results

In human hepatic cells (HepG2), PTP1B expression was silenced by using small interfering RNA (siRNA), in order to be compared with control hepatocytes. They were treated with Thapsigargin (10 μL from stock  1μΜ into 2 ml  media, experiment 1) and Tunicamycin (10 μL from stock 5 mg/mL into 2 ml  media, experiment 2) in order to cause them ER stress . They were cultured in DMEM media (10% Fetal Bovine Syrom, 1% Pen/Strep). They were collected after 0.5h, 1h, 1.5h, 2h, 4h, 6h and 8h, and they were lysed. By using Western Blotting technique the following results were extracted.

Results and Outcomes

Experiment 1: ER stress caused by Thapsigargin on human HepG2 cells +/- PTP1B

Figure 1: the first experiment was conducted for n=2 but due to implications to western blotting it was analysed for n=1. This figure shows that there is an increase in PTP1B levels with ER stress in control cells (CTL siRNA) after the 6^th hour. PTP1B siRNA seems to prevent this increase.

Phosphorylation of eIF2-alpha peaked ~2h of ER stress induction and then reduced to normal levels by 8h. BIP expression levels increased more slowly, peaking at 8h. No significant differences were seen with PTP1B siRNA in this experiment.

Experiment 2: ER stress caused by Tunicamycin on human HepG2 cells +/- PTP1B

Figure 2: in order to support the results of experiment 1 the same procedure was followed, but this time the design was changed for practicing reasons. Each sample was loaded in duplicates. This figure shows that PTP1B siRNA decreases PTP1B levels inconsistently. Also it was concluded that Tunicamycin did not work as well as Thapsigargin in terms of causing ER stress in this experiment.

 Unfortunately, there was not enough time to repeat the experiments, and due to difficulties with western blotting I was not able to present significant results.  However, some conclusions can be extracted. ER stress agents thapsigargin and tunicamycin cause phosphorylation of eIF2-alpha and increase expression of Bip in a time dependant manner. SiRNA technology was inconsistent in knocking down PTP1B levels in HepG2 cells. In order to improve this outcome of this short-time project in the future we should repeat the experiments, change the concentrations of primary and secondary antibodies and finally investigate the role of PTP1B in association with the other ER stress factors, e.g. eIF2a, ATF4, BIP, GADD34.

Thanks to British Biochemical Society and Dr Mody, I had the opportunity to practice my techniques and to apply my knowledge acquired from the taught lectures, as well as to learn new methods and to get involved with the latest technology. Now I feel much more confident in the laboratory. I learned how to extract, analyse and interpret  data, how to efficiently cooperate within a laboratory team, and how to overcome the everyday’s difficulties and frustrations that research reserves, motivating myself to work harder. Until having this opportunity, research was only one of my many career aspirations. Now, after finishing this studentship, I feel that my future belongs to research!

Thank you very much

Petros