FIG 7.

siRNA-mediated silencing of PCGF isoforms increases E. chaffeensis infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with E. chaffeensis at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial dsb to the host cell gapdh in individual PCGF knockdown using real-time qPCR at 48 hpi (n = 3; *, P ≤ 0.05).