Table 2.
Functional Validation of the 14 Selected Proteins in HepG2 Cells
|
Gene reporter activity
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|---|---|---|---|---|---|---|---|---|---|---|
|
mRNA induction assay
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Protein overproduction
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siRNA interference
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Endogenous gene expression
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| Selected proteins | Bait proteins | Rebound screens | TGF β | BMP7 | TGF β | BMP7 | TGF β | BMP7 | TGF β (PAI-1) | BMP7 (AP) |
| LAPTm5 | Smurf2 | + | ▴+++ | - | ▾++ | - | ▴+ | - | ▴++ | - |
| MAPK13 | Smad4 | nd | - | - | - | - | - | - | - | - |
| PTPN12 | Smad5 | - | - | - | - | - | - | - | - | - |
| PPP1CA | SARA | + | - | - | ▴++ | - | ▾+ | - | - | - |
| HIPK3 | SNIP1 | - | - | - | nd | nd | - | - | - | - |
| SnoN | - | |||||||||
| p621 | Smad4 | - | - | - | nd | nd | - | - | - | - |
| PKD2 | Smad1 | - | - | - | - | - | - | - | - | - |
| Smad8 | - | |||||||||
| MAN1 | Smad1 | nd | - | - | - | - | - | ▾+ | - | ▾++ |
| Smad8 | nd | |||||||||
| HYPA | Smad4 | - | - | - | - | - | ▾+ | ▾+ | - | ▾+ |
| LMO4 | Smad8 | - | - | - | - | - | - | ▾+++ | - | ▾+++ |
| RNF11 | Smurf2 | + | - | - | - | - | nd | nd | ▾+++ | - |
| SARA | - | |||||||||
| KIAA1196 | Smad1 | + | - | - | nd | nd | ▾+++ | - | ▾++ | - |
| FLJ20037 | SARA | - | - | - | - | - | - | - | - | - |
| ZNF8 | Smad1 | + | - | - | - | - | - | ▾++ | - | ▾++ |
| Smad5 | - | |||||||||
| Smad8 | - | |||||||||
Proteins used as bait fragments for this selection of prey proteins in yeast two-hybrid screening are indicated (bait proteins). Rebound screening was performed for most of these proteins (nd: not done): a “+” indicates that the selected protein was used in a rebound screen and that interaction was found again. A “-” indicates that the selected protein was used in a rebound screen but that interaction was not found again. The mRNA level of each candidate was monitored for TGFβ or BMP induction using Q-PCR in HepG2 cells. TGFβ and BMP7-responsive luciferase reporters were transfected in HepG2 cells to monitor the effect of the selected proteins' overproduction or siRNA transfection. Reporter assays were normalized using the pRL-TK vector as an internal control. The endogenous expression of the PAI-1 gene or the AP gene was determined in the presence of TGFβ or BMP7 after siRNA transfection, respectively. All Q-PCR results were normalized using GUS as a standard control. Note that reporter or mRNA gene expression was detected above background in each experimental condition. The symbols (▴) and (▾) correspond to up-regulation and down-regulation by the selected protein, respectively. (nd) not done, (-) no significant effect, (+) slight but significant effect (<twofold), (++) a twofold to fourfold effect, and (+++) indicates a strong effect (>fourfold). Triplicates were performed in at least two independent experiments.