Cholesterol-Dependent Anaplasma phagocytophilum Exploits the Low-Density Lipoprotein Uptake Pathway
Figure 8
ERK signaling pathway is involved in the LDLR up-regulation upon A. phagocytophilum infection.
(A) Western blot analysis was performed using antibodies specific to either the phosphorylated or total ERK1/2. Uninfected and A. phagocytophilum–infected HL-60 cells with or without 10 µM ERK inhibitor U0126 treatment were collected at the indicated time points. α-Tubulin was used as the protein loading control to normalize each sample. The values under the bands show the ratios of band intensities of p-ERK and ERK. Ap, A. phagocytophilum. (B) Bacterial numbers per 100 cells treated with the indicated concentrations of U0126 were determined on days 1 and 2 p.i. (C) Western blot analysis of A. phagocytophilum–infected HL-60 cells on day 2 p.i. treated with the indicated concentrations of U0126 was performed using antibodies specific to phosphorylated or total ERK1/2, or A. phagocytophilum outer membrane protein P44. α-Tubulin was used as the protein input control to normalize each sample. The values under the bands show the ratios of band intensities of p-ERK and P44, normalized to total ERK or α-tubulin, respectively. Data are representative of three independent experiments. (D) HL-60 cells were transfected with control (Ctl) or double-stranded siRNA specific targeting the genes encoding MEK1 and MEK2 (MEK1+2) (3 µg/2×106 cells) using the Amaxa Nucleofection system. Two days after transfection, host cell-free A. phagocytophilum was added to the cells and incubated for additional 2 days. One aliquot of samples were lysed and subjected to Western blotting using antibodies against MEK1/2, ERK1/2, phospho-ERK1/2, and A. phagocytophilum P44 protein. The relative protein amount of MEK1/2, phospho-ERK1/2, and P44 were determined using total ERK1/2 as loading control by densitometry analysis. The values under the bands show the relative ratios of band intensities, with the ratios of those from samples nucleofected with control siRNA arbitrarily set as 1. Results are representative of three independent experiments. (E) LDLR expression in A. phagocytophilum–infected HL-60 cells on day 2 p.i. treated with the indicated concentrations of U0126 was determined by quantitative RT-PCR. Data are expressed as mean±standard deviation (n = 3) and are representative of two independent experiments. *, p<0.05 (unpaired two-tailed t test); **, p<0.01 (unpaired two-tailed t-test).