Background
Methods
Cell cultures
Scanning electron microscopy
Experimental set up
RNA extraction, purification and quality control
Target preparation and hybridisation
Data analysis
Real-time kinetic PCR
Caco-2 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | Common | Genbank | 1h | 12h | 24h | 48h | ||||||
RT-PCR | Affymetrix | RT-PCR | Affymetrix | RT-PCR | Affymetrix | RT-PCR | Affymetrix | |||||
matrix metalloproteinase 9* | MMP9 | J05070 | na | nd | no change | up | na | up | na | na | ||
aldo-keto reductase* | AKR1C3 | D17793 | na | nd | no change | up | up (2.8) | nd | na | na | ||
aquaporin 3* | AQP3 | N74607 | no change | down | up (6.8) | up | up (5.0) | up | na | na | ||
plasminogen activator, tissue* | PLAT | M15518 | na | nd | no change | nd | no change | nd | na | na | ||
HMG-box transcription factor 1* | HBP1 | AF019214 | no change | nd | up (2.6) | up | up (2.1) | nd | na | na | ||
Cox-2* | PTGS2 | U04636 | no change | nd | no change | nd | down (2.2) | down | na | na | ||
v-erb-b2* | ERBB3 | M34309 | na | nd | up (3.7) | up | no change | nd | na | na | ||
proline-rich nuclear receptor coactivator 1* | PNRC1 | U03105 | na | nd | no change | up | up (2.4) | nd | na | na | ||
inhibitor of differentiation* | ID1 | AA457158 | down (3.8) | down | na | nd | na | nd | na | nd | ||
E-cadherin** | CDH1 | Z13009 | na | nd | no change | nd | up | nd | no change | na | ||
hsp27** | HSPB1 | U90906 | na | nd | up | nd | up | nd | no change | na | ||
p53** | TP53 | U94788 | na | nd | up | nd | no change | nd | no change | na | ||
N-myc downstream regulated gene 1** | NDRG1 | D87953 | na | nd | up | nd | up | up | up | na | ||
NHEK
| ||||||||||||
30 min
|
3h
|
12h
|
24h
|
48h
| ||||||||
Gene
|
Common
|
Genbank
|
RT-PCR
|
Affymetrix 1 h
|
RT-PCR
|
Affymetrix
|
RT-PCR
|
Affymetrix
|
RT-PCR
|
Affymetrix
|
RT-PCR
|
Affymetrix
|
matrix metalloproteinase 9* | MMP9 | J05070 | no change | nd | no change | nd | na | up | na | up | up (11.1) | nd |
aldo-keto reductase* | AKR1C3 | D17793 | na | nd | down (4.0) | nd | na | nd | na | nd | down (5.3) | nd |
aquaporin 3* | AQP3 | N74607 | no change | nd | no change | nd | na | nd | na | up | up (2.8) | nd |
plasminogen activator, tissue* | PLAT | M15518 | no change | nd | no change | nd | na | nd | na | down | no change | nd |
HMG-box transcription factor 1* | HBP1 | AF019214 | down (6.3) | nd | down (4.0) | nd | na | up | na | nd | no change | nd |
Cox-2* | PTGS2 | U04636 | down (7.7) | nd | down (6.3) | nd | na | nd | na | down | down (8.3) | nd |
v-erb-b2* | ERBB3 | M34309 | na | nd | down (2.4) | nd | na | up | na | nd | down (4.5) | nd |
proline-rich nuclear receptor coactivator 1* | PNRC1 | U03105 | na | nd | down (5.3) | nd | na | up | na | nd | no change | nd |
inhibitor of differentiation* | ID1 | AA457158 | down (8.3) | down | na | nd | na | nd | na | nd | na | nd |
E-cadherin** | CDH1 | Z13009 | na | nd | na | nd | no change | nd | up | nd | up | nd |
hsp27** | HSPB1 | U90906 | na | nd | na | nd | up | nd | up | nd | up | nd |
p53** | TP53 | U94788 | na | nd | na | nd | no change | nd | up | nd | up | nd |
N-myc downstream regulated gene 1** | NDRG1 | D87953 | na | nd | na | nd | no change | nd | no change | nd | no change | nd |
Results
Experimental overview
Analysis
Time points | Caco-2 | NHEK |
---|---|---|
1 hr (repressed) | 8 | 16 |
12 hrs (repressed) | 54 | 10 |
24 hrs (repressed) | 86 | 171 |
1 hrs (induced) | 1 | 26 |
12 hrs (induced) | 69 | 28 |
24 hrs (induced) | 35 | 163 |
Caco-2 analysis
NHEK analysis
NHEK vs. Caco-2 analysis
Time point | Regulation | Probe ID | Gene |
---|---|---|---|
1 hr | repressed | 36618_g_at | Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein |
12 hrs | repressed | none | |
