Cell line A was cultured in the absence and presence of KB9520 or PPT. You then lyse the cells and fractionate them, generating membrane, cytosolic and nuclear protein fractions of the whole cell lysate. Western blots were then run using antibodies against ER (alpha) and ER (beta) for each treatment, and include different lanes for the membrane, cytosolic and nuclear protein fractions.  Draw the expected results for these western blots and briefly discuss the expected diffe

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Chapter1: The Human Body: An Orientation
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Cell line A was cultured in the absence and presence of KB9520 or PPT. You then lyse the cells and fractionate them, generating membrane, cytosolic and nuclear protein fractions of the whole cell lysate. Western blots were then run using antibodies against ER (alpha) and ER (beta) for each treatment, and include different lanes for the membrane, cytosolic and nuclear protein fractions. 

Draw the expected results for these western blots and briefly discuss the expected differences. 

•This question relates to the selectivity of different drugs for the treatment of Ovarian Cancer.
The expression of the two Estrogen Receptors subtypes, ERa and ERß, was investigated in two
human ovarian cancer cell lines, A and B, using Western Blotting of the whole cell fraction (Figure 1).
The inhibitory effect on the cellular growth of the natural hormone estradiol (E2) and two different drugs (KB9520 and PPT) was studied
for both cell lines using a Trypan Blue assay. Figure 2 shows a plot of the inhibition of cell proliferation versus control of the compounds
E2, KB9520 and PPT at increasing concentrations. A full agonist is expected to exert 100% inhibition of the cell proliferation for both cell
lines
A B
ERa
ERB
Tubulin
Figure 1 - Western Blot
% inhibition versus control
60
50
40
30
20
10
0
-10
-20
-30
-40
-50
Cell line A
с 0,1 nM
1 nM 5 nM 10 nM
KB9520 E2 OPPT
% inhibition versus control
60
50
40
30
20
10
0
-10
C
Cell line B
0,1 nM
1 nM
■KB9520
10 nM
5 nM
E2 OPPT
Figure 2 - Inhibition of cell proliferation for cell lines A and B under treatment with KB9520, E2 or PPT
Transcribed Image Text:•This question relates to the selectivity of different drugs for the treatment of Ovarian Cancer. The expression of the two Estrogen Receptors subtypes, ERa and ERß, was investigated in two human ovarian cancer cell lines, A and B, using Western Blotting of the whole cell fraction (Figure 1). The inhibitory effect on the cellular growth of the natural hormone estradiol (E2) and two different drugs (KB9520 and PPT) was studied for both cell lines using a Trypan Blue assay. Figure 2 shows a plot of the inhibition of cell proliferation versus control of the compounds E2, KB9520 and PPT at increasing concentrations. A full agonist is expected to exert 100% inhibition of the cell proliferation for both cell lines A B ERa ERB Tubulin Figure 1 - Western Blot % inhibition versus control 60 50 40 30 20 10 0 -10 -20 -30 -40 -50 Cell line A с 0,1 nM 1 nM 5 nM 10 nM KB9520 E2 OPPT % inhibition versus control 60 50 40 30 20 10 0 -10 C Cell line B 0,1 nM 1 nM ■KB9520 10 nM 5 nM E2 OPPT Figure 2 - Inhibition of cell proliferation for cell lines A and B under treatment with KB9520, E2 or PPT
ERa
ERB
no treatment
MCN
+ KB9520
MCN
+PPT
MCN
Transcribed Image Text:ERa ERB no treatment MCN + KB9520 MCN +PPT MCN
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