Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: Sub-cellular internalization of DOX entrapped VEV micelles (VEVDMs) . MCF7 and U87MG cells were treated with 1 μM DOX formulations. Micelle uptake of VEVDMs by MCF7 and U87MG cells in comparison to free DOX after 2 h of incubation at 37°C is shown by (A) CLSM images showing the internal fluorescence of DOX in cells at a magnification of 60× (B) Comparison of fluorescence intensity by flow cytometry to analyze the extent of internalization.

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Comparison, Incubation, Fluorescence, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: Cytotoxicity study of VEV copolymer . The biocompatibility analysis of empty VEV micelles on MCF7 and U87MG cells at 24, 48 and 72 h on incubation with the concentrations as indicated was analyzed using MTT assay. All the measurements were done in six replicates. The results are expressed as arithmetic mean ± standard error on the mean (S.E.M).

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Incubation, MTT Assay

Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: Cell viability assay . Comparison of the cell viabilities of MCF7 and U87MG cells on treatment with free DOX and equivalent concentrations of VEVDMs as indicated on 24, 48 and 72 h incubation was done by MTT. All the measurements were done in six replicates and the results are expressed as arithmetic mean ± standard error on the mean (S.E.M) with statistical significance *p < 0.05, **p < 0.01.

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Viability Assay, Comparison, Incubation

Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: IC 50 values (in equivalent μM DOX) of MCF7 and U87MG cells cultured with VEVDMs vs. free doxorubicin in 24, 48, 72 h

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Cell Culture

Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: Apoptosis analysis by FACS using Annexin V-FITC stain assay . MCF7 and U87MG cells were incubated with 1 μM of DOX formulations for 24 h. To compare apoptosis, FITC-conjugated annexin binding to phosphatidyl serine, exposed to the outer leaflet, on treatment with DOX formulations was measured by FACS.

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Staining, Incubation, Binding Assay

Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: PARP cleavage determination by western blot analysis . Comparison of PARP cleavage induced by 3 μM of VEVDMs to free DOX in MCF7 and U87MG cells on 24 h incubation. Immunoblotting was carried out using antibodies specific for PARP and detected using enhanced chemiluminescence method.

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Western Blot, Comparison, Incubation

Journal: Journal of Nanobiotechnology

Article Title: Evaluation of triblock copolymeric micelles of δ- valerolactone and poly (ethylene glycol) as a competent vector for doxorubicin delivery against cancer

doi: 10.1186/1477-3155-9-42

Figure Lengend Snippet: Cell cycle arrest analysis by FACS . Effect of 0.05 μM VEVDMs treatment on cell cycle of MCF7 and U87MG cell lines in comparison to a double concentration of 0.1 μM free DOX on 24 h incubation was assessed by FACS.

Article Snippet: Human breast adenocarcinoma (MCF7) and glioblastoma (U87MG) cells were provided from ATCC (USA) and maintained in DMEM medium containing 10% fetal bovine serum (Sigma, USA) and 1% antibiotic antimycotic cocktail (Himedia, India).

Techniques: Comparison, Concentration Assay, Incubation

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: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 1. Analysis of COL16A1 knockdown efficiency. (A) The knockdown was translated to the protein level as shown for the full-length collagen XVI chain in cell lysates in comparison to the scr control and untreated U87MG cells. E-actin. protein expression is unaffected which indicates a specific knockdown of collagen XVI. N = 4 (B) Collagen XVI inhibited cells secreted less collagen XVI into the supernatant in comparison to scr controls. Ponceau Red stained blot membrane served as loading control. N=3 (C) Knockdown efficiency of COL16A1 was analyzed with qRT-PCR and shows an average of 60% lower COL16A1 gene expression in transfected U87MG cells (si1 and si2) compared to control cells transfected with scrambled RNA (scr). N = 19, si1= siRNA 1, si2 = siRNA 2, scr = scrambled RNA = controls.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Knockdown, Comparison, Control, Expressing, Staining, Membrane, Quantitative RT-PCR, Gene Expression, Transfection

