Within the paper, we use numerous means to conclusively demonstrate that Six1 induces a TIC phenotype through both TGF-β and ERK signaling, including examination of surface markers, tumorsphere assays, and in vivo tumor initiating assays.
Interestingly, when cells that had escaped Notch inhibition in the primary assay were reexamined in serial CSC assays, no difference was seen in the secondary tumor-initiating assay, suggesting again the emergence or coexistence of a Notch-independent CSC population.
Abortive initiation assays were initiated using specific dinucleotide primers in the presence of only the next α-P-NTP (for AP3, 250 µM GpU + 50 µM α-P-UTP; for 23S, 250 µM UpG + 50 µM α-P-CTP, see Materials and methods).
To initiate the assay, 20 µl of 0.15 M cupric acetate was added.
The liposome suspension was then sonicated for 2 min. To initiate the assay, 20 μL of 0.15 M cupric acetate were added.
Luciferase readings in untreated and control vector transfected cells were normalized to 1. Experiments were initiated to assay roles of IKKα and IKKβ in inducing IκBα phosphorylation and activation of NF-κB in response to a well-studied NF-κB inducer, TNFα.
Using human-focused molecular initiating event assays to predict higher-level effects across species.
To initiate the assay, MTT reagent was added and cells were incubated for an additional 4 h at 37°C.
The CVC as part of the Sabin Vaccine Institute initiated an Assay Working Group, which has conducted two large-scale ELISPOT proficiency panels over the last 2 years with access for laboratories world-wide.
The plate was preincubated with a lid at 37°C; then, AAPH (50 μL, 160 mM in PBS) was added to initiate the assay and incubated at 37°C in the incubator with shaking.
We initiated the assay by adding xanthine oxidase and monitored it by measuring the change in absorbance at 560 nm in a Lambda-20 ultraviolet/visible spectrophotometer (Perkin Elmer, Waltham, MA, USA).
