Question

Active (uM)	Cell count (% of control)	DNA content (% of control)	DNA fragmentation (% of control)	Caspase-3 activity (% of control)
0	100	100	100	100
1	80	80	200	200
10	50	50	350	350
100	20	20	500	500

1. Create a table and a graph for the effect of the active on DNA fragmentation, give a title that includes whether the active is beneficial or harmful for cancer management/treatment (use Table’s column 4/DNA fragmentation info)

2. Create a table and a graph for the effect of the active on caspase-3 activity, give a title that includes whether the active is beneficial or harmful for cancer management/treatment (use Table’s column 5/ caspase-3 activity info)

3. Section the following procedure for DNA fragmentation into (i) cell lysis buffer (lyses cells), (ii) proteinase K digestion (iii) DNA precipitation and (iv) gel electrophoresis. Illustrate how a gel would look based on column 4 results

Answer 1

DNA fragmentation 100 200 OuM active (Control) 1uM active 10 UM active 100 UM active 350 500 DNA fragmentation % DNA fragmentation compared to control (a.u.) OUM active (Control) UM active 10 M active 100 M active 100 Caspase-3 activity OuM active (Control) 1uM active 200 10 uM active 350 100 UM active 500 Caspase-3 activity % Caspase-3 activity to control (a.u.) OUM active (Control) UM active 10 M active 100 M active

DNA fragmentation and Caspase-3 activity is well known and frequently used method in programmed cell death also known as apoptosis. Since here we are talking about cancer cells so active is beneficial in both cases that is for DNA fragmentation as well as caspase-3 assay. We can clearly evident the effectiveness of the compound in dose dependent manner, a significantly increased DNA fragmentation can be seen in DNA fragmentation as well as in caspase-3 activity, around 5-fold increase can be observed in 500uM active treated cells as compared to control cells (untreated cells).

For DNA fragmentation experiment, first of all extract the DNA from untreated and treated cells through cell lysis, treated with proteinase K, purified DNA samples and run them over 2% (or 1 to 1.5%) agarose gel along with DNA marker, So fragmented DNA can be seen in the form of look like ladder on to the gel which can be visualized in EtBr stained gel under UV light. DNA fragmentation experiment is generally also called as DNA laddering experiment.

Note: Please note that, since this compound is causing damage to the cells so less cells viabity can be seen in dose dependent manner and also fewer cells as compared to the control (assume 100%).