Journal: Cell reports

Article Title: Multiplexed single-cell lineage tracing of mitotic kinesin inhibitor resistance in glioblastoma

doi: 10.1016/j.celrep.2024.114139

Figure Lengend Snippet:

Article Snippet: temozolomide , MedChemExpress , HY-17364.

Techniques: Recombinant, Transfection, DNA Library Preparation, Ex Vivo, Software, Single Cell

Journal: Molecular medicine reports

Article Title: MutL homolog 1 contributes to temozolomide-induced autophagy via ataxia-telangiectasia mutated in glioma.

doi: 10.3892/mmr.2015.3293

Figure Lengend Snippet: Figure 1. TMZ induces autophagy via MLH1. (A) Glioma cells were transfected with 100 nM MLH1 siRNA and harvested at the indicated time points for western blot analysis. (B) Glioma cells and the MLH1‑knockdown glioma cells (100 nM MLH1 siRNA for 48 h) were incubated with 100 µM TMZ or vehicle (DMSO) for 72 h. Following treatment, the cells were collected for western blot analysis. (C) U87MG cells and the MLH1‑knockdown U87MG cells were incubated with 100 µM TMZ or vehicle for 72 h. Following treatment, the cells were collected for AVO detection using flow cytometric analysis. The data are expressed as the mean ± standard deviation (**P<0.01, vs. control group; n=3 for each group). NS, not statistically significant compared with the control group; MLH1, mutL homolog 1; TMZ, temozolomide; siRNA, small interfering RNA; AMPK, adenosine monophosphate‑activated protein kinase; ULK, unc‑51‑like autophagy activating kinase 1; LC3B, microtubule‑associated protein 1 light chain 3 β; AVO, acidic vesicular organelle, p, phosphorylated; FL1, green fluorescence; FL3, red fluorescence.

Article Snippet: China E-mail: gzwangwm0@163.com Abbreviations: TMZ, temozolomide; MMR, DNA mismatch repair; MLH1, mutL homolog 1; ATM, ataxia-telangiectasia mutated; AMPK, adenosine monophosphate-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1 Key words: autophagy, mutL homolog 1, ataxia-telangiectasia mutated, glioma, temozolomide (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Pasching, Austria) at 37 ̊C in a 5% CO2‐humidified atmosphere.

Techniques: Transfection, Western Blot, Incubation, Standard Deviation, Control, Small Interfering RNA, Fluorescence

Journal: Molecular medicine reports

Article Title: MutL homolog 1 contributes to temozolomide-induced autophagy via ataxia-telangiectasia mutated in glioma.

doi: 10.3892/mmr.2015.3293

Figure Lengend Snippet: Figure 2. TMZ induces autophagy via pATM, which is MLH1‑dependent. (A) U87MG, U251 and SHG‑44 cells were treated with 100 µM TMZ and/or 10 µM KU‑55933, as indicated, for 72 h. Subsequently, the cells were collected for western blot analysis. dimethyl sulfoxide was used as a negative control. (B) U87MG cells were treated with 100 µM TMZ and/or 10 µM KU‑55933, as indicated, for 72 h. Following treatments, the cells were collected and the number of AVOs were detected using flow cytometric analysis. (C) Glioma cells and MLH1‑knockdown glioma cells (100 µM MLH1 siRNA for 48 h) were incubated with 100 µM TMZ or vehicle (DMSO) for 72 h. Subsequently, the levels of pATM were assessed using western blot analysis. The data are expressed as the mean ± standard deviation (**P<0.01, vs. control group; n=3 for each group). NS, not statistically significant compared with the control group; MLH1, mutL homolog 1; ATM, ataxia telangiectsia; p, phosphorylated; TMZ, temozolomide; siRNA, small interfering RNA; AMPK, adenosine monophosphate‑activated protein kinase; ULK, unc‑51‑like autophagy activating kinase 1; LC3B, microtubule‑associated protein 1 light chain 3 β; AVO, acidic vesicular organelle; FL1, green fluorescence; FL3, red fluorescence.

Article Snippet: China E-mail: gzwangwm0@163.com Abbreviations: TMZ, temozolomide; MMR, DNA mismatch repair; MLH1, mutL homolog 1; ATM, ataxia-telangiectasia mutated; AMPK, adenosine monophosphate-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1 Key words: autophagy, mutL homolog 1, ataxia-telangiectasia mutated, glioma, temozolomide (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Pasching, Austria) at 37 ̊C in a 5% CO2‐humidified atmosphere.

