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ATCC cancer cell lines a172
Cancer Cell Lines A172, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 2056 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 97 stars, based on 2056 article reviews

cancer cell lines a172 - by Bioz Stars, 2026-06

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Cytotoxicity of GBM cell lines A172 and U251 following prolonged arginine deprivation (A). Average ASS-1 levels, expressed as mean fluorescence intensity (MFI), for each condition (isotype, untreated and treated cells) at 24, 48, 72, 96, and 120 hours post-treatment. Stars indicate a significant increase in ASS-1 expression compared to both isotype control and untreated cells (B) ASS-1 expression of A172 and U251 cell lines at 72 hours post-treatment. MFI is on the X-axis (FL1-H) and forward scatter is on the Y-axis (C).

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Cytotoxicity of GBM cell lines A172 and U251 following prolonged arginine deprivation (A). Average ASS-1 levels, expressed as mean fluorescence intensity (MFI), for each condition (isotype, untreated and treated cells) at 24, 48, 72, 96, and 120 hours post-treatment. Stars indicate a significant increase in ASS-1 expression compared to both isotype control and untreated cells (B) ASS-1 expression of A172 and U251 cell lines at 72 hours post-treatment. MFI is on the X-axis (FL1-H) and forward scatter is on the Y-axis (C).

Article Snippet: Human Glioblastoma cell lines A172 and U251 MG were obtained from the American Type Culture Collection (A172 ATCC, CRL-1620) and from the European Collection of Authenticated Cell Cultures (U251 MG, ECACC, catalog # 09063001).

Techniques: Fluorescence, Expressing, Control

Autophagosome accumulation in GBM cells, expressed as mean fluorescence intensity (MFI), for control untreated cells and cells treated with [HuArgI (Co)-PEG5000] at 24, 48, 72, 96, and 120 hours post-treatment. Stars indicate a significant increase in autophagosome accumulation compared to untreated cells (A). Autophagosome accumulation in A172 and U251 cell lines at 72 hours post-treatment in untreated cells and cells treated with CQ, [HuArgI (Co)-PEG5000] and a combination of both. MFI is on the X-axis (FL1-H) and forward scatter is on the Y-axis (B). Immunohistochemistry staining of autophagosomes in A172 cells untreated and treated with CQ, [HuArgI (Co)-PEG5000], or a combination of both, at 24, 48, and 72 hours, post-treatment (C).

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Autophagosome accumulation in GBM cells, expressed as mean fluorescence intensity (MFI), for control untreated cells and cells treated with [HuArgI (Co)-PEG5000] at 24, 48, 72, 96, and 120 hours post-treatment. Stars indicate a significant increase in autophagosome accumulation compared to untreated cells (A). Autophagosome accumulation in A172 and U251 cell lines at 72 hours post-treatment in untreated cells and cells treated with CQ, [HuArgI (Co)-PEG5000] and a combination of both. MFI is on the X-axis (FL1-H) and forward scatter is on the Y-axis (B). Immunohistochemistry staining of autophagosomes in A172 cells untreated and treated with CQ, [HuArgI (Co)-PEG5000], or a combination of both, at 24, 48, and 72 hours, post-treatment (C).

Techniques: Fluorescence, Control, Immunohistochemistry, Staining

Western blot of Atg13 and phosphor-Atg13 on protein extracts of A172 and U251 untreated and treated with [HuArgI (Co)-PEG5000] for up to 96 hours (Atg13) (A). Histogram of the ratio of p-Atg13 over Atg13 showing a significant decrease in the phosphorylation of Atg13 at 72- and 96-hours post-treatment. Stars indicate statistical significance (A). ULK phosphorylation levels expressed as mean fluorescence intensity (MFI), for untreated cells and cells treated with [HuArgI (Co)-PEG5000] at 24, 48, 72, 96, and 120 hours post-treatment. Double stars indicate significantly lower ULK phosphorylation in treated compared to untreated cells (B). ULK phosphorylation is also shown in A172 and U251 cell lines at 24- and 48-hours post-treatment in isotype controls, untreated cells and cells treated with [HuArgI (Co)-PEG5000]. MFI is represented on the X-axis (FL1-H) and forward scatter is represented on the Y-axis. Last panel on the right is a histogram representing the overlayed phospho-ULK MFI of the isotype control (red), untreated cells (black) and cells treated with [HuArgI (Co)-PEG5000] (blue) (B). Cytotoxicity of [HuArgI (Co)-PEG5000] alone and in combination with chloroquine (CQ) (50 µM) to A172 and U251 cells at 48, 72, 96, and 120 hours (C).

