cell line ipc298  (DSMZ)

Journal: PLoS ONE

Article Title: A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

doi: 10.1371/journal.pone.0185801

Figure Lengend Snippet: ( A ) Melanoma cell lines were grown in the presence of enoxacin (50 μg/ml) or 0.2% NaOH (vehicle, negative control). Relative cell growth was determined using standard MTT assay after 1, 2, 3, 4, and 5 days. Results of three independent experiments are presented (means +/- SD). ( B ) Melanoma cell lines were grown in the presence of 0.2% NaOH (control), enoxacin or ofloxacin (both 50 μg/ml) for four days and relative cell growth was determined by MTT assays. Results of three independent experiments (means + standard deviations) are presented. A significant difference between the effect of enoxacin and ofloxacin **P<0.01 (Student’s t-test, two-tailed). ( C ) A375 cells were grown for 3 days in the presence of 0.2% NaOH (control) or enoxacin in concentrations 25, 50, 75, and 100 μg/ml. Cell viability was determined using propidium iodide exclusion assay and flow cytometry. Results of three independent experiments are presented (means + SD). ** P<0.01 (Student’s t-test, two-tailed).

Article Snippet: The human cancer cell lines A375, Mel-Juso, Mel-Ho, IPC298, H1299, A2780, and MCF7 (obtained from ECACC, DSMZ, and ATCC) were cultured at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin.

Techniques: Negative Control, MTT Assay, Control, Two Tailed Test, Propidium Iodide Exclusion Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

doi: 10.1371/journal.pone.0185801

Figure Lengend Snippet: ( A ) miRNA expression profiling of control and enoxacin-treated A375 cells. Total RNA, including the small RNAs, was analyzed with Affymetrix GeneChip miRNA 3.0 arrays. The heat map shows statistically significant changes in the expression of miRNAs in A375 melanoma cells in response to enoxacin treatment (p<0.05; three independent experiments). ( B ) Matured miRNAs identified as significantly up- or down-regulated in A375 melanoma cells in response to enoxacin (p < 0.05).

Article Snippet: The human cancer cell lines A375, Mel-Juso, Mel-Ho, IPC298, H1299, A2780, and MCF7 (obtained from ECACC, DSMZ, and ATCC) were cultured at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin.

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

doi: 10.1371/journal.pone.0185801

Figure Lengend Snippet: ( A, left ) A375 cells stably transfected with a p53-responsive luciferase reporter construct were grown for 24 hours in the presence of enoxacin in concentrations 25, 50, 75, and 100 μg/ml. Relative p53 transcriptional activity was determined by measuring the activity of luciferase in cell lysates. Results of three independent experiments are presented (means + SD). * P<0.05, ** P<0.01 (Student’s t-test, two-tailed). ( A, right ) A375 cells were treated with enoxacin for 24 hours, lysed, and protein levels of MdmX and p53 in cell lysates were analyzed by Western blotting. PCNA served as a loading control. ( B ) A375 cells stably transfected with a p53 activity reporter construct were treated with 0.2% NaOH (control), etoposide, and three fluoroquinolone antibiotics, and p53 activity was determined. Results of three independent experiments (means + SD) are presented. A significant difference between control and antibiotic-treated cells * P<0.05, ** P<0.01 (Student’s t-test, two-tailed). ( C ) Quantitative real-time PCR analysis of the expression of selected p53 target genes in A375 cells treated for 24 hours with 50 or 100 μg/ml enoxacin or 0.5 μM doxorubicin. The expression of HPRT1/HGPRT served as an endogenous control. ( D ) Mel-Juso cells stably transfected with a p53-responsive luciferase reporter construct were grown for 24 hours in the presence of enoxacin in concentrations 50 and 100 μg/ml. Relative p53 activity was determined by measuring luciferase activity in cell lysates. Results of three independent experiments are presented (means + SD). * P<0.05, (Student’s t-test, two-tailed). MdmX and p53 protein levels in Mel-Juso cell lysates were analyzed by Western blotting. PCNA served as a loading control. ( E ) ( F ) Western blot analysis of A375 cells treated with increasing concentrations of enoxacin or DNA damaging drugs for the indicated time. A mixture of three different primary monoclonal antibodies recognizing various epitopes in human Mdm2 was used in the Mdm2 panels. * Non-specific band.

Article Snippet: The human cancer cell lines A375, Mel-Juso, Mel-Ho, IPC298, H1299, A2780, and MCF7 (obtained from ECACC, DSMZ, and ATCC) were cultured at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin.

Techniques: Stable Transfection, Transfection, Luciferase, Construct, Activity Assay, Two Tailed Test, Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Bioprocessing

Journal: PLoS ONE

Article Title: A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

doi: 10.1371/journal.pone.0185801

Figure Lengend Snippet: ( A ) Schematic representation of MdmX mRNA exon structure and binding sites for PCR primers used in RT-PCR to determine the presence/absence of exon 6 (VI). ( B, left ) Schematic representation of expected MdmX RT-PCR products in the case of presence ( MdmX-FL ) or absence ( MdmX-S ) of exon 6. ( B, right ) RT-PCR analysis of MdmX exon 6 skipping in A375, A2780, and MCF7 cells treated with enoxacin (50 and 100 μg/ml) for 24 hours (primers 1 –top, primers 2 –middle, GAPDH PCR control–bottom). ( C ) RT-PCR analysis of MdmX exon 6 skipping in A375, A2780 and MCF7 cancer cell lines treated with ciprofloxacin and ofloxacin (both 100 μg/ml) for 24 hours (MdmX primers 2 –top, GAPDH PCR control–bottom). * Non-specific band.

Article Snippet: The human cancer cell lines A375, Mel-Juso, Mel-Ho, IPC298, H1299, A2780, and MCF7 (obtained from ECACC, DSMZ, and ATCC) were cultured at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin.

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

doi: 10.1371/journal.pone.0185801

Figure Lengend Snippet: ( A ) Western blot analysis of changes in cellular MdmX protein levels induced by 24 h treatment with three fluoroquinolone drugs at indicated concentrations in A375 melanoma, A2780 ovarian carcinoma, and MCF7 breast carcinoma. PCNA served as a loading control. ( B )( C ) Western blot analysis of changes in cellular MdmX, Mdm2, and p53 protein levels induced by 24 h treatment with enoxacin and DNA damaging agents. PCNA served as a loading control. (B) MCF7, (C) A2780, * non-specific band.

Article Snippet: The human cancer cell lines A375, Mel-Juso, Mel-Ho, IPC298, H1299, A2780, and MCF7 (obtained from ECACC, DSMZ, and ATCC) were cultured at 37°C and 5% CO 2 in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin.

Techniques: Western Blot, Control