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. 2013 Jul;81(7):2426–2436. doi: 10.1128/IAI.00387-13

Fig 1.

Fig 1

Effect of Rab proteins on invasion of heat-inactivated P. aeruginosa in macrophages. (A) J774-Eclone cells alone or J774-Eclone cells expressing GFP were incubated in the presence of heat-inactivated strain PAO1 P. aeruginosa at a ratio of 200:1 (bacteria/cell). At the indicated times during a 60-min incubation period, cells were washed and fixed, and the phagocytic index of P. aeruginosa (PA) was determined as described in Materials and Methods. Inset, J774-Eclone or GFP–J774-Eclone cells were incubated with the indicated ratio of heat-inactivated P. aeruginosa for 60 min, and the phagocytic index was determined and is expressed as a percentage of the maximum, relative to the phagocytic index at the highest bacteria/cell ratio (200/1). (B) J774-Eclone cells expressing GFP or Rab5:WT (WT), Rab5:Q79L (QL), or Rab5:S34N (SN) were incubated with heat-inactivated P. aeruginosa at a ratio of 200:1, and the phagocytic index was determined at the indicated times and is expressed as percent internalization relative to the GFP control at 60 min. Inset, cells expressing the indicated constructs were lysed and subjected to immunoblotting (IB) with anti-GFP or antitubulin antibodies. (C) J774-Eclone cells expressing GFP or Rab7:WT, Rab7:Q67L (QL), or Rab7:S22N (SN) were incubated with heat-inactivated P. aeruginosa at a ratio of 200:1, and the phagocytic index was determined at the indicated times and is expressed as the percentage of the GFP control value at 60 min. Inset, cells expressing the indicated constructs were lysed and subjected to immunoblotting with anti-GFP or antitubulin antibodies. (D) Nontransfected J774-Eclone cells (control), or J774-Eclone cells transfected with a scramble RNAi sequence or RNAi sequences against Rab5 isoforms (Rab5 a, b, and c) were incubated with heat-inactivated P. aeruginosa at a ratio of 200:1 for 60 min, and the phagocytic index was determined and expressed as a percentage of the value for control cells. Inset, cells transfected with the indicated RNAi sequences were lysed and subjected to immunoblotting with antitubulin or anti-Rab5 antibodies. Results represent the mean ± SEM from three independent experiments. Asterisks represent statistically significant differences from control values (*, P < 0.05).