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Western blot of Atg13 and phosphor-Atg13 on protein extracts of A172 and U251 untreated and treated with [HuArgI (Co)-PEG5000] for up to 96 hours (Atg13) (A). Histogram of the ratio of p-Atg13 over Atg13 showing a significant decrease in the phosphorylation of Atg13 at 72- and 96-hours post-treatment. Stars indicate statistical significance (A). ULK phosphorylation levels expressed as mean fluorescence intensity (MFI), for untreated cells and cells treated with [HuArgI (Co)-PEG5000] at 24, 48, 72, 96, and 120 hours post-treatment. Double stars indicate significantly lower ULK phosphorylation in treated compared to untreated cells (B). ULK phosphorylation is also shown in A172 and U251 cell lines at 24- and 48-hours post-treatment in isotype controls, untreated cells and cells treated with [HuArgI (Co)-PEG5000]. MFI is represented on the X-axis (FL1-H) and forward scatter is represented on the Y-axis. Last panel on the right is a histogram representing the overlayed phospho-ULK MFI of the isotype control (red), untreated cells (black) and cells treated with [HuArgI (Co)-PEG5000] (blue) (B). Cytotoxicity of [HuArgI (Co)-PEG5000] alone and in combination with chloroquine (CQ) (50 µM) to A172 and U251 cells at 48, 72, 96, and 120 hours (C).

Techniques: Western Blot, Phospho-proteomics, Fluorescence, Control

Intracellular ROS accumulation in GBM cells following treatment with [HuArgI (Co)|-PEG5000], expressed as both percent positive cells (left panel) and mean fluorescence intensity (MFI) (right panel). Stars indicate a significant increase in ROS accumulation. The bottom histograms show ROS accumulation in treated cells (red) compared to controls (black) in A172 and U251 cells. (A) Autophagosome accumulation in GBM cells, expressed as MFI, for control untreated cells and cells treated with [HuArgI (Co)-PEG5000] alone or a combination of [HuArgI (Co)-PEG5000] and N-acetylcysteine (NAC) at 24, 48, 72, 96, and 120 hours post-treatment (B). Autophagosome formation in A172 and U251 cell lines. Control untreated cells (left panel) and cells treated with [HuArgI (Co)-PEG5000] (10 −7 M) (middle panel) or treated with [HuArgI (Co)-PEG5000] and NAC (0.82 g/L) (right panel) at 24, 48, 72, 96, and 120-hours post-treatment. In the histogram (right panel), control cells are in black, cells treated with [HuArgI (Co)-PEG5000] are in red and cells treated with the combination with NAC are in blue (C). Cytotoxicity of A172 and U251 cells treated with [HuArgI (Co)-PEG5000] alone or in combination with N-acetylcysteine (NAC) (0.82 g/L) (D).

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Intracellular ROS accumulation in GBM cells following treatment with [HuArgI (Co)|-PEG5000], expressed as both percent positive cells (left panel) and mean fluorescence intensity (MFI) (right panel). Stars indicate a significant increase in ROS accumulation. The bottom histograms show ROS accumulation in treated cells (red) compared to controls (black) in A172 and U251 cells. (A) Autophagosome accumulation in GBM cells, expressed as MFI, for control untreated cells and cells treated with [HuArgI (Co)-PEG5000] alone or a combination of [HuArgI (Co)-PEG5000] and N-acetylcysteine (NAC) at 24, 48, 72, 96, and 120 hours post-treatment (B). Autophagosome formation in A172 and U251 cell lines. Control untreated cells (left panel) and cells treated with [HuArgI (Co)-PEG5000] (10 −7 M) (middle panel) or treated with [HuArgI (Co)-PEG5000] and NAC (0.82 g/L) (right panel) at 24, 48, 72, 96, and 120-hours post-treatment. In the histogram (right panel), control cells are in black, cells treated with [HuArgI (Co)-PEG5000] are in red and cells treated with the combination with NAC are in blue (C). Cytotoxicity of A172 and U251 cells treated with [HuArgI (Co)-PEG5000] alone or in combination with N-acetylcysteine (NAC) (0.82 g/L) (D).

