Question

For each of the following, indicate what experimental approach you would use to answer the given scenarios (a-e). You must include the following:

1. Experimental approach

2. General outline of procedure for that approach. That is, an overview of how the protocol is conducted, including critical reagents or steps.

3. Expected results, including any specific marker proteins associated with the assay that would change or vary.

Here is an example:

Protein Z is important in the migration of prostate cancer cells.

1. Migration scratch assay

2. Transfect siRNA for Protein Z and control siRNA into prostate cancer cells. Grow to confluence, generate scratch, photograph scratches at different time points, measure movement of cells into scratch. Determine relative rate of movement of cells over time.

3. Result: Knockdown of Protein Z reduces the rate of movement of cells

a. Protein X can cause cellular transformation when expressed in a normal lung cell line.

b. Tumor cells cause changes in cell surface marker protein B of associated immune cells (TAM’s or tumor associated macrophages).

c. Transcription Factor Y is critical for the EMT phenotype of breast cancer during progression.

d. High expression of Protein Q decreases patient survival.

e. A CAF (cancer associated fibroblast) can cause greater migration of breast tumor cells.

Answer 1

e. i) CAFs are lysed

ii) Cell debris are removed by centrifugation

iii) Take the supernanant

iv) seed brest cancer cells in two separate dishes.

V) at time of 1st media change in one culture CAF supernatant is mixed with normal DMEM media and in other normal DMEM media added.

V)after 24 hr scratch assays are done.

Vi) apart from strach assay matrix metalloproteinases (MMPs) and the urokinase-type plasminogen activator expression level can be checked by western blot or zymography

C. i) liposome transfection of transcription factor Y done on some epidermal cell line (Epidermal Keratinocytes).

ii) Through western blot cytokeratin, vimentin & phosphatidylinositol 3' kinase expression is checked

if EMT happens cytokeratin level decreases vimentin phosphatidylinositol 3' kinase level increases

a)

i) liposome transfection of Protein X in normal lung cell line.

ii) by western blot important cellular transformation markar is checked.