Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Prostatic cell lines were grown in culture dishes for two days. (A) Representative immunoblot displaying MLH1 protein expression in DU145 and normal prostate epithelial PWR-1E and RWPE-1 cells. GAPDH was used as loading control. (B) Relative mRNA expression levels of MLH1 in cell lines as determined by real-time PCR. Expression levels are normalized to PWR-1E. Data are presented as mean±SEM of three experiments. Asterisks denote differences between the compared values: *P<0.05, **P<0.01.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: (A) Ectopic expression of MLH1. DU145 cells stably transfected with either MLH1 or empty vector (pCMV) along with mock (parental DU145 cells treated with transfection reagent alone) were grown for 48 hours and underwent Western analyses. GAPDH was used as loading control. (B) Cell proliferation as analyzed by the MTS cell proliferation assay 48 hours after plating cells. Results are expressed as % and normalized to pCMV control. (C) Cell migration as measured by wound healing assay. A wound was formed by scraping culture dishes using a pipet tip and closure measured after 24 hours. Left: Representative images of wound healing assay are shown. Right: Migration expressed as % closure of wound. (D) Cell invasiveness as measured using Matrigel. Cells were placed onto transwell membrane and allowed to invade for 24 hours. Left: Representative images of invading cells are shown. Right: Cell invasiveness as measured by absorbance (Abs) at 560 nm. Data are presented as mean±SEM of at least three experiments; **P<0.01 MLH1 versus pCMV.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Proliferation Assay, Migration, Wound Healing Assay

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Athymic nude mice were injected subcutaneously with stable MLH1 or pCMV-transfected DU145 cells and growth monitored over time. Left: Representative image of tumors in mice five weeks after injection of cells. Right: Growth of tumor size plotted over time. Data are presented as mean±SEM of five mice per group; *P<0.05 MLH1 versus pCMV for each time point.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Injection, Transfection

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Stable MLH1-expressing and vector control DU145 cells were grown over time. (A) Apoptosis as measured by flow cytometric analyses. Representative biparametric histogram showing cell population in early (bottom right quadrant) and late (top right quadrant) apoptotic, and viable (bottom left quadrant) states. Left: pCMV control, Middle: MLH1-expressing, Right: Total apoptosis %, Upper: 48 hours of growth, Lower: 72 hours of growth. Bar graph of total apoptosis % is presented as mean±SEM of three experiments, *P<0.05 MLH1 versus pCMV. (B) Protein levels of MLH1, cleaved PARP (cl-PARP), c-Abl, and p-c-Abl were determined by Western blot analyses after growing pCMV and MLH1 cells for 48 hours. GAPDH was used as loading control.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Expressing, Plasmid Preparation, Western Blot

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: Three MLH1 siRNAs (siMLH1s) were transfected individually along with a non-specific siRNA control (siControl) into stable MLH1-expressing DU145 cells for 48 hours. pCMV control cells treated with Lipofectamine are also included. (A) Protein levels of MLH1, c-Abl, and p-c-Abl in cells as determined by Western blot analyses. GAPDH was used as loading control. (B) Total apoptotic cells were analyzed by flow cytometry. Data are expressed as mean±SEM of three experiments; *P<0.05 **P<0.01 siMLH1s versus siControl.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Transfection, Expressing, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

doi:

Figure Lengend Snippet: MLH1-expressing and pCMV control DU145 transfectants were treated with either DMSO or STI571 and maintained for 48 hours. Total apoptotic cells were then analyzed by flow cytometry. Data are expressed as mean±SEM of three experiments; **P<0.01 MLH1+/STI571+ versus MLH1+/STI571-.

Article Snippet: DU145 cells were transfected with pCMV6-ENTRY vector expressing the C-terminally Myc and Flag-tagged human MLH1 cDNA as well as empty pCMV6-ENTRY vector as a control (OriGene Techologies, Rockville, MD) using X-treme Gene HD transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Techniques: Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer

doi:

Figure Lengend Snippet: The graphs indicate growth inhibition curves of OTSSP167 for various types of human cancel cell line; (A) A549 (lung cancer), (B) T47D (breast cancer), (C) DU4475 (breast cancer), and (D) 22Rv1 (prostate cancer) cells, in which MELK is highly expressed, as well as (E) HT1197 (bladder cancer) cell line, in which MELK expression is hardly detectable.

Article Snippet: DU145 cells were injected subcutaneously in the left flank of male BALB/cSLC-nu/nu mice (Japan SLC, Inc.).

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer

doi:

Figure Lengend Snippet: Nude mice bearing (A,B) MDA-MB-231 (triple-negative breast cancer), (C,D) A549 (lung cancer), (E) DU145 (prostate cancer), or (F) MIAPaCa-2 (pancreatic cancer) were treated with either vehicle control or OTSSP167 of given concentrations for 14 days. The administration doses were (A) 20 mg/kg intravenously once every two days or (B) 10 mg/kg orally once a day for MDA-MB-231; (C) 1, 5, or 10 mg/kg intravenously once a day or (D) 5 or 10 mg/kg orally once a day for A549; (E) 10 mg/kg orally once a day for DU145; and (F) 10 mg/kg orally once a day for MIAPaCa-2. Mean tumor volumes ± SD (n = 6 for each treatment group) are shown. (G) Lysates of tumor samples taken from A549 and PC-14 xenograft mice were immunoblotted with anti-MELK and anti-ACTB antibodies. (H) OTSSP167 was administered to nude mice bearing PC-14 (MELK-negative bladder cancer cells) at a dose of 10 mg/kg orally once a day. Mean tumor volumes ± SD (n = 3 per group) are shown. i.v. q.2d; intravenously once every two days, i.v. q.d.; intravenously once a day, p.o. q.d.; orally once a day.

Article Snippet: DU145 cells were injected subcutaneously in the left flank of male BALB/cSLC-nu/nu mice (Japan SLC, Inc.).

Techniques: