Week 8 – Plot Twist!

Anita M -

Hello everybody,

This week we ran our qPCR to measure gene expression. After analyzing our data we noticed something off…

Just as a quick reminder, previous literature has proved that ATF6 is activated in response to ER Stress. Tunicamycin (TM) causes ER Stress in Cells. Therefore, cells treated with TM should have increased expression of ATF6.

However, when we look our data, we noticed that in both the GRASP55 and control groups, the TM treated cells had significantly lower gene expression than the control group. Here is a picture for reference:

Just to help make some things clear:
— DMSO is a control treatment, TM is an experimental treatment
— The absolute control of this experiment is the first bar. It has no GRASP55 overexpression and no TM treatment. Therefore, every other bar is compared to the first bar
— the * symbol indicates a significant difference between groups
— The two short bars you see are the TM treated ones
Now, here’s the graph of the second gene, GRP78, expression
Notice a pattern?
The TM treated cells are significantly lower in both cases.
Now what can we conclude from this?
Well, I want to remind you of the relation between ER stress and ATF6. ER stress (aka a TM treatment) should increase ATF6 gene expression. We know that certainly. Look at the red bar in the first image and compare it to the brown bar. It is significantly lower. This means we can’t really conclude much from our cells that had the TM treatment AND overexpression of GRASP55 (purple bar) because our control TM treatment (red bar) did not act the way we expected it too. This means we cannot reliably compare our data and draw a certain conclusion.
However, we can hypothesize what went wrong. We suspect the TM dose was too high and/or left on for too long. To mitigate this in the future, we would run a dose curve to determine what the ideal concentration of TM is. Unfortunately, that would take another two weeks and re-running the experiment afterwards would exceed the timeframe of the senior projects. I do look forward to potentially continuing this research after the conclusion of these 10 weeks, but for now we are still left in a bit of suspense.

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    Anshul Baddi
    Hey Anita, I love what you got going on. Given that the expected increase in ATF6 expression under ER stress was not observed, how might you refine the experimental conditions—such as TM concentration or exposure time—to ensure a more physiologically relevant response while staying within your project’s timeframe?
    April 2, 2025 at 3:34 pm - Reply
    aashi_h
    Interesting research, Anita! I wonder why the ATF6 did not perform as expected. What do you think are the future implications of this result?
    April 3, 2025 at 6:34 pm - Reply
    anita_m
    Hey Aashi, Thanks for asking! I think the major reason for ATF6 not performing as expected was the TM dosage being too high and/or being left on the cells for too long. Therefore, I wouldn't necessarily say this has any implications on ATF6 gene expression itself, but it does imply that when cells are under prolonged stress, they end up dying.
    April 9, 2025 at 10:13 am - Reply
    anita_m
    Hi Anshul, Great question! I would prioritize modify concentration rather than modifying time just because it would be a lor easier and quicker. So, I would run a TM dose response curve, which is essentially treating our HEK cells with different concentrations of TM to see at which concentration TM begins to cause ER stress without actually killing the cells. And each concentration of TM would be left on the cells for the same amount of time (let's say 24 hours). Then I would repeat my experiment and use the determined ideal dose of TM. Unfortunately, bringing up cells and running a dose response curve would take another week. Then on top of that, my experiment would take another 2 weeks, so we can't run the experiment again. However in hindsight, I would've run the TM dosage curve within the first few weeks of my project, which would have helped with achieving results within the project's timeframe.
    April 9, 2025 at 10:26 am - Reply

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