human leukemia cell lines karpas 299  (DSMZ)

Journal: Oncotarget

Article Title: Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

doi: 10.18632/oncotarget.24465

Figure Lengend Snippet: ( A ) Quantitative real-time PCR (qRT-PCR) analysis of miR-125b expression in two NPM-ALK(+) ALCL cell lines (KARPAS-299 and COST) and CD4 lymphocytes stimulated (S) or not (NS) with CD3/CD28 antibodies. SNORD44 expression was used as an internal control. Relative human miR-125b expression was expressed as 2 –ΔCt relative to SNORD44 expression. ( B ) Assessment of miR-125b expression by qRT-PCR in wild type mice (WT, N = 6) or NPM-ALK transgenic mice containing a Tet-OFF system treated (+) or not (−) with doxycycline ( N = 6; B). SNORD202 expression served as the internal control, and the relative ratio of mmu-miR-125b expression was expressed as 2 –ΔΔCt relative to WT mice. ( C ) MiR-125b expression in KARPAS-299 and COST cells treated for 72 hours or not (PBS) with crizotinib or transfected with either an irrelevant siRNA as the negative control (si-CTL) or a siRNA targeting ALK mRNA (si-ALK). SNORD44 expression served as the internal control and the relative ratio of hsa-miR-125b expression was expressed as 2 –ΔΔCt relative to untreated cells or to the si-CTL conditions. Data represent means ± SEM (bars) from 3 independent experiments. ** P < 0.001, and *** P < 0.0001; unpaired 2-tailed Student's t test.

Article Snippet: The human ALCL cell lines KARPAS-299 was obtained from DSMZ (German Collection of Microorganisms and Cell Culture) and COST was established in our laboratory.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control, Transgenic Assay, Transfection, Negative Control

Journal: Oncotarget

Article Title: Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

doi: 10.18632/oncotarget.24465

Figure Lengend Snippet: ( A ) Schematic representation of the position of 13 CpG dinucleotides in the promoter region of MIR125B1 . ( B ) Percentage of DNA methylation, assessed by bisulfite conversion and pyrosequencing in two ALCL NPM-ALK(+) cell lines, KARPAS-299 (KARPAS) and COST, and in CD4 lymphocytes stimulated (S) or not (NS) with CD3/CD28 antibodies. ( C ) Quantitative real-time PCR analysis of miR-125b expression in KARPAS and COST cells treated for 96 hours or not (PBS) with decitabine. SNORD44 was used as an internal control and the relative ratio of miR-125b expression was expressed as 2 –ΔΔCt relative to untreated cells. Data represent means ± SEM (bars) from 3 independent experiments, * P < 0.05 and *** P < 0.0001; unpaired 2-tailed Student's t test.

Article Snippet: The human ALCL cell lines KARPAS-299 was obtained from DSMZ (German Collection of Microorganisms and Cell Culture) and COST was established in our laboratory.

Techniques: DNA Methylation Assay, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Oncotarget

Article Title: Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

doi: 10.18632/oncotarget.24465

Figure Lengend Snippet: ( A ) NPM-ALK(+) ALCL KARPAS-299 cells were transfected with biotinylated forms of human miR-125b (hsa-miR125b) or C. elegans miR-39 (cel-miR39, an irrelevant microRNA, used as a negative control). Quantitative real-time PCR (qRT-PCR) analysis of BAK1, MCL1, PUMA, P53 or GAPDH mRNA was performed after pull-down of the biotinylated microRNAs using streptavidin beads. The results are presented as the percentage of input. ( B and C ) Quantitative RT-PCR analysis of (B) miR-125b expression relative to RNU1A and (C) BAK1 expression relative to GAPDH in reactive lymph nodes (RLN, n = 14) and NPM-ALK(+) primary biopsies ( n = 65). RNU1A or GAPDH were used as internal controls and the relative ratios of miR125b or BAK1 expression were expressed as 2 –ΔΔCt relative to those in the reactive lymph nodes. Data represent means ± SD (bars); ** P < 0.001 and *** P < 0.0001; unpaired two-tailed Student's t -test with Welch's correction.

