes the rapid transactivation of EGFRvIII by EGFRwt, which in turn triggers ERK-mediated feedback inhibition of EGFRvIII. EGFRvIII, epidermal growth factor receptor variant III; TPA, 12-O-tetradecanoylphorbol-13-acetate; wt, wild-type.

Article Snippet: Cell lines and culture conditions Human U87MG glioblastoma cells that overexpress EGFRwt and EGFRvIII were provided by Professors Webster K. Cavenee (University of California San Diego) and Motoo Nagane (Kyorin University) ( 18 , 19 ).

Techniques: Phospho-proteomics, Inhibition, Variant Assay

Journal: PLoS ONE

Article Title: Anticancer effects of a non-narcotic opium alkaloid medicine, papaverine, in human glioblastoma cells

doi: 10.1371/journal.pone.0216358

Figure Lengend Snippet: (A) Chemical structure of papaverine. (B) Protein levels of MGMT, RAGE, and GAPDH analyzed by immunoblotting. GAPDH used as an internal control. Data are representative of at least three independent experiments. (C) T98G cells treated with 10 μg/mL bovine HMGB1 protein or vehicle (PBS) and then incubated for 72 h. Cells were counted by trypan blue dye exclusion assay using the TC20 automated cell count system. Cell proliferation (%) represents the mean ± SE of three independent experiments. P values were calculated against vehicle control with the Student’s t -test. **p < 0.05, **p < 0.01. (D) The migration ability of T98G cells was analyzed in an in vitro scratch assay. T98G cells were treated with papaverine at the indicated concentration or water as a vehicle for 24 h. The migration ratio (%) represents the mean ± SE of triplicate experiments. Similar results were obtained in two independent experiments. P values were calculated against vehicle control with the Student’s t -test. *p < 0.05, **p < 0.01. (E) U87MG and (F) T98G cells were examined for cell activity in a WST-8 assay after 72 h papaverine treatment. Results are the averages of three independent experiments with error bars showing SE from triplicates.

Article Snippet: For heterotrophic/subcutaneous xenografts, 1 × 10 6 U87MG cells resuspended in 100 μL phosphate-buffered saline (PBS) were subcutaneously injected into the right leg of 5-week-old male BALB/c nude mice (CLEA Japan, Inc., Tokyo, Japan) weighing approximately 20–22 g with four mice in each group.

Techniques: Western Blot, Control, Incubation, Exclusion Assay, Cell Counting, Migration, In Vitro, Wound Healing Assay, Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: Anticancer effects of a non-narcotic opium alkaloid medicine, papaverine, in human glioblastoma cells

doi: 10.1371/journal.pone.0216358

Figure Lengend Snippet: Summary of the anticancer effects of papaverine in human GBM U87MG and T98G cells.

Article Snippet: For heterotrophic/subcutaneous xenografts, 1 × 10 6 U87MG cells resuspended in 100 μL phosphate-buffered saline (PBS) were subcutaneously injected into the right leg of 5-week-old male BALB/c nude mice (CLEA Japan, Inc., Tokyo, Japan) weighing approximately 20–22 g with four mice in each group.

Techniques:

Journal: PLoS ONE

Article Title: Anticancer effects of a non-narcotic opium alkaloid medicine, papaverine, in human glioblastoma cells

doi: 10.1371/journal.pone.0216358

Figure Lengend Snippet: (A) Experimental schedule. (B) To assess the effect of papaverine in tumors in a xenograft model, 1 × 10 6 U87MG human GBM cells were subcutaneously injected into the right leg of 5-week-old male BALB/c nude mice. After 11–13 d of inoculation, four mice per group were treated with papaverine (40 mg/kg) or saline (vehicle control, solvent alone) twice a day for 4 d via i.p. administration. Tumor size was measured once every 3–4 d. Tumor volume ( V ) was calculated as described in Materials and methods. Results are the averages for groups of four mice each with error bars showing SE. White circle, control; black circle, papaverine.

Article Snippet: For heterotrophic/subcutaneous xenografts, 1 × 10 6 U87MG cells resuspended in 100 μL phosphate-buffered saline (PBS) were subcutaneously injected into the right leg of 5-week-old male BALB/c nude mice (CLEA Japan, Inc., Tokyo, Japan) weighing approximately 20–22 g with four mice in each group.

Techniques: Injection, Saline, Control, Solvent

Journal: PLoS ONE

Article Title: Anticancer effects of a non-narcotic opium alkaloid medicine, papaverine, in human glioblastoma cells

doi: 10.1371/journal.pone.0216358

Figure Lengend Snippet: Summary of the anticancer effects of papaverine in a human GBM U87MG xenograft mouse model.

Article Snippet: For heterotrophic/subcutaneous xenografts, 1 × 10 6 U87MG cells resuspended in 100 μL phosphate-buffered saline (PBS) were subcutaneously injected into the right leg of 5-week-old male BALB/c nude mice (CLEA Japan, Inc., Tokyo, Japan) weighing approximately 20–22 g with four mice in each group.

