Fig 2. 3CD-mediated induction of PI4P does not require host transcription but uses GBF1 and a type III PI4 kinase(s).

(A) Effect of actinomycin D (AMD) on nucleolin localization. HeLa cells were treated with AMD (5 μg/mL) for 4 h then immunostained for nucleolin (red) [37]. (B) Effect of actinomycin D (AMD) on 3CD-mediated induction of PI4P. HeLa cells were maintained in the absence or presence of AMD (5 μg/mL) prior to transfection with 3CD mRNA. Cells were immunostained for PI4P (red) or 3CD (green); the nucleus was stained with DAPI (blue). Subsequent experiments were performed as described for AMD but used the following inhibitors: (C) brefeldin A (BFA, 2 μg/mL), (D) golgicide A (GCA, 10 μM), and (E) PIK93 (15 μM). All of these treatments interfered with induction of PI4P. PIK93 was the least effective. (F) Quantification of the PI4P levels per cell in the absence (1.8 ± 0.1 x 10⁸) and presence (7.1 ± 0.5 x 10⁷) of PIK93. Error bars represent SEM; Student’s t-test gave a P value of <0.0001.