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 2. Cell morphology and number of focal adhesion contacts. (A) U87MG cells were stained using vinculin antibodies to visualize cell morphology and focal adhesion contacts. Representative images (magnification 200x and 400x) demonstrate that in collagen XVI-knockdown cells FACs were found more concentrated in the cellular periphery while in control cells FACs (white arrows) appeared to be more evenly distributed. N = 4. (B) The number of FACs per cell is significantly decreased in collagen XVI knockdown cells. The evaluation of the number of focal adhesion contacts yielded a significant decrease of 40-45% of FACs per cell after inhibition of collagen XVI. A similar reduction of focal adhesion contacts was observed for both siRNAs. N = 4; * = p < 0.05.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Staining, Knockdown, Control, Inhibition

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 3. Invasive potential of collagen XVI inhibited cells. (A) Downregulation of collagen XVI expression resulted in a reduced invasion of U87MG cells in a Boyden Chamber assay compared to scr-control cells. The number of cells migrated to the lower compartment after 4 hours was significantly decreased in knockdown cells compared to the control. N = 4; * = p< 0.05; ** = p<0.01. (B, C) Adhesion capacity after 4 hours and 6 hours was not influenced by reduced collagen XVI expression. N = 6.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Expressing, Boyden Chamber Assay, Control, Knockdown

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 4. Downstream gene expression analysis. (A) Gene expression of PCDH18, kindlin-1 and kindlin-2 was determined by qRT-PCR and evaluated using the ''Ct method with GAPDH as endogenous loading control set to 0 and U87MG cells transfected with scrambled RNA as calibrator. PCDH18 gene expression was profoundly upregulated whereas gene expression of kindlin-1 and kindlin-2 was clearly downregulated. N = 6. ***= p< 0.001. Protein expres- sion of PCDH 18 (B), Kindlin-1 (C) and Kindlin-2 (D) was determined with western blotting analysis of cell lysates. E-Actin was used as internal loading control. N=3. Control: cell lysate from mouse lung.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Gene Expression, Quantitative RT-PCR, Control, Transfection, Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Inhibition of collagen XVI expression reduces glioma cell invasiveness.

doi: 10.1159/000327947

Figure Lengend Snippet: Fig. 6. Flow cytometric analysis of E1-integrin expression and activation status. FACS analysis was performed to determine E1-integrin expression and activation on collagen XVI-knock- down U87MG cells and scr controls 96 hours post transfection. (A) The antibody integrin beta1D (MAB2259Z) recognizes activated E1-integrin only. Compared to controls E1-integrin was about 50% less activated on collagen XVI-knockdown cells. (B) The antibody integrin beta1 (MAB1900) binds to to- tal E1-integrins (both activated and inactivated forms). There- fore, the result demonstrates that total expression of E1-integrin was not altered on collagen XVI-knockdown cells. black line: Isotype control which is the secondary antibody without first antibody. si1= siRNA 1, si2 = siRNA 2, scr = scrambled RNA.

Article Snippet: Materials and Methods Cell Culture The human glioblastoma cell line U87MG (ATCC: HTB- 14) was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (Sigma, St. Louis, USA) and 1% penicillin/ streptomycin (PAA, Coelbe, Germany) at 37°C in 5% CO2.

Techniques: Expressing, Activation Assay, Knockdown, Transfection, Control

Journal: Nature Communications

Article Title: Iontronic click-to-release enables electrically controlled delivery of drugs and biomolecules beyond charge and size limitations

