GSK3β facilitates IFNγ signaling Supplementary Figure Legends Figure S1. The effects of inhibiting GSK3β on IFNγinduced TNFα expression. A, A549 human epithelial cells were pretreated with control sirna (5 nm) or GSK3β sirna (5 nm) for 48 h. Cells were then treated with IFNγ for 4 h. GSK3α and GSK3β were analyzed with Western blotting. βactin was the internal control. B, ELISA was used to determine TNFα production. The data are means ± SD obtained from three individual cultures. p <.5 compared with the IFNγ group. Figure S. The effects of inhibiting ceramide synthesis on IFNγinduced inos/no biosynthesis. RAW64.7 cells were pretreated with the neutral SMase inhibitor Sph4 (A and B), the acid SMase inhibitor desipramine (C and E) and CHL (D and E), or the ceramide synthase inhibitors myriocin (F and H) and FB1 (G and H) for.5 h and then treated with IFNγ for 4 h. Griess reagent and Western blotting were used to determine the production of NO and the expression of inos, respectively. For nitrite detection, the data are means ± SD obtained from three individual cultures. p <.5. For Western blot analysis, βactin was the internal control. Data shown are representative of three individual experiments. Figure S3. IFNγ induces ceramide generation and ceramide increased IFNγinduced inos/no biosynthesis. A, RAW64.7 cells were treated with ng/ml of IFNγ for.5 h. We used immunostaining to determine the expression of ceramide using anticeramide antibodies, and then Alexa Fluor 488conjugated secondary antibodies (green). DAPI (blue) was used for nuclear staining (A). The scale bar is 75 μm. RAW64.7 cells were pretreated with ceramide analogue C ceramide (B and C) or glucosylceramide synthase inhibitor PDMP (D and E) for.5 h and then treated with IFNγ for 4 h. Griess reagent and Western blotting were used to determine the production of NO and the expression of inos, respectively. For nitrite detection, 1
GSK3β facilitates IFNγ signaling the data are means ± SD obtained from three individual cultures. p <.5. For Western blot analysis, βactin was the internal control. Data shown are representative of three individual experiments. Figure S4. The effects of inhibiting OAsensitive PPase on IFNγinduced inos/no biosynthesis. RAW64.7 cells were pretreated with or without OA for.5 h and then treated with IFNγ for 4 h. Griess reagent and Western blotting were used to determine the production of NO (A) and the expression of inos (B), respectively. For nitrite detection, the data are means ± SD obtained from three individual cultures. p <.5. For Western blot analysis, βactin was the internal control. Data shown are representative of three individual experiments. Figure S5. The effects of inhibiting PCPLC, PIPLC, or PKC on IFNγinduced inos/no biosynthesis. RAW64.7 cells were pretreated with D69 (A and C), U731 (B and C), or CalC and Gö6976 (D, E, and F) for.5 h and then treated with IFNγ for 4 h. Griess reagent and Western blotting were used to determine the production of NO and the expression of inos, respectively. For nitrite detection, the data are means ± SD obtained from three individual cultures. p <.5. For Western blot analysis, βactin was the internal control. Data shown are representative of three individual experiments. Figure S6. IFNγ activates Src through PCPLC and PKC. RAW64.7 cells were pretreated with D69 or Gö6976 for.5 h and then treated with IFNγ for 1 h. The phosphorylation of Src (Tyr416) was analyzed with Western blot. βactin was the internal control. Data shown are representative of three individual experiments. Figure S7. The effects of inhibiting bioactive lipids and their enzymatic generators on IFNγinduced STAT1 activation. RAW64.7 cells were pretreated with a series of inhibitors D69, Gö6976, PP1, A9, BEL, Sph4, or OA for.5 h and then treated with IFNγ
