Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: mTOR activity and metabolic differences in U251, U87 and U373-U human glioma cells. a mTOR activity related proteins and other metabolic enzyme expressions characterise and show some individual differences in the studied human glioma cell lines—representative figures of Western blot results; b the enzyme expression profiles could correlate to mTOR inhibitor sensitivity of glioma cells (rapamycin—Rapa 50 ng/mL; NVP-BEZ235—BEZ 1 µM; PP242 1 µM for 72-h treatments), which were monitored by Alamar Blue and SRB proliferation tests—the cell proliferation of untreated controls was considered 100%; rapamycin inhibited the proliferation in all studied cells, significantly; Significant differences compared to rapamycin were labelled by *p < 0.05

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Western Blot, Expressing

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: The anti-proliferative effects and alterations in mTOR activity and other metabolism related protein levels in response to mTORI and temozolomide treatments in human glioma cell lines. a mTOR inhibitors (rapamycin—Rapa 50 ng/mL; NVP-BEZ235—BEZ 1 µM; PP242 1 µM) and temozolomide (TMZ 100 µM) 72-h combination treatments effectively inhibited the proliferation of U251, U87 and U373-U cells—Alamar Blue and SRB proliferation test results, the cell proliferation of untreated controls were considered 100% (temozolomide slightly reduced the proliferation and had no significant growth inhibitory effect on U251, mTORIs had similar effects as in Fig. a, and all temozolomide + mTORI combined treatments had significant anti-proliferative effects, p < 0.05; the additive (A) or synergistic (S) effects of inhibitor combinations were given based on CI calculation, SD was added); b altered expressions of mTORC1 and C2 activity related proteins and other metabolic enzymes were also shown after different mono-treatments (results of representative Western blots after 72-h treatments)

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Activity Assay, Western Blot

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: Metabolic drugs induced proliferative and protein expression profile changes in human glioma cells. a Doxycycline (Doxy 10 µM), etomoxir (Etom 50 µM), chloroquine (Chl 50 µM) 72-h treatments have slight growth inhibitory effects in U251, U87 and U373-U (Alamar Blue proliferation test data; *p < 0.05); b ACSS2, FASN, CPT1a and p-(Ser473)-Akt protein expressions were influenced by rapamycin, doxycycline, etomoxir and chloroquine after 72-h mono-treatments; c FASN, β-F1-ATPase, p-S6, p-(Ser473)-Akt and CPT1a protein expression compared to the untreated controls after applying two-drug combinations in U251 glioma cells (50 ng/mL rapamycin + 100 µM temozolomide—Rapa + TMZ; 50 ng/mL rapamycin + 10 µM doxycycline—Rapa + Doxy; 100 µM temozolomide + 10 µM doxycycline—TMZ + Doxy (Western blot results)—representative Western blot figures

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Western Blot

Journal: Cancer Cell International

Article Title: Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

doi: 10.1186/s12935-018-0710-0

Figure Lengend Snippet: Rapamycin and temozolomide combined with other metabolic inhibitors have different in vitro anti-proliferative effects in human glioma cell lines. Alamar Blue test results after 72-h different two-drug combined in vitro treatments in U251 ( a ), U87 ( b ) and U373-U ( c ) glioma cells (rapamycin—Rapa 50 ng/mL, temozolomide—TMZ 100 µM, doxycycline—Doxy 10 µM; etomoxir—Etom 50 µM; chloroquine—Chl 50 µM). The additive (A) or synergistic (S) effects of combinations were given based on CI calculation, SD was added, the anti-proliferative effects were significant (p < 0.05) in almost all combined treatments compared to untreated cultures except for doxycycline + etomoxir and temozolomide + etomoxir treatments

Article Snippet: U251 (ECACC-09063001, characteristic glioma cell specific mutations in PTEN, NF-1, p53, MSH2), U87 (ATCC-HTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and Notch-2), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO 2 atmosphere.