Techniques: Fluorescence, Control

GB cells were exposed to GEM for 1h or 24 h at the indicated concentrations. After GEM exposure, cells were maintained in drug-free fresh culture medium for 72 h before a viability assay with Crystal Violet. IC50 of GEM on the A172, U87-MG and U118-MG human GB cell lines were indicated in the legend of . Phase contrast microscopy images of GB cells treated for 24 h at IC 90. Cells were maintained in culture in drug free medium and observed 24 h or 5 days after GEM treatment. Note the presence of enlarged / flattened cells.

Journal: bioRxiv

Article Title: Intraventricular infusion to circumvent the blood-brain barrier to gemcitabine

doi: 10.64898/2026.05.01.722145

Figure Lengend Snippet: GB cells were exposed to GEM for 1h or 24 h at the indicated concentrations. After GEM exposure, cells were maintained in drug-free fresh culture medium for 72 h before a viability assay with Crystal Violet. IC50 of GEM on the A172, U87-MG and U118-MG human GB cell lines were indicated in the legend of . Phase contrast microscopy images of GB cells treated for 24 h at IC 90. Cells were maintained in culture in drug free medium and observed 24 h or 5 days after GEM treatment. Note the presence of enlarged / flattened cells.

Article Snippet: Human GB cell lines A172, U87-MG and U118-MG were purchased from American Type Culture Collection (ATCC) (LGC Standards, Strasbourg, France) and cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (Jacques Boy, Reims, France), 2 mM glutamine and penicillin / streptomycin.

Techniques: Viability Assay, Microscopy

Phenotypic space of ERK activity in glioblastoma cells. A ERK activity was measured by the cytoplasmic/nuclear (C/N) ratio of the green fluorescence of cells expressing ERK-KTR and 53BP1-Apple. B Four levels of ERK activity were defined as very inactive (VI), inactive (I), active (A), and very active (VA) based on the distributions of ERK activity of A172 cells grown in 10% FBS (orange line) or 0.5% FBS (blue line) for 48 h and C 20 nM trametinib for 2 h (green line) or 2.5 ng/ml of EGF for 15 min (red line) after 48 h of serum deprivation. Relative Shannon Index of 4 groups (rSI4 or SI4 ERK ) calculations are indicated. D Average ERK activity and E phenotypic space (rSI4) occupied by cells treated as in B and C . Each dot represents an image field with at least 20 cells (10% n = 2085 cells; 0.5% n = 4466 cells; TRAM n = 362 cells). F Representative image field of A172 glioma cells and I MRC5 fibroblast cells before (left) and after (right) 15 min of 2.5 ng/ml of EGF treatment (20× magnification). The numbers in the image represent the C/N ratio of each cell. The distribution of cells’ phenotypes and SI4 ERK for this group of cells is shown in the graphs below the images. G Distribution of cells treated with EGF after 48 h of serum deprivation in A172 glioma cells and J MRC5 cells. H SI4 ERK occupied by A172 glioma cells and K MRC5 cells after 15 min of EGF treatment. Each dot represents an image field with at least 20 cells (A172 n = at least 350 cells per EGF dose; MRC5 n = at least 186 cells per EGF dose) One-way ANOVA. EGF, epidermal growth factor; TRAM, trametinib; rSI4 or SI4 ERK , relative Shannon Index of 4 groups of ERK activity); * p < 0.05; ** p < 0.01; *** p < 0.001

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells

doi: 10.1007/s00109-026-02644-2

Figure Lengend Snippet: Phenotypic space of ERK activity in glioblastoma cells. A ERK activity was measured by the cytoplasmic/nuclear (C/N) ratio of the green fluorescence of cells expressing ERK-KTR and 53BP1-Apple. B Four levels of ERK activity were defined as very inactive (VI), inactive (I), active (A), and very active (VA) based on the distributions of ERK activity of A172 cells grown in 10% FBS (orange line) or 0.5% FBS (blue line) for 48 h and C 20 nM trametinib for 2 h (green line) or 2.5 ng/ml of EGF for 15 min (red line) after 48 h of serum deprivation. Relative Shannon Index of 4 groups (rSI4 or SI4 ERK ) calculations are indicated. D Average ERK activity and E phenotypic space (rSI4) occupied by cells treated as in B and C . Each dot represents an image field with at least 20 cells (10% n = 2085 cells; 0.5% n = 4466 cells; TRAM n = 362 cells). F Representative image field of A172 glioma cells and I MRC5 fibroblast cells before (left) and after (right) 15 min of 2.5 ng/ml of EGF treatment (20× magnification). The numbers in the image represent the C/N ratio of each cell. The distribution of cells’ phenotypes and SI4 ERK for this group of cells is shown in the graphs below the images. G Distribution of cells treated with EGF after 48 h of serum deprivation in A172 glioma cells and J MRC5 cells. H SI4 ERK occupied by A172 glioma cells and K MRC5 cells after 15 min of EGF treatment. Each dot represents an image field with at least 20 cells (A172 n = at least 350 cells per EGF dose; MRC5 n = at least 186 cells per EGF dose) One-way ANOVA. EGF, epidermal growth factor; TRAM, trametinib; rSI4 or SI4 ERK , relative Shannon Index of 4 groups of ERK activity); * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: In this study, we used A172 glioblastoma cell line (ATCC, catalog number CRL-1620), U-251 MG glioma cell line (STR validated in October 2018 by the Banco de Células do Rio de Janeiro), U138 MG glioblastoma cell line (ATCC HTB-16), and MRC5 lung fibroblast cell line (ATCC, catalog number CCL-171).

