Fig. 6.

rhSlit2 attenuates bTSH induction of IL-23/p19 gene transcription while enhancing that of the IL-12p35 gene in fibrocytes. In contrast, knocking-down Slit2 in GD-OF enhances IL-23p19 gene transcription while attenuating that of the IL-12p35 gene. These effects are divergently affected by JNK inhibitor (JNKi). (A) Fibrocytes were incubated with nothing or rhSlit2 (50 ng/ml) for 7–9 d. (B) GD-OF monolayers were subjected to treatment with either control (3μg) siRNA or SLIT2-targeting siRNAs (3μg) for 3 d. (A, B), Cells were treated with nothing or bTSH (5 mIU/mL) for 2 h, harvested, and subjected to a Pol II Chip transcription assay as described in “Methods”. Values were normalized to respective monolayer protein content. (C) Confluent fibrocyte cultures treated without or with Slit2 for 7 d. were pre-treated with bTSH for 2 h. At time “0”, all cultures received 5,6-dichlorobenzimidazole (20 μg/mL) without or continued in the presence of bTSH. Monolayers were harvested at the times indicated along the abscissas, mRNA was reversed transcribed and subjected to real-time PCR for IL-23p19.. (D) Fibrocytes were pretreated without or with rhSlit2 (50 ng/ml) for 7 d, and were then incubated without or with bTSH (5mIU/mL) for 30 m. Cell layers were collected, proteins subjected to Western blot analysis for pJNK and then re-probed with anti-β-actin. Densitometric analysis: Control 5.4 AU, bTSH : 52.9 AU, rhSlit2 10.1 AU, bTSH plus rhSlit2 127.4 AU. rhSlit2 (50 ng/ml) treated fibrocyte cultures incubated with, nothing or JNKi (50μM) for 18 h, and then treated without or with bTSH (5mIU/mL) for 6 h. mRNA was reversed transcribed from the cell layers and subjected to real-time PCR for IL-23p19 and IL-12p35. Data are expressed as mean ± SD of triplicate determinations from a single experiment, representative of three studies performed. *, p<0.05; **, p<0.01