Journal:

Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells

doi: 10.1073/pnas.251383598

Figure Lengend Snippet: Allele discrimination. A Molt-4 cell in which a single nucleotide (G to A) difference in two alleles of the Tp53 gene was detected by RCA in situ. Two probes were used, which differed in that the 3′ termini of the normal and the mutant versions were complementary to the normal (green signal) and the mutant (red signal) sequences, respectively.

Article Snippet: Molt-4 cells are reported by ATCC to express no normal Tp53 and to have one normal and one or more abnormal copies of the Tp53 gene, in which there is a G→A transition in codon 248 of exon 7.

Techniques: In Situ, Mutagenesis

Journal:

Article Title: Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells

doi: 10.1073/pnas.251383598

Figure Lengend Snippet: mRNA detection. Normal HLB cells (A) and Molt-4 cells (B) stained with AO, which labels single-stranded nucleic acid (RNA) red and double-stranded nucleic acid (nuclear DNA) yellow. (A and B) The presence of RNA in each of the cell types. RCA was performed on replicate cell preparations using a probe with 3′ and 5′ DNA binding sites complementary to the probe described in Fig. ​Fig.3.3. (C) The green fluorescence signal surrounding the nuclei of the HLB cells demonstrates the presence of Tp53 transcript detected by RCA. (D) No such signal is seen in the Molt-4 cells, demonstrating the lack of normal Tp53 transcript.

Article Snippet: Molt-4 cells are reported by ATCC to express no normal Tp53 and to have one normal and one or more abnormal copies of the Tp53 gene, in which there is a G→A transition in codon 248 of exon 7.

Techniques: Staining, Binding Assay, Fluorescence

Journal: Oncotarget

Article Title: Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

doi:

Figure Lengend Snippet: (A) Flow cytometric analysis of Annexin-V-FITC/PI stained Molt-4 cells treated with SKi (6.9 μM) for 6, 16, 24 and 40 h documented a time-dependent increase in apoptotic cells with respect to untreated cells. (B) After SKi treatment, cells were collected, lysed and analyzed by western blotting for cleaved caspase-9, -3, and -8. (C) Molt-4 cells were treated with SKi (6.9 μM) for the indicated time, then processed for TEM analysis that documented apoptosis induction. Arrows point to condensed apoptotic chromatin. Scale bar 2 μm. (D) Western blot analysis for the autophagic marker LC3 and p-mTOR (Ser2448) in cells treated with SKi for the indicated times. (E) Western blot analysis for SK1 expression in T-ALL cell lines upon SKi treatment for 40 h. In (B) (D) and (E) 50 μg of protein was loaded for each lane. β-tubulin was used as a loading control.

Article Snippet: The T-ALL cell lines Molt-4, Jurkat, CEM-R (CEM VBL100, drug-resistant cells overexpressing 170-kDa P-glycoprotein [ ], and CEM-S were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Techniques: Staining, Western Blot, Marker, Expressing

Journal: Oncotarget

Article Title: Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

doi:

Figure Lengend Snippet: and stress response. (A) Molt-4, Jurkat and CEM-R cells were treated with SKi for 40 h. Then, cells were collected, lysed, and analyzed by western blotting. Protein (50 μg) was loaded in each lane.β-tubulin was used as a loading control. (B-C) Primary cells from T-ALL patients were treated with SKi (10 μM) for 24 h, stained with various antibodies or Annexin-V/PI and subjected to cytometric analysis.

Article Snippet: The T-ALL cell lines Molt-4, Jurkat, CEM-R (CEM VBL100, drug-resistant cells overexpressing 170-kDa P-glycoprotein [ ], and CEM-S were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Techniques: Western Blot, Staining

Journal: Oncotarget

Article Title: Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

doi:

Figure Lengend Snippet: MTT assays of Molt-4 and Jurkat cells treated for 40 h with increasing concentrations of SKi and/or vincristine (VCR). The combined treatment resulted in strong synergism (CI < 0.3). Data represent the mean of at least three independent experiments ± s.d.

Article Snippet: The T-ALL cell lines Molt-4, Jurkat, CEM-R (CEM VBL100, drug-resistant cells overexpressing 170-kDa P-glycoprotein [ ], and CEM-S were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Techniques:

Journal: Oncotarget

Article Title: Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

doi:

Figure Lengend Snippet: (A) MTT assays of Molt-4, Jurkat, CEM-R, and CEM-S cells treated with increasing concentrations of ROMe for 40 h. The results are the mean of three different experiments ± s.d. The table shows IC 50 values of each cell line. (B) Western blot analysis documented that incubation with ROMe for 4, 6, 24, and 40 h activated caspases only after very short times of drug incubation, and a sustained autophagy in Molt-4, Jurkat and CEM-R cell lines. Cells were treated with ROMe at a concentration which corresponds to the IC 50. Protein (50 μg) was loaded for each lane.β-tubulin was used as a loading control. (C) TEM analysis of CEM-R cells documented the presence of large cytoplasmic vacuoles containing various degraded organelles (arrow). Scale bar 2 μm.

Article Snippet: The T-ALL cell lines Molt-4, Jurkat, CEM-R (CEM VBL100, drug-resistant cells overexpressing 170-kDa P-glycoprotein [ ], and CEM-S were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Techniques: Western Blot, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

doi:

Figure Lengend Snippet: (A) Inhibition of autophagy by chloroquine (Chlor) (20 μM), bafilomycin A1 (baf A1) (5 nM), or 3-MA (200 μM) impairs the cytotoxic effect of ROMe (40 h). Inhibition of apoptosis through Z-VAD-FMK (20 μM) does not affect ROMe-induced cell death. Cell viability was assessed by MTT assay on Molt-4, Jurkat, and CEM-R cells. (B) Cells were treated with ROMe for 4, 6, 24 and 40 h, collected, lysed and subjected to western blotting for ER stress/UPR markers. Protein (50 μg) was loaded for each lane.β-tubulin was used as a loading control. (C) Molt-4, Jurkat and CEM-R were treated with ROMe for 40 h and western blot analysis was performed. ROMe does not interfere with the analyzed signaling pathways, with the exception of a down-regulation of Raf/MEK/ERK in Jurkat cells.

Article Snippet: The T-ALL cell lines Molt-4, Jurkat, CEM-R (CEM VBL100, drug-resistant cells overexpressing 170-kDa P-glycoprotein [ ], and CEM-S were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Techniques: Inhibition, MTT Assay, Western Blot