Techniques: Activity Assay, Fluorescence, Expressing

Impact of TMZ on ERK phenotype heterogeneity. A Average and B distribution of ERK activity of A172 cells treated with TMZ (100 mM for 3 h). Cells were imaged every 10 min for 12 h prior to TMZ treatment, during 3 h of treatment, and for 12 h 3 or 10 days after treatment withdrawal. Each dot represents the time-averaged ERK activity of an image field with at least 20 cells (prior to TMZ n = 1834 reads, during TMZ n = 268 reads, 3 days after n = 9683 reads, 10 days after n = 3671 reads). DMSO was used in the same volume as TMZ for control of mechanical ERK stimulation. One-way ANOVA. C Distribution of ERK states frequency prior to TMZ treatment, and 3, 6, or 10 days after treatment withdrawal in U-251 MG cells. D Phenotypic space occupied by A172 or E U-251 MG cells treated as in A . One-way ANOVA. F Average nuclear area of untreated and TMZ treated A172 cells 3, 5, and 10 days after drug removal. Each dot represents the average of a field with at least 30 cells (10× magnification). One-way ANOVA. ** p < 0.005, **** p < 0.0001. G Relation of phenotypic space of ERK activity (SI4 ERK ) and nuclear area heterogeneity (SI4 NucArea ) of A172 glioma cells before TMZ treatment and 3, 5, and 10 days after treatment withdrawal. Each dot represents the SI4 ERK /SI4 NucArea of a field with at least 30 cells (10× magnification). Unpaired t -test. H Phenotypic space of ERK activity occupied by the cells of A172 colonies immediately before (day 4) and 3 days after treatment withdrawal (day 7). Each dot represents the SI4 ERK of a colony of cells imaged every 10 min for 3 h (10× magnification) (untreated n = 32; TMZ treated n = 26). Unpaired t -test. I Change in SI4 ERK of each colony after TMZ treatment and its relationship with colony growth. Each dot represents the SI4 ERK of a colony of cells imaged every 10 min for 3 h (10× magnification). Arrows indicate the pathway from initial to final SI4 ERK and colony size of each colony. Blue dots indicate homogeneous colonies, and red dots indicate heterogeneous colonies. J Correlations among colony size (CS), average ERK activity of the colony (AV ERK), and phenotypic space of ERK activity (SI4 ERK ), together with the deltas (∆CS, ∆AV ERK , and ∆SI4 ERK ) of these features from day 4 (B, before TMZ) and day 7 (A, 3 days after TMZ withdrawal). Relevant positive and negative correlations are shown in detail ( J (a, b, c, d))

