Transcript LDH - hwdsb

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, assessment
of 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 -/-
TDAG51 +/+
TDAG51 Expression in Mouse Embryonic
Fibroblast used for Experiments
TDAG51
b-actin
Series1
70
60
*
% Cell Death
50
40
30
20
10
0
Wild type veh
Wild type 1uM Thap
tdko veh
tdko 1uM Thap
Series1
250
*
Percentage (%)
200
150
100
50
0
Wild type veh
Wild type 1uM Thap
tdko veh
tdko 1uM Thap
Series1
180
160
140
% Cell Death
120
100
80
60
40
20
0
Wild type veh
Wild type 1uM Thap
tdko veh
tdko 1uM Thap
Series1
400.00
*
350.00
300.00
Percentage (%)
250.00
*
200.00
150.00
100.00
50.00
0.00
Wild type veh
Wild type 1uM Thap
tdko veh
tdko 1uM Thap
1. TDko P20 0 Hrs
KD
1
2 3
4
5
6
7
8
2. TDwt E3 P2 0 Hrs
9
3.
KDEL
(GRP 78) 4.
TDko E2 P1
TDko P19 DMSO 18 Hrs
5. TDwt E3 P1 DMSO 18
Hrs
CHOP
37
Β -Actin
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
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Series1
tdko tdwt tdko tdko tdwt tdwt tdko
Un Un Un veh veh 1uM 1uM
E2 18h 18h Thap Thap
18h 18h
tdwt
1uM
Thap
48h
GRP78/Beta Actin
3.5
3
O.D. ratio
O.D. ratio
CHOP/Beta Actin
2.5
2
Series1
1.5
1
0.5
0
tdko Un tdwt Un tdko Un tdko
E2
veh
18h
tdwt
veh
18h
tdwt
1uM
Thap
18h
tdko
1uM
Thap
18h
tdwt
1uM
Thap
48h
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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?
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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
 Dr.
Research Centre
Austin
 Dr. Dickhout
 Dr. Hiam Al-Bayati
 Hill Park Coop Program
 Mr. Watts