doi: 10.1038/s41467-026-70985-0

Figure Lengend Snippet: a Schematic of the synthesis and immobilization strategy for biotinylated CA4-prodrug ( 6 ), synthesized via sequential decoration of cTCO-bis-NHS ( 5 ). The biotin handle allows immobilization onto streptavidin-coated magnetic beads, forming a localized, iontronically activatable prodrug reservoir. IPs were positioned above the beads in a transwell insert containing U-87 cells in the lower chamber. Grey pills indicate inactive (bound) drug, blue (colored) pills indicate active drug. b 3D-printed platform designed to precisely position iontronic devices in standard 96-well transwell plates. c Cell viability of U-87 cells after operating iontronic devices delivering 1 for 4 h at +20 nA (Active, n = 8) and at −20 nA (Reverse, n = 7) into transwells holding CA4-immobilized beads from ( a ). Points represent individual biological replicates; bars indicate mean ± SD. GC growth control (untreated, n = 32), “No Beads” = operation of iontronic devices delivering 1 at +20 nA, no beads in transwell present ( n = 6), “No Tz 1 ” = CA4-immobilized beads in transwell present with iontronic delivery of K + (0.1 M KCl) instead of Tz 1 ( n = 8). Statistical analysis was done via a Brown–Forsythe and Welch ANOVA tests (one-way) and Dunnett’s T3 multiple comparisons test. α = 0.05. p -values: GC vs. No Beads (ns) = 0.2377; GC vs. No Tz 1 (ns) = 0.3827; GC vs. Reverse, 4 h (ns) = 0.5739, GC vs. Active, 4 h (****) = <0.0001. Certain graphical elements in ( a ) were created in BioRender. Hecko, S. (2026) https://www.BioRender.com/z8yr1eu .

Article Snippet: The human GBM cell line U-87 (provided by the MUG Cell Bank, catalogue no. 300367, CLS) was cultured at 37 °C and 5% CO 2 in Eagle’s minimum essential medium (E-MEM) containing 10% fetal bovine serum (FBS), 4.5 g/L glucose, 2 mM L -glutamine, and 1% MEM Non-Essential Amino Acids.

Techniques: Synthesized, Magnetic Beads, Control

Journal: Nature Communications

Article Title: Iontronic click-to-release enables electrically controlled delivery of drugs and biomolecules beyond charge and size limitations

doi: 10.1038/s41467-026-70985-0

Figure Lengend Snippet: a Schematic overview of the bioorthogonal click-to-release (C2R) cascade of CA4-prodrug: Aminoethyl tetrazine (Tz 1 ) triggers the release of the cytotoxic agent combretastatin A-4 ( CA4 ) from sulfo-cTCO-DMEDA-CA4 ( 4 ) via a tetrazine-triggered elimination from TCO caged payload and self-immolation. b Dose-dependent cell viability of human glioblastoma cell line U-87 after 72 h incubation with the key components of the C2R reaction. Data represent mean ± SD of n = 3 independent biological replicates. Curves were fit using four-parameter logistic regression. Prodrug 4 (0.01–1000 nM, grey curve); parent CA4 (0.01–1000 nM, blue curve); released CA4 (0.01–1000 nM 4 + 5 µM 1, dashed blue curve); Tz 1 (5 µM, purple data point). c Cell viability of U-87 cells after operating iontronic devices delivering 1 for 1 h at +20 nA (Active, n = 9) and at −20 nA (Reverse, n = 11). Points represent individual biological replicates; bars indicate mean ± SD. GC growth control (untreated, n = 12). Statistical analysis was done via a Brown–Forsythe and Welch ANOVA tests (one-way) and Dunnett’s T3 multiple comparisons test. α = 0.05. p -values: GC vs. Reverse, 1 h (ns) = 0.5339; GC vs. Active, 1 h (****) = <0.0001. Certain graphical elements in ( a ) were created in BioRender. Hecko, S. (2026) https://www.BioRender.com/z8yr1eu .

Article Snippet: The human GBM cell line U-87 (provided by the MUG Cell Bank, catalogue no. 300367, CLS) was cultured at 37 °C and 5% CO 2 in Eagle’s minimum essential medium (E-MEM) containing 10% fetal bovine serum (FBS), 4.5 g/L glucose, 2 mM L -glutamine, and 1% MEM Non-Essential Amino Acids.

Techniques: Incubation, Control

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