Techniques: In Vitro

Journal: Viruses

Article Title: Beclin1 Binds to Enterovirus 71 3D Protein to Promote the Virus Replication

doi: 10.3390/v12070756

Figure Lengend Snippet: Knockdown of BECN1 attenuates EV71 replication in RD and U251 cells. ( A , B ) RD cells were transfected with pLKO.1-shNC, pLKO.1-shBeclin1-1, pLKO.1-shBeclin1-2, and the efficiency of knockdown was testified by Real-time PCR ( A ). The transfected RD cells were infected with EV71 (MOI = 1), and the protein levels of EV71-VP1 and Beclin1 were detected by Western blot with β-actin as an internal control ( B ). ( C ) RD cells were transfected with Flag-vector or Flag-Beclin1, pLKO.1-shNC or pLKO.1-shBeclin1 and infected with EV71 for 12h, and supernatants were collected for TCID50 assay. ( D – F ) The stable RD cells were infected with EV71 (MOI = 1) for different periods. The RNA and protein levels of EV71-VP1 were detected by Real-time PCR with GAPDH ( D ) and Western blot with β-actin as controls ( E ), and the LC3-II/LC3-I ratio was measured( F ), respectively. ( G – I ) The stable U251 cells were infected with EV71 (MOI = 2) for different periods. The RNA and protein levels of EV71 VP1 were detected by Real-time PCR ( G ) and Western blot ( H ), and the LC3-II/LC3-I ratio was measured( I ), respectively. Graph expressed as mean ± SD, ns, not-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Human embryonic kidney (HEK293T) cells, human rhabdomyosarcoma (RD) cells, and human astroglioma (U251) cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China).

Techniques: Knockdown, Transfection, Real-time Polymerase Chain Reaction, Infection, Western Blot, Control, Plasmid Preparation, TCID50 Assay

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: DRD2 involved in the PD-L1 expression in GBM cells in the GBM-macrophage co-culture system. ( A ) U87-GFP and U251-GFP cells were co-cultured with the THP-1 differentiated macrophage for 48 h. PD-L1 expression was determined by flow cytometry analysis. ( B ) U87-GFP and U251-GFP cells were treated with paliperidone (20 μM, PAL) for 30 min, and cells were then co-cultured with THP-1 macrophages (HM) after wash-out of paliperidone for 48 h. PD-L1 expression on GBM was determined by flow cytometry analysis. * p < 0.05 compared with GBM alone group. # p < 0.05 compared with THP-1 group. ( C ) THP-1 macrophages (HM) were treated with paliperidone (20 μM, PAL) for 30 min, washed out for 48 h, and then co-cultured with U87-GFP and U251-GFP cells. PD-L1 expression on cell surface of HM was determined by flow cytometry analysis. * p < 0.05 compared with THP-1 alone group. # p < 0.05 compared with U87 or U251 groups ( n = 3–4).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Expressing, Co-Culture Assay, Cell Culture, Flow Cytometry

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: GBM primed macrophage potentiates PD-L1 expression in GBMs. Human U87 ( A , C , E ) and U251 ( B , D , F ) GBM were incubated with GBM cultured medium, THP-1 cultured medium, HM CM, or HM/GCM CM for 48 h. PD-L1 expression was determined by flow cytometry analysis ( A , B ), Western blot ( C and D ), and real time-PCR ( E , F ). * p < 0.05 compared with GBM alone group. # p < 0.05 compared with HM CM group. ( n = 3–4).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Expressing, Incubation, Cell Culture, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: DRD2 is involved in the interaction of GBM-macrophage-induced PD-L1 expression in GBM. ( A , C , E ) Human U251 GBMs were incubated with human macrophage (HM) cultured medium, HM CM or HM/GCM CM, and then treated with paliperidone (20 μM, PAL), risperidone (20 μM, RS), or L741626 (1 μM) for 48 h. ( B , D , F ) Mouse ALTS1C1 GBMs were incubated with BMDMs cultured medium, BMDMs CM or BMDM/ACM CM, and then treated with or without PAL (20 μM), risperidone (20 μM, RS), or L741626 (1 μM) for 48 h. PD-L1 expression on the cell surface was determined by flow cytometry analysis ( A – D ) and Western blot ( E , F ). * p < 0.05 compared with HM medium group. # p < 0.05 compared with HM/GCM CM group. ( n = 3–4) ( G , H ) mRNA levels of PD-L1 and DRD2 were determined by real-time PCR. * p < 0.05 compared with HM medium or BMDMs medium groups. # p < 0.05 compared with HM/GCM CM or BMDM/ACM CM groups ( n = 3).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Expressing, Incubation, Cell Culture, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression

doi: 10.3390/cancers13174357

Figure Lengend Snippet: ERK and STAT3 signaling pathways are involved in the interaction of GBM-macrophage-induced PD-L1 expression in GBMs. Human U251 ( A ) and mouse ALTS1C1 ( B ) GBM were incubated with HM/GCM CM or BMDM/ACM CM for the indicated time periods (5, 10, 30, 60, or 120 min). Human U251 ( C ) and mouse ALTS1C1 ( D ) GBM were treated with PAL (20 μM), RS (20 μM), or L741626 (1 μM) and incubated with HM/GCM CM or BMDM/ACM CM. Phosphorylated-STAT3 and phosphorylated ERK expressions were determined by using Western blot analysis. Human U251 ( E ) and mouse ALTS1C1 ( F ) GBM were treated with S31-201 (30 μM) or U0126 (1 μM) and incubated with HM/GCM CM or BMDM/ACM CM for 48 h. PD-L1 expression was determined by using Western blot analysis. ( n = 3–4).

Article Snippet: Human glioma U251 cells were purchased from the (JCRB NO. IFO50288, Tokyo, Japan).

Techniques: Protein-Protein interactions, Expressing, Incubation, Western Blot

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Scatter plot of PCR array data showing relative gene expression levels in UTMZ cells relative to U251 cells. Genes upregulated by more than 2-fold are shown in black circles, genes downregulated by more than 2-fold are shown in black squares. Arrow shows the data point representing BMI1 . (B) Representative western blot (top) and quantitative analysis (bottom graph) showing the relative BMI1 expression levels in U251 and UTMZ cells. (C) Analysis of RNA-sequencing data provided by TCGA depicting co-expression of COX4I1 mRNA and BMI1 mRNA in patients with high-grade GBM. (D) Representative western blots depicting COX4-1 and BMI1 expression in a panel of 24 primary human GBM tumors. (E) Quantification of relative band intensities in (D) Numbers in parentheses indicate the mean value from all tumors. (F) OS for patients with high and low tumor expression levels of COX4-1 ( P < 0.0001 by the log-rank test; hazard ratio for death in patients with high tumor COX4-1 expression, 54.99; 95% CI, 11.02 to 274.3) or BMI1 ( P = 0.0113 by the log-rank test; hazard ratio for death in patients with high tumor BMI1 expression, 2.59; 95% CI, 2.107 to 3.073). Numbers in parentheses indicate the median survival time for each group.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Gene Expression, Western Blot, Expressing, RNA Sequencing

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) COX4-1 and COX4-2 constructs ( pCMV6-COX4-1-FLAG and pCMV6-COX4-2-FLAG) were transfected into U251-COX4-2 depleted cells to create U251-TgCOX4-1 and U251-TgCOX4-2 stable cell lines. Expression of COX4 isoforms and BMI1 was detected in each cell line by western blot analysis. Citrate synthase (CS) expression is shown as mitochondrial loading control and actin expression is shown as nuclear loading control. (B) Proliferation rates of each cell line. (C) Representative pictures of clonogenic assays with each cell line, showing anchorage-independent cell growth. (D) Representative images of tumors from athymic nude mice inoculated with the cell lines. Tumors were excised 4 weeks after inoculation. (E) Analysis of tumor volumes in mice over the course of the experiment. (F) Comparison of tumor weights upon excision. Graphs represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Construct, Transfection, Stable Transfection, Expressing, Western Blot, Control, Comparison