Techniques: Western Blot, Negative Control, Incubation, Standard Deviation, Control, Small Interfering RNA, Fluorescence

Journal: Molecular medicine reports

Article Title: MutL homolog 1 contributes to temozolomide-induced autophagy via ataxia-telangiectasia mutated in glioma.

doi: 10.3892/mmr.2015.3293

Figure Lengend Snippet: Figure 3. Knock down of MLH1 results in TMZ resistance, whereas inhibition of ATM augments TMZ cytotoxicity in inherently TMZ‑sensitive cells. (A) Glioma cells and MLH1‑knockdown glioma cells (100 µM MLH1 siRNA for 48 h) were incubated with 100 µM TMZ or vehicle for 72 h. The cells were then collected to detect the expression of γH2AX by western blot analysis. (B) Glioma cells and MLH1‑knockdown glioma cells were seeded at a density of 3,000 cells/well in 96‑well microplates and treated with 100 µM TMZ or vehicle (DMSO) for 72 h. MTT assays were performed to assess the cell viability (n=6 for each group). (C) U87MG cells were treated as in (A) and apoptosis was detected by annexin‑V‑fluorescin isothiocyanate/propidium iodide double staining (n=3 for each group). (D and E) U87MG and U87TMZ cells were treated with 100 µM TMZ and/or 10 µM KU‑55933, as indicated, for 72 h. (D) Western blot analysis was then performed to detect the expression of γH2AX. Dimethyl sulfoxide was used as a negative control. (E) MTT assays were performed to assess the cell viability (n=6 for each group). The data are expressed as the mean ± standard deviation (*P<0.05 and **P<0.01, vs. control group). NS, not statistically significant; MLH1, mutL homolog 1; TMZ, temozolomide; siRNA, small interfering RNA.

Article Snippet: China E-mail: gzwangwm0@163.com Abbreviations: TMZ, temozolomide; MMR, DNA mismatch repair; MLH1, mutL homolog 1; ATM, ataxia-telangiectasia mutated; AMPK, adenosine monophosphate-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1 Key words: autophagy, mutL homolog 1, ataxia-telangiectasia mutated, glioma, temozolomide (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Pasching, Austria) at 37 ̊C in a 5% CO2‐humidified atmosphere.

Techniques: Knockdown, Inhibition, Incubation, Expressing, Western Blot, Double Staining, Negative Control, Standard Deviation, Control, Small Interfering RNA

Journal: Molecular medicine reports

Article Title: MutL homolog 1 contributes to temozolomide-induced autophagy via ataxia-telangiectasia mutated in glioma.

doi: 10.3892/mmr.2015.3293

Figure Lengend Snippet: Figure 4. AMPK inhibition decreases levels of autophagy and augments TMZ cytotoxicity in inherently TMZ‑sensitive glioma cells. (A) U87MG, U251 and SHG‑44 cells were treated with 5 µM compound C or vehicle (DMSO) for 72 h. The cells were then harvested for western blot analysis to determine the levels of pAMPKα (Thr172). (B‑D) U87MG, U251 and SHG‑44 cells were treated with 100 µM TMZ and/or 5 µM compound C for 72 h. (B) MTT assays were per formed to assess the cell viability (n=6 for each group) and (C) western blot analysis was performed to detect the expression of LC3B and γH2AX. (D) U87MG cells were treated with 100 µM TMZ and/or 5 µM compound C for 72 h. Apoptosis was detected using annexin‑V‑fluorescin isothiocyanate/propidium iodide double staining (n=3 for each group). U87TMZ cells were treated with 100 µM TMZ and/or 5 µMm compound C for 72 h. (E) Western blot analysis was performed to detect the expression levels of pULK1 and LC3B. (F) MTT assays were performed to assess the cell viability (n=6 for each group). The data are expressed as the mean ± standard deviation (*P<0.05 and **P<0.01, vs. control group or indicated group). NS, not statistically significant, MLH1, mutL homolog 1; TMZ, temozolomide; siRNA, small interfering RNA; AMPK, adenosine monophosphate‑activated protein kinase; ULK, unc‑51‑like autophagy activating kinase 1; LC3B, microtubule‑associated protein 1 light chain 3 β; p, phosphorylated.

Article Snippet: China E-mail: gzwangwm0@163.com Abbreviations: TMZ, temozolomide; MMR, DNA mismatch repair; MLH1, mutL homolog 1; ATM, ataxia-telangiectasia mutated; AMPK, adenosine monophosphate-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1 Key words: autophagy, mutL homolog 1, ataxia-telangiectasia mutated, glioma, temozolomide (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Pasching, Austria) at 37 ̊C in a 5% CO2‐humidified atmosphere.

Techniques: Inhibition, Western Blot, Expressing, Double Staining, Standard Deviation, Control, Small Interfering RNA

