Figure 4.

TLR4 Activation by Whole E. coli Induces Interferon-Independent G0 Arrest

(A) pHrodo-labeled E. coli were added to MDMs for 1 h. MDMs were washed and fixed. 10⁴ cells were recorded and analyzed. Percentage of E. coli-positive cells was determined using automated cell imaging system Hermes WiScan and ImageJ.

(B) IRF3/NF-κB translocation assay. Cells were exposed to pHrodo E. coli in the presence or absence of inhibitors and 2 h later stained for IRF3/NF-κB. The percentage of cells with nuclear staining was determined (n = 3, mean ± SEM). No IRF3 or NF-κB translocation was detected in un-treated cells.

(C and D) MDMs were treated with (C) TAK242 or RUXO 6 h or (D) BX795 2 h before addition of pHrodo E. coli. Cells were infected by VSV-G-pseudotyped HIV-1 18 h later. The percentage of infected cells was determined 48 h post-infection (n = 3 donors, mean ± SEM). Cells from a representative donor were used for immunoblotting.

(E and F) Relative expression levels (fold changes) of ISGs (E) and cell-cycle-associated transcripts (F). MDMs were treated with RUXO 6 h before addition of pHrodo E. coli. Cells were collected 24 h later (n = 3 donors, mean ± SEM).

(G) Diagram of G0 arrest following exposure to E. coli and TLR4/TRIF activation resulting in block to HIV-1 infection.

^∗∗∗p ≤ 0.001; ^∗∗p ≤ 0.01; ^∗p ≤ 0.1, paired t test.