TABLE 3.

Effects of PGIS overexpression on cytokine-induced NFκB, activator protein 1, and iNOS promoter activation and nitrite production in RINm5F insulin-producing cell clones

For estimation of transcription factor and iNOS promoter activation, RINm5F insulin-producing cells were transfected 24 h prior to cytokine incubation and then treated with IL-1β (600 units/ml) alone or a cytokine mixture (60 units/ml IL-1β, 185 units/ml TNF-α, 14 units/ml IFNγ) for 6 h. Thereafter the medium was collected, and a SEAP-reporter gene assay was performed. For nitrite measurements, RINm5F insulin-producing cells were incubated with IL-1β (600 units/ml) alone or a cytokine mixture (60 units/ml IL-1β, 185 units/ml TNF-α, and 14 units/ml IFNγ) for 72 h. Thereafter the medium was collected, and the nitrite concentration was measured. The numbers of experiments are given in parentheses. The data are mean values for the indicated numbers (n) of independent experiments, each measured in at least three repetitions (analysis of variance followed by Bonferroni).

RINm5F cell clone	RINm5F	RIN-PGIS K4
NFκB (% of untreated)
Untreated	100 ± 2 (4)	100 ± 1 (4)
IL-1β	217 ± 23 (4)a	111 ± 5 (4)b
Mix	190 ± 19 (4)a	119 ± 15 (4)c
AP-1 (% of untreated)
Untreated	100 ± 1 (6)	100 ± 1 (6)
IL-1β	125 ± 6 (6)	110 ± 20 (6)
Mix	129 ± 10 (6)	94 ± 18 (6)
iNOS promoter (% of untreated)
Untreated	100 ± 1 (4)	100 ± 3 (4)
IL-1β	422 ± 49 (4)a	144 ± 9 (4)b
Mix	531 ± 91 (4)a	182 ± 19 (4)c
Nitrite (pmol/μg protein)
Untreated	0 ± 0 (6)	0 ± 0 (6)
IL-1β	3.9 ± 0.5 (6)a	0 ± 0 (6)b
Mix	5.5 ± 0.7 (6)a	0 ± 0 (6)c

^a p < 0.05 versus untreated.

^b p < 0.05 IL-1β versus control clone.

^c p < 0.05 cytokine mixture versus control clone.