sirna duplex solution  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

doi: 10.1371/journal.pone.0084611

Figure Lengend Snippet: ( A ) Westerns and Northerns were performed on cells before and after selection for DOX resistance. ( B ) Cells before and after DOX selection were stained with antibodies against TFPI1 (red) or DAPI for DNA (blue). Open arrows indicate perinuclear TFPI1 localization and closed arrows indicate nucleolar staining. ( C ) ELISA was used to detect thrombin protein in the spent media of MCF7 parental and DOX Sel cells. ( D ) Parental MCF7 cells were stained before and after a 48-hour treatment of 20 µM Argatroban, a direct thrombin inhibitor (DTI), with antibodies against thrombin and TFPI1. Cells were stained with DAPI to visualize nuclear DNA. E. Four parental and drug resistant sets of cancer cells (K562 leukemia cells, Colo201 colorectal cells, MCF7 breast cancer cells and C6/F98 rat glioma cells) were prepared for Western analyses using antibodies shown.

Article Snippet: For TFPI1 RNA silencing, siRNA duplex solutions (fluorescein-conjugated, scrambled siRNA control and TFPI1 siRNAs) were prepared by adding 50 nM of TFPI1 siRNA to the transfection reagent LipofectamineRNAiMax (Invitrogen).

Techniques: Selection, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: PLoS ONE

Article Title: TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

doi: 10.1371/journal.pone.0084611

Figure Lengend Snippet: ( A ) Parental and DOX selected MCF7 cells were treated with scrambled (S) or TFPI1 siRNA (+). A Western analysis using antibodies against TFPI1 show that silencing of TFPI1 was effective. eGFP was transfected along with the siRNA constructs and show that transfection efficiency was consistent. αTubulin was used as a load control. ( B ) Following 24 hours of siRNA treatment in DOX Sel cells, 1 µM DOX was added for 48 hours. MTT was performed to determine cell killing. ( C ) Parental MCF7 cells were treated with 1 µM DOX for 48 hours, then maintained in 100 nM DOX for an additional 3 days. Protein samples were prepared every 24 hours and analyzed by Western blotting with the antibodies shown. ( D ) Parental MCF7 cells were incubated in 100 nM DOX for 5 days with samples removed every 24 hours for Western blotting with the antibodies indicated.

Article Snippet: For TFPI1 RNA silencing, siRNA duplex solutions (fluorescein-conjugated, scrambled siRNA control and TFPI1 siRNAs) were prepared by adding 50 nM of TFPI1 siRNA to the transfection reagent LipofectamineRNAiMax (Invitrogen).

Techniques: Western Blot, Transfection, Construct, Incubation

Journal: PLoS ONE

Article Title: TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

doi: 10.1371/journal.pone.0084611

Figure Lengend Snippet: ( A ) Parental MCF7 cells were transfected with an empty vector construct, or a construct overexpressing TFPI1. After 24 hours, the cells were harvested and prepared for protein analyses using the antibodies shown. ( B ) Cells were transfected with a TFPI1 expressing vector or the empty vector, and left for 24 hours. Next, the cells were treated with 1 µM DOX for an additional 24 hours. MTT was performed to determine cell killing. The MTT assay was done in triplicate with the standard error of the mean indicated. ( C ) The lysates used above were used to assess levels of the proteins shown. ( D ) A schematic representation of a possible model for how DOX exposure leads to DOX resistance. Increased TFPI1 protein could be p53-dependent (see ), while elevated HIF1α protein could be through HIF1α stabilization.

Article Snippet: For TFPI1 RNA silencing, siRNA duplex solutions (fluorescein-conjugated, scrambled siRNA control and TFPI1 siRNAs) were prepared by adding 50 nM of TFPI1 siRNA to the transfection reagent LipofectamineRNAiMax (Invitrogen).

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, MTT Assay