Coculture supernatant induces iNOS in astrocytes. Total RNA was extracted from MDM/HFA cocultures at day 18 postinfection and from HFA cultures incubated for 7 days with different supernatants. Levels of iNOS mRNA in HFA were analyzed by RT-PCR. The cytokine-stimulated A172 cell line was used as a positive control. Lane 1, positive control; lane 2, HFA; lane 3, HFA cocultured with uninfected MDM; lane 4, HFA cocultured with infected MDM; lane 5, HFA incubated with HIV-1; lane 6, HFA incubated with day 18 to 21 supernatant of uninfected MDM culture; lane 7, HFA incubated with day 18 to 21 supernatant of infected MDM culture; lane 8, HFA incubated with day 18 to 21 supernatant of coculture with uninfected MDM and HFA; lane 9, HFA incubated with day 3 to 6 supernatant of coculture with infected MDM and HFA; lane 10, HFA incubated with day 18 to 21 supernatant of coculture with infected MDM and HFA.