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: Representative images of tumors resulting from intracranial implantation of U251 and U251-TgCOX4-1 glioma cells, stained for (A) H&E, (B) Ki-67, (C) COX4-1, (D) COX4-2, and (E) BMI1. Scale bar, 100 μm.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Staining

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Representative western blot depicting BMI1 expression in nuclear extracts of U251-TgCOX4-1 cell following 24-h PTC-209 treatment (0–10 μM). (B) Cell proliferation in control and PTC-209-treated (5 μM) U251-TgCOX4-1 cells. (C) Representative western blot depicting BMI1 expression in U251-TgCOX4-1 cells expressing shRNA control or one of four different vectors expressing shRNA against BMI1. (D) Quantification of the relative expression levels of BMI1 detected in (C). (E) Cell proliferation in clones expressing shRNA against BMI1. (F) Representative western blot depicting BMI1 expression levels (inset) and the cell proliferation rates of control and pCMV6-BMI1-transfected U251 cells. Graphs represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Western Blot, Expressing, Control, shRNA, Clone Assay, Transfection

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Relative activity of CcO normalized to citrate synthase (CS) activity. (B) Oxygen consumption rates were determined using a respirometer. Representative traces of cellular respiration rates of U251 (black) and U251-TgCOX4-1 (red) cells (blue line, oxygen concentration). (C) Kinetic characterization of glutamate/malate, succinate, and fatty acid-dependent respiration of U251-TgCOX4-1 and U251-TgCOX4-2 cells. (D) Kinetic characterization of FCCP-dependent respiration in U251-TgCOX4-1 and U251-TgCOX4-2 cells. (E) Dose-response analyses of glucose uptake in cell lines expressing different COX4 isoforms. Graphs represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Activity Assay, Concentration Assay, Expressing

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Representative histograms from flow cytometric analysis of total cellular ROS (left, DCFDA fluorescence) and mitochondrial ROS (right, MitoSOX fluorescence) in parental and U251-TgCOX4-1 cells. Bar graphs provide quantitative analysis of fluorescence intensity. (B) Quantitative graphs showing the relative levels of catalase activity, superoxide dismutase activity, NAD + /NADH ratio, and GSH/GSSG ratio in U251-TgCOX4-1 cells. (C) Representative histograms from flow cytometric analysis of total cellular or mitochondrial ROS production in U251-TgCOX4-2 cells treated with NAC (300 μM) or PTC-209 (5 μM) (left) and in U251-TgCOX4-1 cells treated with NMP (10 μM) or PTC-209 (right). (D) Representative western blots depicting BMI1 expression in the nuclear extracts of parental cells or U251-TgCOX4-1 cells after treatment with NAC or PTC-209 for 24 h (top) and quantitative analysis of expression levels (bottom). Bars represent the average from triplicate determinations from at least three independent experiments.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: Fluorescence, Activity Assay, Western Blot, Expressing

Journal: Oncotarget

Article Title: Nuclear-encoded cytochrome c oxidase subunit 4 regulates BMI1 expression and determines proliferative capacity of high-grade gliomas

doi:

Figure Lengend Snippet: (A) Representative phase contrast photomicrographs (10× magnification) of parental U251, U251-shRNA-COX4-2, U251-TgCOX4-1, and U251-TgCOX4-2 cells after 10 days of culture in serum-free Neurobasal medium supplemented with EGF and FGF. (B) Spheroids of U251-TgCOX4-1 cells were immunostained with antibodies against COX4-1, BMI1, or CD133 or with control antibodies. (C) In vitro limiting dilution assays and quantification of COX4-1 and BMI1 expressing cells. Results represent the average from two independent experiments. (D) Spheroid multipotency was assessed by immunofluorescence for neuronal (neurofilament, CNPase, and βIII-tubulin) and glial (GFAP) markers.

Article Snippet: U251 COX4-2-KO or -knockdown cells were electroporated with CMV6 plasmids containing FLAG-epitope-tagged COX4-2 or COX4-1 (Catalog # RC209204 and RC209374, OriGene Technologies).

