Fig. 8.

Knockdown of Rab10 markedly reduces generation of infectious progeny and aph1235 transcription.

A. Rab10 knockdown (KD) reduces generation of infectious progeny. HEK-293T cells were treated with Rab10-targeting, GAPDH-targeting or non-targeting siRNA for 72 h and subsequently infected with A. phagocytophilum (Ap). At 48 h post-infection, a time point at which infectious DC organisms would be present in the media if the bacterial developmental cycle had proceeded normally, media from the infected siRNA-treated cells was added to naïve HEK-293T cells. At 24 h, the recipient cells were screened with antibody against Ap P44 and examined by immunofluorescence microscopy to determine the percentages of infected cells (A) and the mean number (± standard deviation) of ApVs per cell (B). Data presented are representative of three experiments with similar results.

C. Knocking down Rab10 inhibits transcription of the Ap DC marker, aph1235. Total RNAs isolated from non-targeting siRNA-treated or Rab10-targeting siRNA-treated, Ap-infected cells at 24, 28 and 32 h were subjected to qRT-PCR using primers specific for aph1235. Relative aph1235 transcript levels were normalized to Ap 16S rRNA gene transcript levels using the 2^−ΔΔCT method. Data presented are representative of two experiments with similar results. Statistically significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001) values are indicated.