24 hrs | repressed | 33720_at 1069_at | Putatative 28 kDa protein, Human cyclooxygenase-2 (hCox-2) gene, complete cds |
1 hr | induced | none | |
12 hrs | induced | 39809_at 36980_at 1585_at | HMG-box transcription factor 1 proline-rich nuclear receptor coactivator 1 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) |
24hrs | induced | 39248_at | Aquaporin 3 |
Gene ontology analysis of Caco-2 and NHEK
Gene Category | Down (genes) | Up (genes) | Combined list Affy probes | EASE score |
---|---|---|---|---|
mitotic cell cycle
| 41 | 4 | 49 | 1.96E-14 |
cell cycle
| 52 | 12 | 76 | 2.03E-12 |
M phase
| 22 | 1 | 24 | 7.73E-09 |
DNA replication and chromosome cycle
| 22 | 1 | 24 | 7.73E-09 |
cell proliferation
| 56 | 22 | 100 | 0.000000024 |
nuclear division
| 20 | 1 | 22 | 7.23E-08 |
M phase of mitotic cell cycle
| 19 | 1 | 21 | 0.000000217 |
DNA replication
| 19 | 1 | 21 | 0.000000217 |
S phase of mitotic cell cycle
| 19 | 1 | 21 | 0.000000217 |
mitosis
| 19 | 1 | 21 | 0.000000217 |
DNA metabolism
| 29 | 6 | 41 | 0.000000403 |
DNA dependent DNA replication
| 13 | 0 | 13 | 0.00000641 |
cytokinesis
| 12 | 0 | 12 | 0.0000199 |
metabolism | 100 | 79 | 256 | 0.000126 |
regulation of cell cycle | 26 | 11 | 48 | 0.00137 |
cell growth and/or maintenance | 79 | 62 | 202 | 0.00241 |
nucleobase\, nucleoside\, nucleotide and nucleic acid metabolism | 53 | 36 | 124 | 0.00262 |
biosynthesis | 15 | 5 | 24 | 0.00475 |
protein metabolism | 43 | 29 | 101 | 0.00992 |
physiological process | 132 | 130 | 389 | 0.0103 |
obsolete biological process | 17 | 7 | 31 | 0.0125 |
macromolecule biosynthesis | 11 | 4 | 18 | 0.0267 |
protein modification | 22 | 14 | 50 | 0.0639 |
cytoplasm organization and biogenesis | 12 | 6 | 23 | 0.0693 |
cell organization and biogenesis | 17 | 13 | 42 | 0.211 |
phosphorus metabolism | 16 | 12 | 40 | 0.244 |
phosphate metabolism | 16 | 12 | 40 | 0.244 |
protein amino acid phosphorylation | 12 | 9 | 30 | 0.326 |
phosphorylation | 12 | 10 | 32 | 0.424 |
macromolecule catabolism | 10 | 8 | 26 | 0.435 |
protein catabolism | 10 | 8 | 26 | 0.435 |
proteolysis and peptidolysis | 10 | 8 | 26 | 0.435 |
cell adhesion
| 4 | 16 | 36 | 1 |
cell motility
| 2 | 6 | 14 | 0.995 |
Gene Category | Down (genes) | Up (genes) | Combined list Affy probes | EASE score |
---|---|---|---|---|
cell motility
| 8 | 1 | 10 | 0.0459 |
cytoplasm organization and biogenesis
| 8 | 1 | 10 | 0.0459 |
muscle development
| 7 | 1 | 9 | 0.0872 |
morphogenesis
| 18 | 7 | 33 | 0.0879 |
development
| 22 | 10 | 43 | 0.107 |
cell adhesion
| 11 | 3 | 18 | 0.112 |
cell surface receptor linked signal transduction
| 11 | 3 | 18 | 0.112 |
organelle organization and biogenesis
| 6 | 1 | 8 | 0.158 |
cytoskeleton organization and biogenesis
| 6 | 1 | 8 | 0.158 |
cell organization and biogenesis
| 11 | 4 | 19 | 0.159 |
organogenesis
| 16 | 7 | 31 | 0.179 |
response to stress
| 8 | 3 | 14 | 0.282 |
macromolecule biosynthesis
| 9 | 4 | 17 | 0.331 |
cell-cell signaling
| 6 | 2 | 10 | 0.346 |
signal transduction | 19 | 5 | 30 | 0.0118 |
immune response | 9 | 1 | 11 | 0.0233 |
protein metabolism | 15 | 4 | 23 | 0.0235 |
cellular process | 58 | 34 | 126 | 0.0316 |
defense response | 10 | 2 | 14 | 0.0467 |
transcription\, DNA-dependent | 17 | 10 | 37 | 0.358 |
transcription | 17 | 10 | 37 | 0.358 |
response to biotic stimulus | 10 | 5 | 20 | 0.374 |
nucleobase\, nucleoside\, nucleotide and nucleic acid metabolism | 24 | 16 | 56 | 0.434 |
regulation of transcription\, DNA-dependent | 16 | 10 | 36 | 0.442 |
regulation of transcription | 16 | 10 | 36 | 0.442 |
cell cycle | 9 | 5 | 19 | 0.486 |
nucleotide metabolism
| 3 | 2 | 7 | 0.835 |
nuclear division
| 3 | 3 | 9 | 0.927 |
mitosis
| 3 | 3 | 9 | 0.927 |
mitotic cell cycle
| 5 | 3 | 11 | 0.691 |
cytokinesis
| 2 | 2 | 6 | 0.952 |