Heat shock factors (HSFs) regulate genes that protect cells from stress. This experiment studied the effect of the anti-inflammatory drug indomethacin on HSF1 and HSF2 in HeLa cells. Cells were treated with various concentrations of indomethacin and subjected to heat shock or normal temperatures. Nuclear extracts containing HSFs bound to radiolabeled DNA were run on a gel. The gel image shows that heat shock increased HSF binding (Band B) and indomethacin decreased HSF1 and HSF2 binding in a dose dependent manner (Band C). Antibodies added showed this effect was on HSF binding rather than expression levels.
Using your understanding of the method above answer the questions rel.pdf
1. Using your understanding of the method above answer the questions related to the experimental
data below. Heat shock factors (HSF) are transcription factors that induce genes endcoding for
proteins that protect against cellular stress. 2 HSFs (HSF-1 and HSF-2), recognize the same
binding site in DNA. The figure shows the result of an experiment in which the effect of an anti-
inflammatory drug, indomethacin, was studied. HeLa cells were cultured at 37C (lane 1[C,
control] and lanes 3-10), or at 42C (lane 2; HS, heat-shocked). The cultures for samples 3-10
were also treated with various concentrations of indomethacin that are indicated in the figure
(100, 250, 500, 1000 etc...) After treatment nuclear protein extracts were prepared and mixed
with 32-P radioactively labeled double stranded oligonucleotides (small DNA fragments)
containing the HSF binding site. Anti-HSF1 (samples 7 and 8) or anti HSF2 (samples 9 and 10 )
antibodies were also added to some of the samples. Note: You are not permitted to re-post in
another forum, distribute, or reproduce content from this course without the express written
permission of the instructor. Any violation of this course rule will be reported to the appropriate
university offices and officials, including to the Dean of Students as academic misconduct. See
IP Policy UA-05 for details.
The samples were subjected to a non-denaturing polyacrylamide gel and imaged. The data
obtained is shown to the right. a. What molecules are present in regions of gel corresponding to
A? (1 point) b. Describe the effect of heat shock on the cells (compare samples 1 and 2) (1.5
points). Hint: Think of what Band B represents. c. Describe the effect of indomethacin on the
cells (compare samples 1-6). (1.5pts) d. What was the aim of using the antibodies? What is the
role of HSF1 and HSF2 in the effect of indomethacin? Hint: think of what band C represents
(1.5) e. We cannot conclude from this experiment whether indomethacin affects the expression
of HSFs or the activity of HSFs. What technique could be used to identify whether HSF
expression is affected by indomethacin? (1.5pts)
![Using your understanding of the method above answer the questions related to the experimental
data below. Heat shock factors (HSF) are transcription factors that induce genes endcoding for
proteins that protect against cellular stress. 2 HSFs (HSF-1 and HSF-2), recognize the same
binding site in DNA. The figure shows the result of an experiment in which the effect of an anti-
inflammatory drug, indomethacin, was studied. HeLa cells were cultured at 37C (lane 1[C,
control] and lanes 3-10), or at 42C (lane 2; HS, heat-shocked). The cultures for samples 3-10
were also treated with various concentrations of indomethacin that are indicated in the figure
(100, 250, 500, 1000 etc...) After treatment nuclear protein extracts were prepared and mixed
with 32-P radioactively labeled double stranded oligonucleotides (small DNA fragments)
containing the HSF binding site. Anti-HSF1 (samples 7 and 8) or anti HSF2 (samples 9 and 10 )
antibodies were also added to some of the samples. Note: You are not permitted to re-post in
another forum, distribute, or reproduce content from this course without the express written
permission of the instructor. Any violation of this course rule will be reported to the appropriate
university offices and officials, including to the Dean of Students as academic misconduct. See
IP Policy UA-05 for details.
The samples were subjected to a non-denaturing polyacrylamide gel and imaged. The data
obtained is shown to the right. a. What molecules are present in regions of gel corresponding to
A? (1 point) b. Describe the effect of heat shock on the cells (compare samples 1 and 2) (1.5
points). Hint: Think of what Band B represents. c. Describe the effect of indomethacin on the
cells (compare samples 1-6). (1.5pts) d. What was the aim of using the antibodies? What is the
role of HSF1 and HSF2 in the effect of indomethacin? Hint: think of what band C represents
(1.5) e. We cannot conclude from this experiment whether indomethacin affects the expression
of HSFs or the activity of HSFs. What technique could be used to identify whether HSF
expression is affected by indomethacin? (1.5pts)](https://image.slidesharecdn.com/usingyourunderstandingofthemethodaboveanswerthequestionsrel-230331064924-08fbd60e/85/Using-your-understanding-of-the-method-above-answer-the-questions-rel-pdf-1-320.jpg)