Journal: Cancer Gene Therapy

Article Title: Divergent transcriptomic signatures from putative mesenchymal stimuli in glioblastoma cells

doi: 10.1038/s41417-023-00724-w

Figure Lengend Snippet: G816 ( A ) or G559 ( B ) proneural GICs were plated on Matrigel and treated with 10 ng/mL TGFβ or TNFα, or both ligands, for 4 days. Expression of the mesenchymal markers FN1 , CD44 , CHI3L1 , and COL1A2 and the proneural marker OLIG2 were measured by RT-qPCR. Error bars represent the mean ± standard deviation of three biological replicates. * indicates p < 0.05, as determined by one-way ANOVA and Tukey’s post-hoc test. Only significant differences with respect to the untreated condition are shown. G816 ( C ) or G559 ( D ) cells were plated on Matrigel and treated with DMSO (vehicle) or 100 μM temozolomide (TMZ) for 4 days, and the indicated transcripts were measured by RT-qPCR. Error bars represent the mean ± standard deviation of three biological replicates. * indicates p < 0.05 using two-tailed unpaired t -test. E G816 cells were plated on Matrigel-coated glass coverslips and treated with 10 ng/mL TGFβ + TNFα or 100 μM temozolomide (TMZ) for four days. Immunofluorescence microscopy was performed for N-cadherin to identify cell boundaries, and the indicated metrics (definitions in Materials and Methods) were extracted using automated image analysis, n = 3. * indicates p < 0.05 as determined by one-way ANOVA and Games-Howell post-hoc test. Scale bars = 100 μm.

Article Snippet: Temozolomide (Santa Cruz Biotechnology) was reconstituted in DMSO at 20 mg/mL, according to manufacturer recommendations.

Techniques: Expressing, Marker, Quantitative RT-PCR, Standard Deviation, Two Tailed Test, Immunofluorescence, Microscopy

Journal: British Journal of Cancer

Article Title: Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

doi: 10.1038/sj.bjc.6600711

Figure Lengend Snippet: Volume growth of ( A ) GaMG and ( B ) U87MG spheroids. Spheroids treated with temozolomide show a concentration-dependent inhibition of volume growth. Error bar=s.e.m. ( C , D ) Radial migration of tumour cells. Area covered by cells migrating away radially from disassembling ( C ) GaMG spheroids and ( D ) U87 spheroids after placement on an uncoated cell culture dish. No difference in migration ability was observed between treated and control cells. After 5 days, differences of proliferative activity add to the migrational behaviour. Error bar=s.d.

Article Snippet: Temozolomide was obtained from Essex Pharma (Munich, Germany).

Techniques: Concentration Assay, Inhibition, Migration, Cell Culture, Activity Assay

Journal: British Journal of Cancer

Article Title: Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

doi: 10.1038/sj.bjc.6600711

Figure Lengend Snippet: Invasion into foetal brain aggregates. Confrontation of U87MG spheroids (T) with a 21-day-old foetal rat brain aggregate ( B ). ( A–C ) Volume growth of the tumour spheroids is noticeably reduced at 20 μ g ml −1 temozolomide concentration compared to untreated controls ( D–F ). As a consequence of this, reduced invasion capacity is observed.

Article Snippet: Temozolomide was obtained from Essex Pharma (Munich, Germany).

Techniques: Concentration Assay

Journal: British Journal of Cancer

Article Title: Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

doi: 10.1038/sj.bjc.6600711

Figure Lengend Snippet: Repetitive treatment of multicellular glioblastoma spheroids with temozolomide

Article Snippet: Temozolomide was obtained from Essex Pharma (Munich, Germany).

Techniques:

Journal: British Journal of Cancer

Article Title: Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

doi: 10.1038/sj.bjc.6600711

Figure Lengend Snippet: TUNEL labelling. Cross-sections of paraffin-embedded spheroids, stained for fragmented DNA by TUNEL. The more loosely associated U87 spheroids contain significantly more apoptotic nuclei 2 days after temozolomide treatment ( A , B ). It takes about 6 days until there is a significant number of apoptotic nuclei labelled in GaMG spheroids ( C , D ). Nuclei are counterstained with nuclear fast red.

Article Snippet: Temozolomide was obtained from Essex Pharma (Munich, Germany).

Techniques: TUNEL Assay, Staining

Journal: British Journal of Cancer

Article Title: Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

doi: 10.1038/sj.bjc.6600711

Figure Lengend Snippet: DNA fragmentation. Laddering phenomenon of fragmented DNA resolved on a 1.5% agarose gel. Alkylation of DNA by temozolomide results in induction of apoptosis in U87 cells during the first 48 h postexposure. After 7 days, untreated U87 cells also undergo apoptosis because of unfavourable culture conditions. GaMG is not initiating apoptosis after 2 days and a slight induction is observed after 7 days in culture.

Article Snippet: Temozolomide was obtained from Essex Pharma (Munich, Germany).

Techniques: Agarose Gel Electrophoresis

Journal: British Journal of Cancer

Article Title: Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids

doi: 10.1038/sj.bjc.6600711

Figure Lengend Snippet: Senescence-associated galactosidase activity in glioma spheroids treated with temozolomide. X-Gal staining revealed that GaMG spheroids never accumulate a detectable galactosidase activity as a response to temozolomide treatment. However, U87 cells clearly exhibit a drug-dependent stimulation of galactosidase activity. This accumulation of lysosomal galactosidase enzyme is already observed shortly after drug application.

Article Snippet: Temozolomide was obtained from Essex Pharma (Munich, Germany).

Techniques: Activity Assay, Staining