Journal: Cancer cell

Article Title: MAST1 drives cisplatin resistance in human cancers by rewiring cRaf independent MEK activation

doi: 10.1016/j.ccell.2018.06.012

Figure Lengend Snippet: (A) Cell viability, colony formation, apoptosis induction, and MEK1 phosphorylation in KB-3-1 and 212LN with vehicle control, lestaurtinib, cisplatin and the combination. (B) Lestaurtinib effect on cell viability and cisplatin sensitivity of TKO-002 cells. Combination index (CI) values for synergistic effect are shown. (C) Effect of lestaurtinib, cisplatin and the combination effect on tumor growth of cisR HNSCC, lung cancer, and ovarian cancer PDX mice. Pt-8 tumor and Pt-11 tumor in Figure 5C were used for lung cancer PDX and ovarian cancer PDX, respectively. Error bars represent SEM. Scale bars for the dissected tumors represent 5 mm. (D) Ki-67 expression was determined by IHC staining. Scale bars represent 50 μm. (E) MAST1 activity was assessed by MAST1 in vitro kinase assay using MBP as a substrate. (F) MEK1 inhibition by lestaurtinib and cisplatin combination in PDX tumor lysates. (G) Proposed model for the role of MAST1 in cisplatin resistance in human cancer. Left: Cancer cells rely on cRaf-dependent MEK1 activation to promote tumor growth in the absence of cisplatin. Right: Cisplatin treatment dissociates cRaf from MEK1, while MAST1 phosphorylates MEK1 to activate the MAPK pathway in cRaf-independent manner, inhibiting BIM and providing a proliferative advantage to cancer cells. Targeting MAST1 by lestaurtinib restores cisplatin sensitivity to cells. Data are mean ± SD from three technical replicates of each sample except panel (C). p values were determined by two-tailed Student’s t test (*: 0.01 < p < 0.05; **: p < 0.01). See also Figures S7 and S8.

Article Snippet: Ovarian cancer PDX donor mouse was obtained from Jackson Labs and serially transplanted to 6-week-old NOD scid gamma (NSG) mice.

Techniques: Phospho-proteomics, Control, Expressing, Immunohistochemistry, Activity Assay, In Vitro, Kinase Assay, Inhibition, Activation Assay, Two Tailed Test

Journal: Mobile DNA

Article Title: Dynamic silencing of somatic L1 retrotransposon insertions reflects the developmental and cellular contexts of their genomic integration

doi: 10.1186/s13100-017-0091-2

Figure Lengend Snippet: New L1 integrants in mouse embryonic stem (mES) cells undergo dense cytosine methylation. Dense cytosine methylation at new L1 integrants in mouse ES cells was revealed by bisulfite sequencing. Initially, Bruce 4 cells were transfected with pJH435, encoding an inactivated L1 ORFeus donor element marked with GFPuv-AI reporter regulated by a respiratory syncytial virus (RSV) promoter. Upon activation of L1 donor expression by transient infection of the culture using adenoviral Cre recombinase, individual colonies were picked and cultured on feeder cells for > 2 months. Of these mES subclones, most harbored newly retrotransposed L1 reporter integrants, as shown by a PCR-based assay documenting spliced integrated copies of the reporter GFPuv-AI (not shown). Their cytosine methylation status was assessed either in bulk or at individual loci using bisulfite sequencing. a For mES subclone 1B6-A07, we used primers DES3301 x DES3314, which anneal within the gene encoding GFPuv. This PCR amplicon does not cross the AI splice site, so unspliced donor L1 sequences also can be amplified. b For mES subclone 1B6-A08, primers DES3298 x DES3299 were used. c For mES clones 1B06/B02, 1C6 and 2D4, primers DES3321 x DES3322 were used to assay 15 CpG dinucleotides in a 234 nt amplicon across the GFPuv-AI splice junction

Article Snippet: Mouse ES cells including Bruce4 cells (provided by Dr. Colin Stewart, NCI; [ ]), and the resulting Truck_305 cells containing a mouse synthetic L1 ORFeus donor transgene (purchased from Ozgene [ ]), were grown in high-glucose DMEM, supplemented with 15% ES-cell-qualified FBS, 1% L-glutamine, 1% non-essential amino acids, 0.1 mM 2ME, 50 U/ml of P/S and ESGRO in 10% CO 2 .

Techniques: Methylation, Methylation Sequencing, Transfection, Virus, Activation Assay, Expressing, Infection, Cell Culture, Amplification, Clone Assay