Article Figures & Data
Figures
- Fig. 1.
Time course of O. tsutsugamushi-stimulated chemokine induction by the J774A.1 cell line. (A) Before and after incubation of J774A.1 cells with O. tsutsugamushi. The levels of chemokine mRNAs at each time point were assayed by the RNase protection assay. (B) Normalized expression level of each chemokine mRNA. (C) mRNA levels of chemokine genes induced by the infection ofO. tsutsugamushi, analyzed by semiquantitative RT-PCR at each time point. M, φX174 DNA digested with HaeIII; N, negative control (reactions performed without cDNA).
- Fig. 2.
(A) Determination of chemokine mRNA induction in J774A.1 cells treated with polystyrene beads or CHX, by semiquantitative RT-PCR. (B) The band intensities were determined with TINA software, and the level of each chemokine mRNA expression was normalized with mRNA level of β-actin. J774A.1 cells stimulated for 6 h with medium alone (C), L-929 cell lysate (Lysate), O. tsutsugamushi (OT), polystyrene beads (PS), LPS derived fromE. coli (LPS), cycloheximide (CHX), or cycloheximide andO. tsutsugamushi (CHX + OT).
- Fig. 3.
(A) Semiquantitative RT-PCR to determine the effect of polymyxin B on the levels of O. tsutsugamushi-induced chemokine mRNAs in J774A.1 cells. (B) The band intensities were determined and normalized as for the experiment in Fig. 2. J774A.1 cells were stimulated for 6 h with medium (C), O. tsutsugamushi (OT), and LPS derived from E. coli (LPS) in the absence or presence of polymyxin B (PB).
- Fig. 4.
Effect of PDTC and TPCK on the levels of O. tsutsugamushi-induced chemokine mRNAs in J774A.1 cells. (A) Levels of each chemokine mRNA were analyzed in total RNA samples prepared from uninfected cells (C), O. tsutsugamushi-infected cells (OT), and infected cells in the presence of PDTC (PDTC + OT) or TPCK (TPCK + OT) by RT-PCR analysis. (B) The intensities of bands were determined and normalized as specified for Fig. 2.
- Fig. 5.
Activation of the transcription factor NF-κB byO. tsutsugamushi and effect of PDTC and TPCK on O. tsutsugamushi-induced activation of NF-κB. (A) NF-κB activation was analyzed by EMSA for nuclear extracts prepared from J774A.1 cells treated for 2 h with medium (C), L-929 cell lysate (Lysate), and O. tsutsugamushi (OT). The nuclear extracts from the cells pretreated with PDTC (PDTC + OT) or TPCK (TPCK + OT) for 1 h before infection with O. tsutsugamushiwere also analyzed. A competitive inhibition assay was performed on nuclear extracts preincubated with the unlabeled NF-κB consensus oligonucleotide (50 × Competitor). (B) A supershift assay was also performed. Nuclear extract was preincubated with antibodies against the p65 subunit of NF-κB. N. S., nonspecific.
- Fig. 6.
Chemokine responses to inactivated or active O. tsutsugamushi. (A) The levels of chemokine mRNAs were compared by semiquantitative RT-PCR after incubation of the J774A.1 cells for 6 h with medium (C), L-929 cell lysate (Lysate), or heat-inactivated (HOT) or live (LOT) O. tsutsugamushi. (B) The intensities of bands were determined and normalized as specified for Fig. 2.
