Yousaf Malik

Mouse Embryonic Fibroblast (MEF)

◦ TDwt (cells containing the TDAG 51 gene)

◦ TDko (cells lacking the TDAG 51 gene)

It is programmed cell death in which a series of biochemical events that lead to a variety of morphological changes such as changes to the cell membrane like the loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation (1-4). In this process the cell is able to dispose of cellular debris that would otherwise result from necrosis in order protect the surrounding from damage.

B, assessmentof cell death by lactate dehydrogenase(LDH) release into the medium, posttransfection(Hossain et al, JBC, 2003)

Causes ER Stress by blocking the ability of cell from pumping calcium into the endoplasmic reticulum

Low level of calcium prevent the folding chaperones from folding the proteins properly

Results in the production of pro-apoptotic genes like chop and TDAG 51 that trigger apoptosis

Cell Culture Techniques: Changing the media Passage the cells Cell counting (5.0 x 105 cells/mL) Preparing the Six Well Plate

◦ Plated and left over night to grow◦ Changed media to 1 % fbs 1 x Antibiotics

Treatment◦ Treated with DMSO (veh.), 1 µM Thapsigargin◦ Another well was treated with 1 % Triton X for 100

% cell death control

Gathering the Results: Lactate dehydrogenase (LDH) Assay

◦ Quantifies the amount of cell death in sample ◦ Chlorometric assay◦ Analysis sample under 492 nm of light to measure

the amount of cell death◦ Not a measure of programmed cell death

(apoptosis)

Western Blotting essential part of this experiment Verified that the TDAG (T-Cell Death

Associated Gene 51) 51 gene was not present in the Tdko cells

Allowed to measure GRP 78 (ER chaperone) CHOP (other pro-apoptotic ER stress response gene)

TDAG51

-actin

TD

AG

51 +

/+

TD

AG

51 -

/-

TDAG51 Expression in Mouse Embryonic TDAG51 Expression in Mouse Embryonic Fibroblast used for ExperimentsFibroblast used for Experiments

*

KDEL (GRP 78)

CHOP

KD

37

Β -Actin

1 2 3 4 5 6 7 8 9

1. TDko P20 0 Hrs

2. TDwt E3 P2 0 Hrs

3. TDko E2 P1

4. TDko P19 DMSO 18 Hrs

5. TDwt E3 P1 DMSO 18Hrs

6. TDwt E3 P1 1 µM Thp. 18 Hrs

7. TDko P19 1 µM Thp. 18 Hrs

8. TDwt E3 P1 1 µM Thp. 48 Hrs

9. Tdko 1 µM Thp. 48 Hrs

The results from this experiment suggest that TDAG 51 is part of the apoptotic mechanism. It is probably one of the trigger genes because the cells that lacked the gene were able to survive longer than the wild type cells.

How exactly does the tdko affect apoptosis? What key role does it play in the mechanism?

We found that the gene TDAG 51 is very important for ER Stress induced cell death at the 24 hour time point.

TDAG 51 is likely a pro-apoptotic gene.

Henderson Research CentreDr. Austin Dr. DickhoutDr. Hiam Al-BayatiHill Park Coop ProgramMr. Watts