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells

doi: 10.1007/s00109-026-02644-2

Figure Lengend Snippet: Impact of TMZ on ERK phenotype heterogeneity. A Average and B distribution of ERK activity of A172 cells treated with TMZ (100 mM for 3 h). Cells were imaged every 10 min for 12 h prior to TMZ treatment, during 3 h of treatment, and for 12 h 3 or 10 days after treatment withdrawal. Each dot represents the time-averaged ERK activity of an image field with at least 20 cells (prior to TMZ n = 1834 reads, during TMZ n = 268 reads, 3 days after n = 9683 reads, 10 days after n = 3671 reads). DMSO was used in the same volume as TMZ for control of mechanical ERK stimulation. One-way ANOVA. C Distribution of ERK states frequency prior to TMZ treatment, and 3, 6, or 10 days after treatment withdrawal in U-251 MG cells. D Phenotypic space occupied by A172 or E U-251 MG cells treated as in A . One-way ANOVA. F Average nuclear area of untreated and TMZ treated A172 cells 3, 5, and 10 days after drug removal. Each dot represents the average of a field with at least 30 cells (10× magnification). One-way ANOVA. ** p < 0.005, **** p < 0.0001. G Relation of phenotypic space of ERK activity (SI4 ERK ) and nuclear area heterogeneity (SI4 NucArea ) of A172 glioma cells before TMZ treatment and 3, 5, and 10 days after treatment withdrawal. Each dot represents the SI4 ERK /SI4 NucArea of a field with at least 30 cells (10× magnification). Unpaired t -test. H Phenotypic space of ERK activity occupied by the cells of A172 colonies immediately before (day 4) and 3 days after treatment withdrawal (day 7). Each dot represents the SI4 ERK of a colony of cells imaged every 10 min for 3 h (10× magnification) (untreated n = 32; TMZ treated n = 26). Unpaired t -test. I Change in SI4 ERK of each colony after TMZ treatment and its relationship with colony growth. Each dot represents the SI4 ERK of a colony of cells imaged every 10 min for 3 h (10× magnification). Arrows indicate the pathway from initial to final SI4 ERK and colony size of each colony. Blue dots indicate homogeneous colonies, and red dots indicate heterogeneous colonies. J Correlations among colony size (CS), average ERK activity of the colony (AV ERK), and phenotypic space of ERK activity (SI4 ERK ), together with the deltas (∆CS, ∆AV ERK , and ∆SI4 ERK ) of these features from day 4 (B, before TMZ) and day 7 (A, 3 days after TMZ withdrawal). Relevant positive and negative correlations are shown in detail ( J (a, b, c, d))

Techniques: Activity Assay, Control

$Reduction of ERK phenotype heterogeneity reduces fractional killing of clonal populations. A Average of ERK activity from cells treated with TMZ (100 mM for 3 h); TRAM (20 nM for 24 h) or a combination of TMZ and TRAM. Cells were imaged every 10 min for 12 h prior to TMZ treatment, during 3 h of treatment and for 12 h 3 or 10 days after treatment withdrawal. Each dot represents the time-averaged ERK activity of an image field with at least 20 cells (TMZ during n = 268, TMZ 3 d n = 9683, TMZ 10 d n = 3671; TRAM during n = 362; TRAM 3 d n = 331; TRAM 10 d n = 245; TMZ + TRAM during n = 428, TMZ + TRAM 3 d n = 650; TMZ + TRAM 10 d n = 599). One-way ANOVA. B Impact of MEK inhibition with TRAM on the SI4 ERK 3 or 10 days after drug withdrawal in A172 and C U-251 MG cells. Cells were treated as in A . One-way ANOVA. D Colony size of A172 cells for each treatment type on day 14. Each dot represents a colony. Unpaired, two-sided Mann–Whitney U test. E Lethal fraction (LF) over time of A172 colonies after TMZ, F TRAM or G TMZ and TRAM treatments. Heterogeneity in LF induction was calculated as the Shannon Index diversity (SI4 LF ) for 4 equal categories. Black lines represent the average of LF of all colonies. H LF of colonies whose phenotypic heterogeneity remained stable (stb), increased (inc) or decreased (dec) 3 days after TMZ treatment. ∆SI4 ERK was calculated as SI4 ERK after TMZ treatment minus SI4 ERK before treatment. ∆SI4 ERK was considered stable when it changed to less than 10%. One-way ANOVA. I Maximum LF observed after TMZ, TRAM, or TMZ and TRAM treatment. Each dot represents a colony (untreated n = 37; TMZ n = 64; TRAM n = 39; TMZ and TRAM n = 33). J Proportion of colonies with > 75%, 25–75% or < 25% of death rate at the end of the experiment for TMZ (above) and TMZ and TRAM treatments (lower). LF (lethal fraction); TMZ (temozolomide); TRAM (trametinib); ns, non-significative; * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001$

