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. Author manuscript; available in PMC: 2016 Jun 4.
Published in final edited form as: Cell. 2015 May 28;161(6):1306–1319. doi: 10.1016/j.cell.2015.05.009

Figure 5. Sequential trafficking of intracellular UPEC before expulsion.

Figure 5

(A) TEM showing intracellular UPEC (i) encased in a single membrane (arrow) vacuole (ii) break their initial vacuole (arrow) or (iii) completely encased in an autophagosome with double-layered membrane (arrow) (iv) encased in a single membrane compartment containing membrane-bound bacteria (arrow) and ILVs (arrow), resembling a MVB. The bacteria associated with each structure is quantified and expressed as the percentage of total examined UPEC, provided in parenthesis. Scale bar: 0.2 µm. n=3 grids

(B) Extracellular UPEC surviving from gentamycin with/without 0.1% Triton X-100 treatment were quantified from BECs treated with rapamycin or ATG5 or Beclin1 shRNA. Error bars, SEM. n=18.

(C) Immunoblot quantification of CD63 associated with EUCV isolated at 12 h.p.i. from infected BECs treated with DMSO vehicle (lane1), 200 nM of rapamycin (lane2) or transfected with control shRNA (lane3) or ATG5 shRNA (lane4). The CD63 detected in the total cell lysate (TCL) was used to suggest similar number of cells were used.

(D) Bacterial expulsion levels at 6 h.p.i. in control or Alix/Tsg101 knockdown BECs, with/without 200 nM rapamycin treatment. Error bars, SEM. n=18.

(E) Immunofluorescence staining of colocalization between autophagosome marker LC3 (green) and MVB marker CD63 (red) when housing UPEC (blue) in infected BECs at 4 h.p.i.. The number of LC3+ and CD63+ compartment-encased UPEC was quantified and expressed as percentage of total ICU examined, shown in the parenthesis. Scale bar: 5 µm. n=3 slides.

(F) The number of CD63+ compartment-encased UPEC was quantified in control KD or ATG5 KD BECs and expressed as percentage of total ICU, n=3 slides.

(G) Bacterial expulsion level from infected control KD, or ATG5KD, Alix KD, VAMP3 KD, Rab7 KD, SYT7 KD BECs, or SYT7 & ATG5 double KD, or SYT7 & Alix double KD were quantified at 4, 6, and 8 h.p.i.. The time point immediately after 1h gentamycin treatment was considered as 0h. Error bars, SEM. n=12.

(H) Immunoblot quantification of CD63 associated with EUCV isolated at 12 h.p.i. from BECs transfected with control shRNA (lane1), SYT7 shRNA (lane2), or Rab7 shRNA (lane 3). CD63 detected in the total cell lysate (TCL) (lane 4, 5, 6) was used to suggest similar number of cells were used.