To examine the function of the Scribble polarity

To examine the function of the Scribble polarity aenetwo shRNA seauences(shScrib1 and shScrib2) have been designed to knockdown Scribble expression. To test their effectiveness,along with a negative control(empty vector)and a positive control(shGFP)(which can also knockdown the Scribble-GFP fusion)they have been tested on HEK293 cells that stably express Scribble GFP In Panel B,Blue represents untransfected cellsRed represents transfected cells.

HEK293-Scribble-GFP cells Empty vector transfection
HEK293-Scribble-GFP cells shGFP vector transfection
HEK293-Scribble-GFP c

C.Which of the shRNA vectors targeted to Scribble(1 or 2)is most effective in knocking down Scribble? Is it as effective as the shRNAtargeting GFP?Why might this be the case?(1 Mark)

D.Does loss of expression of Scribble in the above experiment alter cell shape or cell size? Explain your rationale for the answer.(1 Mark)

E.If the various transfected cell populations above were sorted using the FSC and SSC gate and plated onto a glass slideand examined by fluorescence microscopyfor GFPwhat would be the main difference you would observe between the shGFP alone and the shScrib1 transfected cells in terms of GFP intensity?What chemical stain might you use to identifythe nucleus of each cell? How does it work?(1 Mark)

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