EVALUATE AND CLARIFY THE ROLE OF HYPOXIA (HIF-1ALPHA) IN CHOLESTEROL SYNTHESIS
Different cell lines (HT29, HepG2) will be cultured in different conditions:
1- hypoxia (3% O2)
2- normoxia (21% O2) + dexferal ---> to mimic hypoxia and to induce HIF-1alpha activation
3- normoxia (21% O2) + CoCl2 ---> to mimic hypoxia and to induce HIF-1alpha activation
Evaluation of:
a) HIF-1alpha induction and activation
b) HMG-CoAR activity
c) HMG-CoAR increase (protein and mRNA)
d) cholesterol synthesis
e) cellular proliferation (violet crystal)
f) pentose phosphate pathway activity
g) G6PD activity
h) G6PD increase (protein and mRNA)
Ho cominciato a prendere in considerazione le seguenti
condizioni:
- controllo
- ipossia, CoCl2, desferal (per attivare HIF)
- YC-1 (per inibire HIF)
- rotenone (per inibire la catena respiratoria)
e ad analizzare la proliferazione cellulare e la misura di colesterolo/ubichinone (24-48 h), la traslocazione di HIF al nucleo (24-48 h), l'induzione dell'HMG-CoAR (per adesso solo come mRNA).