GSK3β facilitates IFNγ signaling for 3 h. We used immunocytochemical staining and then fluorescent microscopy to detect the nuclear translocation of STAT1 (A). The scale bar is 75 μm. Western blotting was used to determine the phosphorylation of STAT1 (Tyr71 and Ser77) in IFNγstimulated cells with or without D69 (B), Gö6976 (C), PP1 (D), A9 (E), BEL (F), Sph4 (G), or OA (H) pretreatment. βactin was the internal control. Data shown are representative of three individual experiments. Figure S8. The effects of inhibiting GSK3β on IFNγinduced STAT1 activation. A549 cells were pretreated with control sirna (5 nm) or GSK3 sirna (5 nm) for 48 h and then treated with IFNγ for 3 h. Western blotting was used to determine the expression of GSK3α, GSK3β, and phosphorylation of STAT1 (Tyr71). βactin was the internal control. Data shown are representative of three individual experiments. Figure S9. IFNγ induces Jak, Src, ERK1/, and p38 MAPKregulated STAT1 activation. Western blotting was used to determine the time kinetics of AG49, PP1, PD9859, and SB358 on IFNγinduced phosphorylation of STAT1 at Tyr71 (A) and at Tyr77 (B) in RAW64.7 cells. βactin was the internal control. Data shown are representative of three individual experiments. Figure S. GSK3β negatively regulates SHP. A549 cells were pretreated with control sirna (5 nm) or GSK3 sirna (5 nm) for 48 h and then treated with IFNγ for 3 h. The phosphorylation of SHP (Tyr54) was analyzed with Western blotting. βactin was the internal control. Data shown are representative of three individual experiments. 3
A GSK3α GSK3β βactin IFNγ (ng/ml) GSK3β sirna Control sirna B TNFα (pg/ml) 8 6 4 IFNγ (ng/ml) GSK3β sirna Control sirna Tsai et al. Figure S1
A 5 4 3 IFNγ (ng/ml) Sph4 (μm) B inos IFNγ (ng/ml) Sph4 (μm) C 6.3 1.5 D E 8 6 4 IFNγ (ng/ml) Desprimine (μm) 6 4 IFNγ (ng/ml) CHL (μm) inos.5.1. 1 1.5 F G H 6 4 IFNγ (ng/ml) Myriocin (μm) 6 4 IFNγ (ng/ml) FB1 (μm) βactin inos 1.5 5 5 βactin βactin IFNγ (ng/ml) Desprimine (μm) CHL (μm).5 IFNγ (ng/ml) Myriocin (μm) FB1 (μm) 5 5 Tsai et al. Figure S
A B D Untreated inos inos βactin βactin Ceramide/DAPI IFNγ IFNγ (ng/ml) Ceramide (μm) C 75 5 IFNγ (ng/ml) PDMP (μm) E 6 4 75 μm IFNγ (ng/ml) Ceramide (μm) IFNγ (ng/ml) PDMP (μm) 1.5 Tsai et al. Figure S3
A 5 4 3 IFNγ (ng/ml) OA (nm) B inos βactin IFNγ (ng/ml) OA (nm) Tsai et al. Figure S4
A 8 6 4 D 8 6 4 IFNγ (ng/ml) D69 (μm) B 8 6 4 5 IFNγ (ng/ml) CalC (μm) E 8 6 4.13..5 IFNγ (ng/ml) U731 (μm) C inos.5 5 IFNγ (ng/ml) Gö6976 (μm) F inos.5 1 βactin βactin IFNγ (ng/ml) D69 (μm) U731 (μm) IFNγ (ng/ml) CalC (μm) Gö6976 (μm).5 Tsai et al. Figure S5
Src Tyr416 Src βactin IFNγ (ng/ml) D69 (μm) Gö6976 (μm) Tsai et al. Figure S6
A C F IFNγ IFNγA9 STAT1 Tyr71 STAT1 Tyr71 STAT1 Ser77 STAT1 Ser77 IFNγD69 IFNγBEL STAT1 STAT1 IFNγGö6976 IFNγSph4 D βactin IFNγ (ng/ml) Gö6976 (μm) STAT1 Tyr71 G βactin IFNγ (ng/ml) BEL (μm) STAT1 Tyr71 5 5 IFNγPP1 IFNγOA STAT1 Ser77 STAT1 Ser77 B E H STAT1 Tyr71 75 μm STAT1 βactin IFNγ (ng/ml) PP1 (μm) STAT1 Tyr71 STAT1 βactin IFNγ (ng/ml) Sph4 (μm) STAT1 Tyr71 STAT1 Ser77 STAT1 Ser77 STAT1 Ser77 STAT1 STAT1 STAT1 βactin βactin βactin IFNγ (ng/ml) D69 (μm) IFNγ (ng/ml) A9 (μm) 1.5 1.5 IFNγ (ng/ml) OA (nm) Tsai et al. Figure S7
GSK3α GSK3β STAT1 Tyr71 STAT1 βactin IFNγ (ng/ml) GSK3β sirna Control sirna Tsai et al. Figure S8
A B STAT1 Tyr71 STAT1 βactin Time (min) IFNγ ( ng/ml) AG49 ( μm) PP1 ( μm) 5 15 3 6 5 15 3 6 5 15 3 6 STAT1 Ser77 STAT1 βactin Time (h) IFNγ ( ng/ml) PD9859 ( μm) SB358 ( μm)..5 1 3..5 1 3..5 1 3 Tsai et al. Figure S9
SHP Tyr54 SHP βactin IFNγ (ng/ml) GSK3β sirna Control sirna Tsai et al. Figure S