MCD BIO 165A Lecture 9: Discussion Quiz 3

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Molecular, Cell, and Developmental Biology
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1. A) According to Figure 1B, how does cell density affect proliferation? (1 point) The denser the population of cells, the less proliferation is observed. B) How does knock down of YAPTAZ affect the relationship between cell density and proliferation? (1 point) Knocking down YAPTAZ in the sparsely and confluent plated cells essentially eliminated the cells response to crowding, suggesting that the proliferation due to having room is due to the activation of the YAZTAZ pathway. However, in the densely plated cells there is minimal proliferation before and after YAPTAZ knock down, which could mean that proliferation is not solely dependent on the YAPTAZ pathway. C) How does the data in Figure 1C relate to the findings in Figure 1B? (1 point) The data in Figure 1C shows that when the cells are sparsely plated YAPTAZ are predominantly found in the nucleus. Then, as the cell cultures get denser, there is more YAPTAZ localization in the cytoplasm. This relates to Figure 1B because when YAPTAZ localize in the nucleus they can affect transcription and increase proliferation. In the cytoplasm, YAPTAZ are not affecting transcription factors and are not increasing proliferation. 2. A) Figure 3 depicts an siRNA screen to find factors regulating YAPTAZ activity. What was the assay used to measure YAPTAZ activity in this screen? (1 point) After screening genes for genes important for YAPTAZ mechanical regulation they used a mechanotransduction assay. Then, after the transfected MECs were put on soft hydrogels for 48 hours they were analyzed by quantitative PCR and CTGF mRNA expression was used to measure YAPTAZ activity. The type of PCR used is RT PCR, which utilizes the ability to make DNA from RNA. To do this they take mRNA and use an enzyme to convert it to DNA and then use the DNA in PCR to see how many transcripts of each gene were being expressed. B) Briefly describe another assay that could have been used to measure YAPTAZ activity in this screen. (2 point) You could use a ligandbinding assay in which you mark the negative regulators with ligand molecules. 3. How does knocking down CapZ, Cfl, or Gls affect the stability of the YAP and TAZ proteins? What data (which figure) in this paper most directly addresses this issue? (2 points)
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