Figure 7.
RAB26 activity is essential for CCV biogenesis. (A) U2OS cells infected with Coxiella NMII were transfected with pLVX-GFP, pLVX-GFP-RAB26, pLVX-GFP-RAB26T77N, pLVX-GFP-RAB26Q123L or pLVX-GFP-RAB26N177I (green) 2 d post-infection. We fixed cells 24 h post-transfection and stained with anti-LC3B (red) and stained DNA using Hoechst 33,258 (blue). White arrows indicate Coxiella colonies. (B) Cells were treated as in A, and the CCV area was measured for at least 100 cells per condition. Values are mean ± SD from 3 independent experiments (n.s. = non-significant, **** = P < 0.0001, one-way ANOVA, Dunnett’s multiple comparison test). (C) Immunoblot of lysates from U2OS Cas9 cells expressing non-targeting guides (Guide CTRL1 and guide CTRL2) or RAB26-targeting guides (RAB26 Guide 3, 5 and 7). Immunoblots were probed with antibodies against RAB26, GAPDH and Cas9. (D) U2OS Cas9 cells expressing the non-targeting guide CTRL2 or the RAB26-targeting guide 5 were challenged for 6 d with Coxiella WT GFP (WT). The normalized CCV area for each condition was determined using the Cell Profiler software. (E) U2OS cells were challenged as in D and Genome Equivalents (GE) were determined by quantitative PCR. Values are mean ± SD from 3 independent experiments (n.s. = non-significant, **** = P < 0.0001, ** = P < 0.001, unpaired t test). Scale bars: 10 μm