human mcl 1 expression vector  (OriGene)

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Mcl-1 protein expression in the lineage-related ARCaP E and ARCaP M cells. (B) Ectopic expression of Mcl-1 in ARCaP M cells and the effects on docetaxel cytotoxicity in ARCaP M cells. pCMV: vector control. (C) Left panel: The dose- and time-dependent effects of PDGF-BB on Mcl-1 mRNA expression in ARCaP M cells; middle panel: qRT-PCR analysis of Mcl-1 mRNA expression in response to PDGF-BB treatment in ARCaP M cells (24 h); right panel: The effects of PDGF-BB treatment (20ng/ml, 72 h) on Mcl-1 protein expression in ARCaP M cells. ImageJ was used to quantitate the relative expression of Mcl-1 protein. (D) The effects of exogenous PDGF-BB on the cytotoxicity of docetaxel in ARCaP M cells, as determined by the MTS assay.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, MTS Assay

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Expression profile of PDGFR signaling components in PCa cells, as analyzed by RT-PCR and Western blotting. (B) The effects of PDGF-BB (20 ng/ml) on the phosphorylation of PDGFR-α and -β in ARCaP M cells. (C) The effects of depleting PDGFR-α or/and -β on Mcl-1 protein expression in ARCaP M cells. The cells were transfected with either isotype-specific siRNAs targeting PDGFR-α (left panel, 30 nM) or PDGFR- β (central panel, 100 nM), or a mixture of PDGFR-α and -β siRNAs (right panel) for 48 h, serum-starved overnight, and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 72 h. (D) Upper panel: The time-dependent effects of AG-17 (100 nM) on Mcl-1 mRNA expression in ARCaP M cells; bottom panel: The effects of AG-17 treatment on the expression of Mcl-1 and cleaved PARP in the presence (20 ng/ml) or absence of PDGF-BB (20 ng/ml) in ARCaP M cells. (E) The effects of AG-17 treatment (100 nM, 72 h) on the viability of ARCaP E and ARCaP M cells.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Incubation

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Expression profile of β-catenin-TCF signaling components in PCa cells. (B) TCF reporter activity in the LNCaP-C4-2 and ARCaP E -ARCaP M cells. (C) Upper panel: The effects of PDGF-BB (20 ng/ml) on the nuclear translocation of β-catenin in ARCaP M cells; Bottom panel: The effects of PDGF-BB (20 ng/ml) on TCF reporter activity in the presence (100 nM) or absence of AG-17. (D) The effects of ectopic expression of β-catenin (72 h) on Mcl-1 expression at both mRNA and protein levels. (E) The effects of β-catenin depletion on PDGF-BB regulation of Mcl-1 expression in ARCaP M cells. The cells were transfected with β-catenin siRNA or control siRNA (30 nM) for 48 h, serum-starved overnight, and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 72 h. (F) The effects of β-catenin depletion on Mcl-1 reporter activity in ARCaP M cells. The cells were transfected with β-catenin or control siRNA (30 nM) for 48 h, and further transfected with a human Mcl-1 reporter for 24 h. Following serum starvation overnight, the cells were incubated in the presence or absence of PDGF-BB (20 ng/ml) for 48 h.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Activity Assay, Translocation Assay, Transfection, Incubation

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Expression of c-Abl and p68 in PCa cells. (B) Left panel: The effects of PDGF-BB (20 ng/ml) on the phosphorylation of c-Abl; middle panel: The effects of PDGF-BB (20 ng/ml) on the phosphorylation of p68 and the expression of β-catenin in the p68 immunoprecipitates in ARCaP M cells. Phosphorylation of p68 on the tyrosine sites was detected using a pan-phosphorylated-Tyr antibody; right panel: The effects of PDGF-BB (20 ng/ml) on the expression of p68 in the β-catenin immunoprecipitates in ARCaP M cells. (C) The effects of PDGF-BB (20 ng/ml) on the nuclear translocation of p68 in ARCaP M cells. (D) Confocal microscopy analysis of the effects of PDGF-BB on the co-localization of β-catenin and p68 in the nucleus in a time course experiment in ARCaP M cells. (E) The effects of p68 depletion on PDGF regulation of Mcl-1 in ARCaP M cells. The cells were transfected with p68 or control siRNA (30 nM) for 48 h, serum-starved overnight, and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 24 h (upper panel) or 72 h (bottom panel). Upper panel: RT-PCR analysis of mRNA expression of p68 and Mcl-1; bottom panel: Western blot analysis of protein expression of p68, β-catenin and Mcl-1. (F) The effects of p68 depletion on Mcl-1 reporter activity in ARCaP M cells. The cells were transfected with p68 or control siRNA (30 nM) for 48 h, and further transfected with human Mcl-1 reporter for 24 h. Following serum starvation overnight, the cells were incubated in the presence or absence of PDGF-BB (20 ng/ml) for 48 h.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Translocation Assay, Confocal Microscopy, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) The effects of HIF-1α depletion on Mcl-1 expression in ARCaP M cells. The cells were transfected with HIF-1α or control siRNA (30 nM) for 72 h, and analyzed for Mcl-1 expression by immunoblotting. (B) Western blot analysis of the effects of PDGF-BB (20 ng/ml) on the nuclear translocation of β-catenin and HIF-1α in ARCaP M cells. (C) Co-immunoprecipitation assays of the effects of PDGF-BB (20 ng/ml) on the interaction between β-catenin and HIF-1α in the nucleus in ARCaP M cells. (D) Confocal microscopy of the effects of PDGF-BB (20 ng/ml) on the co-localization of β-catenin and HIF-1α in the nucleus in ARCaP M cells.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Transfection, Western Blot, Translocation Assay, Immunoprecipitation, Confocal Microscopy

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) The effects of PDGF-BB on the HIF-1 reporter activity in ARCaP M cells. The cells were transiently transfected with HIF-1 reporter or pGL3 for 24 h, serum-starved and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 48 h. (B) Schematic diagram of human Mcl-1 promoter and its deletion mutation at the putative HRE site. (C) The effects of deleting the putative HRE site on PDGF regulation of Mcl-1 promoter activity in ARCaP M cells. IL-6 (200 ng/ml) was included as the positive control. (D) ChIP-Re-ChIP assay of the effects of PDGF-BB treatment (20 ng/ml) on the binding of β-catenin and HIF-1α to human Mcl-1 promoter region in ARCaP M cells.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Activity Assay, Transfection, Incubation, Mutagenesis, Positive Control, Binding Assay

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: The engagement of PDGF-BB to PDGFR dimers activates the c-Abl-p68 cascade, which subsequently stablizes β-catenin and promotes its nuclear translocation. In the nucleus, interaction between β-catenin and HIF-1α increases the binding of HIF-1α to the HRE site within Mcl-1 promoter, thereby activating the transcription of Mcl-1 gene. Upregulation of Mcl-1 antagonizes apoptotic signals and confers survival advantages to metastatic PCa cells. Furthermore, tumor-derived and locally expressed PDGF may mediate the interactions between PCa and bone microenvironment. Co-targeting the PDGF signaling in PCa cells (autocrine) and microenvironment (paracrine) could provide a new strategy to disrupt the “vicious cycle” and efficaciously treat metastatic PCa.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Translocation Assay, Binding Assay, Derivative Assay