Techniques: Saline

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Expression of ERα gene ( A ) and ERα protein ( B ) in U87 cells cultured under different conditions. Data are representative of each group cultured in control, nutrient-deficient, hypoxic, and necrotic conditions. Statistical analysis was performed using t -test *, p < 0.005.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Representative images taken with the FV1000 confocal microscope system (Olympus, Hamburg, Germany) show ERα protein expression in U87 cells cultured under specific conditions: control ( A ), nutrient deficiency ( B ), hypoxia ( C ), and necrotic conditions ( D ). FITC (AR) and DAPI (nuclear) markers were used. Microphotographs were taken at ×20 magnification ( A , B , D ) and ×40 magnification ( C ); scale bar 30 µm.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Microscopy, Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Expression of the ERβ gene ( A ) and ERβ protein ( B ) in U87 cells cultured under different conditions. Data are representative of each group cultured in control, nutrient-deficient, hypoxic, and necrotic conditions. Statistical analysis was performed using a t -test * p < 0.05.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model

doi: 10.3390/ijms25074130

Figure Lengend Snippet: Representative images taken with the FV1000 confocal microscope system (Olympus, Hamburg, Germany) show ERβ protein expression in U87 cells cultured under specific conditions: control ( A ), nutrient deficiency ( B ), hypoxia ( C ), and necrotic conditions ( D ). FITC (AR) and DAPI (nuclear) markers were used. Microphotographs were taken at ×20 magnification ( A , B , D ) and ×40 magnification ( C ); scale bar 30 µm.

Article Snippet: Cell culture of the U87 line (obtained from the European Collection of Authenticated Cell Cultures (ECACC)) was performed under standard conditions of 37 °C, 95% humidity, and 5% CO 2 , according to the manufacturer’s instructions.

Techniques: Microscopy, Expressing, Cell Culture, Control

Journal: Scientific Reports

Article Title: A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage

doi: 10.1038/s41598-023-35927-6

Figure Lengend Snippet: IC 50 of SPOPP-3 ( 1 ), SPOPP-5 ( 2 ) and doxorubicin against human cancer cell lines.

Article Snippet: For example, SPOPP-3 has stronger anti-proliferative effect on the faster growing human leukemia cell lines (K562, KG1a, CEM) and glioblastoma cell lines (U251, U87) but has weaker effect on the slower growing HepG2 liver cancer cell line, DU145 prostate cancer cell line, and pancreatic cancer cell lines (MiaPaca2, Panc1) (Table ).

Techniques:

Journal: Journal of Neuro-Oncology

Article Title: GliPR1 knockdown by RNA interference exerts anti‐glioma effects in vitro and in vivo

doi: 10.1007/s11060-021-03737-3

Figure Lengend Snippet: Doxycycline-dependent GliPR1 knockdown in U87-MG glioma cells. a GliPR1 gene expression (measured by qRT-PCR) in U87-MG cell pools transduced with various GliPR1 shRNAs or a non-targeting control shRNA (shNT) using a lentiviral “all-in-one” TetOn-shRNA vector. In this preliminary experiment, GliPR1 shRNAs were compared to find the most potent knockdown sequences, in the presence of doxycycline. The GliPR1 sh#301 (in cell pool 980) had already been established as potent in an earlier experiment. The GliPR1 sh#258 (in cell pool 1361) was found to be the most potent. Data are mean relative to TOP1 reference gene expression, in the absence of doxycycline. b GliPR1 protein expression (measured by Western blot) of clone 980-5 (transduced with GliPR1 sh#301), in presence and absence of doxycycline. Actin is loading control. c–d Confluency assays of clone 980-5 (transduced with GliPR1 sh#301) and a control clone (transduced with shNT), in presence and absence of doxycycline (using CloneSelect Imager), over time. Data are mean. e–h Confluency assays of clones 1361-1 and 1368-7 (transduced with GliPR1 sh#258), polyclonal parental U87-MG cells, and control clones (transduced with shNT), in presence and absence of doxycycline (using CloneSelect Imager), over time. Data are mean ± SD

Article Snippet: Material preparation, data collection, and analysis regarding development of optimal shRNA sequences and in vitro and in vivo experiments with TetOn inducible shRNA-transduced U87-MG cell pools and clones were performed by Urban Scheuring, supported by SIRION Biotech GmbH (Martinsried, Germany) and EPO GmbH (Berlin-Buch, Germany) as service providers.

Techniques: Expressing, Quantitative RT-PCR, Transduction, shRNA, Plasmid Preparation, Western Blot, Clone Assay

Journal: Journal of Neuro-Oncology

Article Title: GliPR1 knockdown by RNA interference exerts anti‐glioma effects in vitro and in vivo

doi: 10.1007/s11060-021-03737-3

Figure Lengend Snippet: Stable GliPR1 knockdown in polyclonal U87-MG, A172, and U343-MG glioma cells transduced with GliPR1sh#301, GliPR1sh#258, or a control luciferase shRNA (shLuc), at 7 days post transduction. a GliPR1 gene expression by qRT-PCR. Data are mean ± SD relative to TBP reference gene expression. * P < 0.05 versus shLuc control in same cell line. b GliPR1 protein expression by Western blot. Data are mean relative to GliPR1 protein expression for shLuc control in same cell line. GADPH is loading control

Article Snippet: Material preparation, data collection, and analysis regarding development of optimal shRNA sequences and in vitro and in vivo experiments with TetOn inducible shRNA-transduced U87-MG cell pools and clones were performed by Urban Scheuring, supported by SIRION Biotech GmbH (Martinsried, Germany) and EPO GmbH (Berlin-Buch, Germany) as service providers.

Techniques: Transduction, Luciferase, shRNA, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Neuro-Oncology

Article Title: GliPR1 knockdown by RNA interference exerts anti‐glioma effects in vitro and in vivo

doi: 10.1007/s11060-021-03737-3

Figure Lengend Snippet: Clonogenic survival following stable polyclonal GliPR1 knockdown in U87-MG, A172, and U343-MG glioma cells transduced with GliPR1sh#301, GliPR1sh#258, or a control luciferase shRNA (shLuc), at 3 weeks post transduction. a Representative images showing clonogenic survival results. b Quantification of clonogenic survival results. Data are mean ± SD number of colonies per well. * P < 0.05 versus shLuc control in same cell line

Article Snippet: Material preparation, data collection, and analysis regarding development of optimal shRNA sequences and in vitro and in vivo experiments with TetOn inducible shRNA-transduced U87-MG cell pools and clones were performed by Urban Scheuring, supported by SIRION Biotech GmbH (Martinsried, Germany) and EPO GmbH (Berlin-Buch, Germany) as service providers.

Techniques: Transduction, Luciferase, shRNA

Journal: Journal of Neuro-Oncology

Article Title: GliPR1 knockdown by RNA interference exerts anti‐glioma effects in vitro and in vivo

doi: 10.1007/s11060-021-03737-3

Figure Lengend Snippet: Induction of apoptosis following stable polyclonal GliPR1 knockdown in U87-MG, A172, and U343-MG glioma cells transduced with GliPR1sh#301, GliPR1sh#258, or a control luciferase shRNA (shLuc), at 7 days post transduction. a Flow cytometry dot plot of cells stained using Annexin V FITC/PI kit, which distinguishes early apoptotic cells (annexin V positive, PI negative) from late apoptotic/necrotic cells (PI positive) and viable cells (annexin V negative, PI negative). b Quantification of apoptosis results. Data are mean ± SD proportion of apoptotic cells relative to shLuc control in same cell line. ** P < 0.001 for U87-MG, and U343-MG glioma cells transduced with GliPR1sh#301, GliPR1sh#258, or a control luciferase shRNA (shLuc)

Article Snippet: Material preparation, data collection, and analysis regarding development of optimal shRNA sequences and in vitro and in vivo experiments with TetOn inducible shRNA-transduced U87-MG cell pools and clones were performed by Urban Scheuring, supported by SIRION Biotech GmbH (Martinsried, Germany) and EPO GmbH (Berlin-Buch, Germany) as service providers.

Techniques: Transduction, Luciferase, shRNA, Flow Cytometry, Staining

Journal: Journal of Neuro-Oncology

Article Title: GliPR1 knockdown by RNA interference exerts anti‐glioma effects in vitro and in vivo

doi: 10.1007/s11060-021-03737-3

Figure Lengend Snippet: In vivo effects of GliPR1 knockdown in U87-MG glioma cells. a, b Tumor growth upon doxycycline-dependent GliPR1 knockdown, using clone 980-5 transduced with GliPR1 sh#301. 8 mice underwent subcutaneous transplantation and 4 additionally underwent cranial transplantation; from day 8, 4 were left untreated (-Dox) and 4 were given doxycycline in drinking water (+ Dox). a Subcutaneous tumor volumes over time. Data are mean ± SD. * P < 0.05 for + Dox group versus -Dox group. b Representative cryosections (stained with cresyl violet) of brains from mice in -Dox group and + Dox group. Arrow shows cerebral tumor at end of experiment in -Dox example. c Cerebral tumor area at end of second experiment with doxycycline-dependent GliPR1 knockdown (clone 980-5; sh#301) using larger cohorts (each n = 5). Data are mean ± SD. P = 0.056 for + Dox group versus -Dox group. d Survival upon stable GliPR1 knockdown, using polyclonal U87-MG cells transduced with GliPR1 sh#258 (n = 14) or a control luciferase shRNA (shLuc; n = 16). Kaplan-Meier plot. P < 0.001 for between-group difference

Article Snippet: Material preparation, data collection, and analysis regarding development of optimal shRNA sequences and in vitro and in vivo experiments with TetOn inducible shRNA-transduced U87-MG cell pools and clones were performed by Urban Scheuring, supported by SIRION Biotech GmbH (Martinsried, Germany) and EPO GmbH (Berlin-Buch, Germany) as service providers.

Techniques: In Vivo, Transduction, Transplantation Assay, Staining, Luciferase, shRNA