Techniques: shRNA, Control, In Vitro, Expressing, Immunofluorescence

Journal: Cell Death & Disease

Article Title: The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

doi: 10.1038/s41419-022-05528-8

Figure Lengend Snippet: A Process for high-throughput drug screening. B , C U87MG, U251, GBM60, and GBM38 cell viability was determined using a CCK-8 assay after treatment with various concentrations of AT7519. * P < 0.05, *** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with the control using one-way ANOVA followed by Dunnett’s multiple test. D U87MG and U251 cell viability was determined using a CCK-8 assay after treatment with 0.4 µM AT7519 for 6, 12, 24, 48, and 60 h. * P < 0.05, **** P < 0.0001 compared with the control using one-way ANOVA followed by Dunnett’s multiple test. E Colony formation assays to verify the effect of AT7519 on glioblastoma cell proliferation. * P < 0.05, *** P < 0.001, and **** P < 0.0001 based on one-way ANOVA followed by Dunnett’s multiple test. F , G The level of DNA synthesis of U87MG and U251 cells was determined using an EdU assay after treatment with increasing concentrations of AT7519. The nuclei were stained with Hoechst (blue), and the proliferating cells were stained with EdU (yellow). * P < 0.05, *** P < 0.001 and **** P < 0.0001 based on one-way ANOVA followed by Dunnett’s multiple test. The results are presented as the mean ± SD from three independent experiments.

Article Snippet: Human glioblastoma cell lines (U87MG and U251) were purchased from ATCC.

Techniques: High Throughput Screening Assay, Drug discovery, CCK-8 Assay, Control, DNA Synthesis, EdU Assay, Staining

Journal: Cell Death & Disease

Article Title: The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

doi: 10.1038/s41419-022-05528-8

Figure Lengend Snippet: A U87MG and U251 cells were treated with AT7519 and morphological features of pyroptosis in SEM (red arrows, membrane pore-forming). B After U87MG and U251 cells were treated with AT7519 for 48 h, cytotoxicity was detected by lactate dehydrogenase (LDH) released into the cell culture medium. **** P < 0.0001 by Student’s t -test. C , D Full-length GSDME (GSDME-FL) and N-terminal GSDME (GSDME-N) were detected in glioblastoma cells after treatment with AT7519 for 48 h by western blot analysis. E , F U87MG and U251 cells were pretreated with Z-VAD-FMK for 2 h and then treated with AT7519 for 48 h. Cytotoxicity was detected by LDH release assay. *** P < 0.001 and **** P < 0.0001 as assessed by Student’s t -test. The apoptosis marker cleaved PARP and pyroptosis marker GSDME-N were detected by western blot. G U87MG and U251 cells were treated with Z-DEVD-FMK combined with AT7519 for 48 h, and western blot analysis of cleaved caspase-3, GSDME-FL and GSDME-N proteins was performed.

Article Snippet: Human glioblastoma cell lines (U87MG and U251) were purchased from ATCC.

Techniques: Membrane, Cell Culture, Western Blot, Lactate Dehydrogenase Assay, Marker

Journal: Cell Death & Disease

Article Title: The CDK inhibitor AT7519 inhibits human glioblastoma cell growth by inducing apoptosis, pyroptosis and cell cycle arrest

doi: 10.1038/s41419-022-05528-8

Figure Lengend Snippet: A Image of the subcutaneous xenograft tumors formed in nude mouse models. B Tumor volumes were measured and calculated every week. **** P < 0.0001 as assessed by two-way ANOVA followed by Sidak’s multiple test. C Tumors were excised and weighed at the end of the experiment. *** P < 0.001 by Student’s t -test. D Body weight of nude mice during administration of AT7519. E Representative H&E-stained images of the intracranial xenograft model in the AT7519 treatment group and control group. * p < 0.05 as assessed by Student’s t -test. F Western blot assay of the apoptosis, pyroptosis, and cell cycle-related key protein expression levels in tumor tissue. G Schematic model of the antitumor mechanism of AT7519 in glioblastoma cells.

Article Snippet: Human glioblastoma cell lines (U87MG and U251) were purchased from ATCC.

Techniques: Staining, Control, Western Blot, Expressing