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells

doi: 10.1007/s00109-026-02644-2

Figure Lengend Snippet: Reduction of ERK phenotype heterogeneity reduces fractional killing of clonal populations. A Average of ERK activity from cells treated with TMZ (100 mM for 3 h); TRAM (20 nM for 24 h) or a combination of TMZ and TRAM. Cells were imaged every 10 min for 12 h prior to TMZ treatment, during 3 h of treatment and for 12 h 3 or 10 days after treatment withdrawal. Each dot represents the time-averaged ERK activity of an image field with at least 20 cells (TMZ during n = 268, TMZ 3 d n = 9683, TMZ 10 d n = 3671; TRAM during n = 362; TRAM 3 d n = 331; TRAM 10 d n = 245; TMZ + TRAM during n = 428, TMZ + TRAM 3 d n = 650; TMZ + TRAM 10 d n = 599). One-way ANOVA. B Impact of MEK inhibition with TRAM on the SI4 ERK 3 or 10 days after drug withdrawal in A172 and C U-251 MG cells. Cells were treated as in A . One-way ANOVA. D Colony size of A172 cells for each treatment type on day 14. Each dot represents a colony. Unpaired, two-sided Mann–Whitney U test. E Lethal fraction (LF) over time of A172 colonies after TMZ, F TRAM or G TMZ and TRAM treatments. Heterogeneity in LF induction was calculated as the Shannon Index diversity (SI4 LF ) for 4 equal categories. Black lines represent the average of LF of all colonies. H LF of colonies whose phenotypic heterogeneity remained stable (stb), increased (inc) or decreased (dec) 3 days after TMZ treatment. ∆SI4 ERK was calculated as SI4 ERK after TMZ treatment minus SI4 ERK before treatment. ∆SI4 ERK was considered stable when it changed to less than 10%. One-way ANOVA. I Maximum LF observed after TMZ, TRAM, or TMZ and TRAM treatment. Each dot represents a colony (untreated n = 37; TMZ n = 64; TRAM n = 39; TMZ and TRAM n = 33). J Proportion of colonies with > 75%, 25–75% or < 25% of death rate at the end of the experiment for TMZ (above) and TMZ and TRAM treatments (lower). LF (lethal fraction); TMZ (temozolomide); TRAM (trametinib); ns, non-significative; * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001

Techniques: Activity Assay, Inhibition, MANN-WHITNEY

( A ) qRT-PCR analysis showing that IPA3 treatment rescued stmn1a and stmn4l expression in CRISPR-mediated betaPix F 0 knockouts at 48 hpf. Each dot represents one embryo. Data are presented in mean ± SEM; one-way ANOVA with Dunnett’s test, individual p-values mentioned in the figure. ( B ) Schematic diagram illustrates four-guide RNAs-mediated F 0 knockout strategy in the loci of stmn1a , stmn1b, and stmn4l . ( C ) Whole-mount RNA in situ hybridization confirming that stmn1a, stmn1b, and stmn4l decreased in CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Dorsal views with anterior to the left. ( D ) qRT-PCR analysis confirming that stmn1a, stmn1b, and stmn4l decreased in mutant embryos of CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Each dot represents one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( E ) qRT-PCR analysis confirming that stmn1a, stmn1b, and stmn4l decreased in the FACS-sorted glia of CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Each dot represents sorted cells from one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( F ) Optical sections of glial structure in the hindbrains of siblings and CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Lateral view with anterior to the left. Abnormal glial structures with disoriented arrangements (yellow arrows) in stmn1a/1b/4l mutant embryos. ( G ) Quantification of average CtA and glial length in . Each dot represents one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( H ) Whole-mount RNA in situ hybridization revealed that nestin increased while pax2a decreased in CRISPR-mediated stmn1a/1b/4 l F0 knockout embryos at 48 hpf. Dorsal views with anterior to the left. ( I ) 3D reconstruction and whole-mount RNA in situ hybridization showing that gfap:GFP-stmn1b transgenic overexpression rescued glial defects in bbh fn40a ; Tg( gfap:GFP-stmn1b ) embryos at 48 hpf. Lateral views with anterior to the left. ( J ) Whole-mount RNA in situ hybridization showing that nestin and pax2a were normally expressed in bbh fn40a ; Tg( gfap:GFP-stmn1b ) embryos at 48 hpf. Dorsal views with anterior to the left. ( K ) Quantification of average CtA and glia length in . Each dot represents one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( L ) qRT-PCR analysis confirming the efficacy of betaPix transgenic overexpression and betaPix siRNA knockdown in U251 cells. Data are presented in mean ± SEM; one-way ANOVA with Dunnett’s test, individual p-values mentioned in the figure. ( M ) Representative stereomicroscopy images of A172 cells at 0 and 18 hours after wounding. betaPIX siRNA treatment decreased A172 cell migration, which was rescued by either betaPIX or STMN1 overexpression. The wound edges are highlighted by dashed lines, with arrow lines indicating the wound width. ( N ) Quantification of wound closures in ( M ). Data are presented in mean ± SEM; one-way ANOVA with Dunnett’s test. ** p<0.01 compared to negative control siRNA and empty vector transfection. ## p<0.01, ### P <0.005 compared to betaPix knockdown and empty vector transfection. Individual scale bars are indicated in the figure.

