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. 2019 Aug 6;17(7):741–752. doi: 10.1038/s41423-019-0265-6

Fig. 2.

Fig. 2

ZFYVE1 is important for TLR3-mediated signaling. a Effects of ZFYVE1-RNAi on the expression of ZFYVE1. As shown in the upper two panels, HEK293 cells (1 × 105) were transfected with HA-ZFYVE1 (0.1 μg), HA-β-actin (0.01 μg), and the indicated ZFYVE1-RNAi plasmids (0.5 μg) for 24 h before immunoblotting analysis. As shown in the lower two panels, HEK293 cells (1 × 105) were transfected with the indicated ZFYVE1-RNAi plasmids (0.5 μg) for 12 h. The cells were then selected with puromycin (1 μg/mL) for 24 h before immunoblotting analysis. b Effects of ZFYVE1-RNAi on poly(I:C)-induced activation of the IFN-β promoter, ISRE, and NF-κB. 293-TLR3 cells (1 × 105) were transfected with the indicated reporter and ZFYVE1-RNAi plasmids (0.5 μg) for 36 h and then were or were not treated with poly(I:C) (20 μg/mL) for 6 h before luciferase assays. c Effects of ZFYVE1-RNAi on the poly(I:C)-induced transcription of downstream genes. 293-TLR3 cells (1 × 105) were transfected with control or ZFYVE1-RNAi plasmid (1 μg) for 12 h. The cells were selected with puromycin (1 μg/mL) for 24 h and then were or were not treated with poly(I:C) (50 μg/mL) for 3 h before qPCR analysis. d Effects of ZFYVE1 deficiency on the poly(I:C)-induced transcription of downstream genes. ZFYVE1-deficient HT1080 cells were generated by the CRISPR-Cas9 method. ZFYVE1-KO and control HT1080 cells (1 × 105) were or were not treated with poly(I:C) (50 μg/mL) for the indicated times before qPCR analysis. e Effects of ZFYVE1 deficiency on the LPS-induced transcription of downstream genes. ZFYVE1-deficient 293-TLR4 cells were generated by the CRISPR-Cas9 method. ZFYVE1-KO and control 293-TLR4 cells (1 × 105) were or were not treated with LPS (100 ng/mL) for 2 h before qPCR analysis. f Effects of ZFYVE1 deficiency on the poly(I:C)- and LPS-induced phosphorylation of TBK1, IRF3, and p65. ZFYVE1-KO and control cells (1 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before immunoblotting analysis. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test)