Fig. 2.

Effects of Tip110 knockdown on the IL-8 mRNA stability. a. 1205Lu were transfected with pIL-8.Luc.pro reporter vector plus si-Tip110 and si-Ctrl and cultured for 48 h. The cells were treated with 10 ng/ml TNF-α for 20 h. pTK-βgal was used to normalize the transfection efficiency variations among all transfections. ### P < 0.001 compare to si-RNA untreated control. *** P < 0.001; 2-way ANOVA. b. Schematic for the primer sets on the IL-8 (indicated by arrow) used for qRT-PCR to determine the levels of IL-8 pre-mRNA and mRNA. c. 1205Lu were transfected with si-Tip110 or si-Ctrl, cultured for 48 h, and harvested for total RNA isolation and qRT-PCR to determine the level of IL-8 pre-mRNA and mRNA. The pre-mRNA levels in si-Ctrl-transfected cells were set to 1. ### P < 0.001, compare to si-Ctrl pre-mRNA, *** P < 0.001; 2-way ANOVA. d-g. 1205Lu were transfected same as above, treated with 5 μg/ml actinomycin D (ActD) for 0, 2, 3, 6, 8, or 12 h, and harvested for RNA isolation and qRT-PCR to determine the mRNA levels of IL-8 (d) and TNF-α (f). β-actin and GAPDH were included in the qRT-PCR and used as a relative reference. The mRNA levels of si-Ctrl-transfected cells at 0-time point were set to 1. ** P < 0.01, *** P < 0.001; Student t-test. The percentage of the remaining IL-8 mRNA (e) and TNF-α mRNA (g) were calculated by setting their respective mRNA level of the transfected cells at 0 h to be 100. The dashed lines indicated the 50% mRNA half-life. h. 1205Lu were transfected as above, and treated with 5 μM p38 MAPK inhibitor SB203580, or the vehicle control DMSO for 5 h, and harvested for RNA isolation and qRT-PCR for the IL-8 mRNA level. ### P < 0.001, compare to si-Ctrl control, *** P < 0.001; 2-way ANOVA. All the data are representative of triplicate independent experiments and the data presented as the mean ± SE. UT, Untreated