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. 2018 Feb 20;9(1):e01800-17. doi: 10.1128/mBio.01800-17

FIG 4 .

FIG 4 

Rab4a is essential for Y. pestis to avoid phagosome acidification and LAMP-1 acquisition. (A) Representative confocal microscopy images of RAW264.7 macrophages transfected with the indicated siRNAs that were treated with Lysotracker and infected with Y. pestis CO92 pCD1(-) pGEN222, which expresses EGFP (MOI of 7.5). The different colors indicate the following: Lysotracker (red), Y. pestis (green), and colocalization (yellow). Bars = 10 µm. (B and C) Yersinia-containing vacuole (YCV) colocalization with Lysotracker was calculated using IMARIS at 20 min (B) and 80 min (C) postinfection. (D) Representative confocal microscopy images of RAW264.7 macrophages transfected with scrambled or Rab4a siRNAs that were infected with Y. pestis CO92 pCD1(-) pGEN222 (MOI of 3) and stained with anti-LAMP-1 antibody. LAMP-1 is shown in red, and Y. pestis is shown in green. Yellow arrows indicate locations of bacteria that do not colocalize with LAMP-1 and white arrowheads indicate bacteria that colocalize with LAMP-1 based on Imaris COLOC function. Bars = 5 µm. (E and F) YCV colocalization with LAMP-1 was calculated using IMARIS at 20 min (E) and 80 min (F) postinfection. One-way ANOVA with Tukey’s posthoc test was performed, and the results are indicated as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. The treatments are indicated as follows: Scr, scrambled siRNA; Killed, untransfected macrophages infected with PFA-killed Y. pestis CO92 pCD1(-) pGEN222; Rab4a, Rab4a siRNA; Rab11b, Rab11b siRNA; Myo5b, Myo5b siRNA.