Article Snippet: The human ALCL cell lines KARPAS-299 was obtained from DSMZ (German Collection of Microorganisms and Cell Culture) and COST was established in our laboratory.

Techniques: Transfection, Negative Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Oncotarget

Article Title: Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

doi: 10.18632/oncotarget.24465

Figure Lengend Snippet: In NPM-ALK(+) ALCL KARPAS-299 and COST cells transfected with a mimic miR-125b (miR-125b) or negative control microRNA (miR-CTL). ( A ) Western blotting analysis of BAK1 and GAPDH protein levels in cells transfected with a mimic miR-125b (miR-125b) or negative control microRNA (miR-CTL). Measurement of caspase 3/7 activity ( B ) and western blotting analyses of BAK1 and GAPDH protein levels ( C ) in cells treated with doxorubicin and transfected with either miR-CTL or miR-125b mimics. Densitometric analysis was performed using GeneTools software from Syngene. * P < 0.05 and ** P < 0.001; unpaired 2-tailed Student's t test.

Article Snippet: The human ALCL cell lines KARPAS-299 was obtained from DSMZ (German Collection of Microorganisms and Cell Culture) and COST was established in our laboratory.

Techniques: Transfection, Negative Control, Western Blot, Activity Assay, Software

Journal: Oncotarget

Article Title: Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

doi: 10.18632/oncotarget.24465

Figure Lengend Snippet: ( A ) Analysis in NPM-ALK(+) ALCL KARPAS-299 and COST cells of BAK1 and GAPDH expression by western blotting in cells transfected with an irrelevant siRNA as the negative control (si-CTL) or an siRNA targeting BAK1 mRNA (si-BAK1). ( B ) Assessment of caspase 3/7 activity in NPM-ALK(+) ALCL KARPAS-299 and COST cells transfected with si-CTL or si-BAK1 and treated for 48 h with 200 or 400 nM doxorubicin. Data represent means ± SEM (bars) from 3 independent experiments. * P < 0.05 and ** P < 0.001; unpaired 2-tailed Student's t test.

Article Snippet: The human ALCL cell lines KARPAS-299 was obtained from DSMZ (German Collection of Microorganisms and Cell Culture) and COST was established in our laboratory.

Techniques: Expressing, Western Blot, Transfection, Negative Control, Activity Assay

Journal: Oncotarget

Article Title: Doxorubicin-induced loss of DNA topoisomerase II and DNMT1- dependent suppression of MiR-125b induces chemoresistance in ALK-positive cells

doi: 10.18632/oncotarget.24465

Figure Lengend Snippet: ( A and B ) Quantitative real-time PCR (qRT-PCR) analysis of miR-125b expression in NPM-ALK(+) ALCL KARPAS-299 cells treated for 48h or not (PBS) with 400 nM doxorubicin (A) or etoposide at a final concentration ranging from 100 to 300 nM (B). ( C ) Quantitative real-time PCR (qRT-PCR) analysis of miR-29a expression in NPM-ALK(+) ALCL KARPAS-299 cells treated for 48h or not (PBS) with doxorubicin or etoposide. SNORD44 served as an internal control and the level of hsa-miR-125b or miR-29a were expressed as 2 –ΔΔCt relative to untreated cells. ( D and E ) ChIP experiments were performed using antibodies against DNMT1 (D) or histone H3 trimethylated on Lys-27 (H3K27me3) (E) on KARPAS-299 cells treated for 48h or not (PBS) with doxorubicin. Results are expressed as a ChIP enrichment relative to the untreated condition (PBS). ( F ) qRT-PCR analysis of the expression of pri-miR125b1 in KARPAS-299 cells treated or not (PBS) with doxorubicin. Actin mRNA was used as the internal control and the relative ratio of pri-miR125b1 expression was expressed as 2 –ΔΔCt relative to untreated cells. (Data represent means ± SEM (bars) from 3 independent experiments. * P < 0.05, ** P < 0.001, *** P < 0.0001; unpaired 2-tailed Student's t test.

Article Snippet: The human ALCL cell lines KARPAS-299 was obtained from DSMZ (German Collection of Microorganisms and Cell Culture) and COST was established in our laboratory.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Concentration Assay, Control