Figure 5

PGRN counteracted IL1β and LPS-induced nitric oxide (NO) production and inhibited NOS2 expression in cultured ATDC-5 cells, at least in part, through TNFR1.

(a,b) Cells were treated with 0.1 ng/ml of IL1β (a) and 250 ng/ml of LPS (b) alone or in combination PGRN (200 ng/ml) during 48 h. Culture medium was subsequently analyzed for nitrite levels. NO concentration (μM) was determined using the Griess reaction. Values are the mean ± SEM of at least 3 independent experiments (a) **P < 0.01 PGRN + IL1β vs. IL1β; (b) **P < 0.01 PGRN + LPS vs. LPS). Cell lysates underwent Western blotting analysis using NOS2 antibody. GAPDH was used as a loading control. The blots were run under the same experimental conditions. The full blots are shown in Supplementary Fig. 3. Blots are representative of at least 3 independent experiments. (c,d) Cells were transfected with negative control siRNA or TNFR1-targeted siRNA (siTNFR1) and stimulated with 0.1 ng/ml of IL1β (c) and 250 ng/ml of LPS (d) in absence or presence of PGRN 200 ng/ml for 48 h. Relative mRNA levels of NOS2 were measure by RT-PCR. Values are the mean ± SEM of at least 3 independent experiments (c) **P < 0.01 siC- PGRN + IL1β vs. siC- IL1β; (d) **P < 0.01 siC- PGRN + LPS vs. siC-LPS).