Journal: eLife

Article Title: Glial betaPix is essential for blood vessel development in the zebrafish brain

doi: 10.7554/eLife.106665

Figure Lengend Snippet: ( A ) qRT-PCR analysis showing that IPA3 treatment rescued stmn1a and stmn4l expression in CRISPR-mediated betaPix F 0 knockouts at 48 hpf. Each dot represents one embryo. Data are presented in mean ± SEM; one-way ANOVA with Dunnett’s test, individual p-values mentioned in the figure. ( B ) Schematic diagram illustrates four-guide RNAs-mediated F 0 knockout strategy in the loci of stmn1a , stmn1b, and stmn4l . ( C ) Whole-mount RNA in situ hybridization confirming that stmn1a, stmn1b, and stmn4l decreased in CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Dorsal views with anterior to the left. ( D ) qRT-PCR analysis confirming that stmn1a, stmn1b, and stmn4l decreased in mutant embryos of CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Each dot represents one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( E ) qRT-PCR analysis confirming that stmn1a, stmn1b, and stmn4l decreased in the FACS-sorted glia of CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Each dot represents sorted cells from one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( F ) Optical sections of glial structure in the hindbrains of siblings and CRISPR-mediated stmn1a/1b/4 l F0 knockouts at 48 hpf. Lateral view with anterior to the left. Abnormal glial structures with disoriented arrangements (yellow arrows) in stmn1a/1b/4l mutant embryos. ( G ) Quantification of average CtA and glial length in . Each dot represents one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( H ) Whole-mount RNA in situ hybridization revealed that nestin increased while pax2a decreased in CRISPR-mediated stmn1a/1b/4 l F0 knockout embryos at 48 hpf. Dorsal views with anterior to the left. ( I ) 3D reconstruction and whole-mount RNA in situ hybridization showing that gfap:GFP-stmn1b transgenic overexpression rescued glial defects in bbh fn40a ; Tg( gfap:GFP-stmn1b ) embryos at 48 hpf. Lateral views with anterior to the left. ( J ) Whole-mount RNA in situ hybridization showing that nestin and pax2a were normally expressed in bbh fn40a ; Tg( gfap:GFP-stmn1b ) embryos at 48 hpf. Dorsal views with anterior to the left. ( K ) Quantification of average CtA and glia length in . Each dot represents one embryo. Data are presented in mean ± SEM; unpaired Student’s t -test with individual p-values mentioned in the figure. ( L ) qRT-PCR analysis confirming the efficacy of betaPix transgenic overexpression and betaPix siRNA knockdown in U251 cells. Data are presented in mean ± SEM; one-way ANOVA with Dunnett’s test, individual p-values mentioned in the figure. ( M ) Representative stereomicroscopy images of A172 cells at 0 and 18 hours after wounding. betaPIX siRNA treatment decreased A172 cell migration, which was rescued by either betaPIX or STMN1 overexpression. The wound edges are highlighted by dashed lines, with arrow lines indicating the wound width. ( N ) Quantification of wound closures in ( M ). Data are presented in mean ± SEM; one-way ANOVA with Dunnett’s test. ** p<0.01 compared to negative control siRNA and empty vector transfection. ## p<0.01, ### P <0.005 compared to betaPix knockdown and empty vector transfection. Individual scale bars are indicated in the figure.

Article Snippet: U251 and A172 human glioblastoma cell lines, kindly provided by Dr. Jian Chen at Chinese Institute for Brain Research, Beijing, were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China), cultured in high glucose DMEM medium (Hyclone) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, at 37°C in 5% CO 2 humidified incubator.

Techniques: Quantitative RT-PCR, Expressing, CRISPR, Knock-Out, RNA In Situ Hybridization, Mutagenesis, Transgenic Assay, Over Expression, Knockdown, Migration, Negative Control, Plasmid Preparation, Transfection

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