Figure 1.
Induction of autophagosomes by NTZ and TIZ.
(A)Structures of NTZ and TIZ. (B) Punctate EGFP-LC3 levels in MCF-7 cells stably expressing EGFP-LC3. Accumulation was measured quantitatively by automated microscopy in cells incubated for 3 h or 24 h with different concentrations of NTZ or TIZ. (C) Representative images of cells showing EGFP-LC3 (green) and DNA (blue).Scale bar, 10 µm.
Figure 2.
Increased EGFP-LC3 processing and inhibition of mTORC1 signaling by NTZ and TIZ.
Cells were treated with the indicated concentrations of NTZ, TIZ or rapamycin for 4 h (A) or 24 h (B). In panel C, cells were treated with 10 µM NTZ, 10 µM TIZ, 30 nM rapamycin, or DMSO without or with 0.1 µM bafilomycin A1 for 4 h. EGFP-LC3 processing was examined by immunoblotting with antibodies against GFP, mTORC1 activity using antisera against phospho-S6K Thr389 and total S6K, and mTORC2 activity with antisera against phospho-AKT Ser473 and total AKT. Total AKT was also used as a loading control.
Figure 3.
Time-course and reversibility of mTORC1 inhibition and EGFP-LC3 processing by NTZ and TIZ.
(A) Cells were incubated with 10 µM NTZ or 10 µM TIZ for the indicated times. (B) Cells were incubated with 10 µM NTZ, 10 µM TIZ or 30 nM rapamycin for 4 h. The drugs were then washed away and cells were incubated in drug-free medium for the indicated times post-washout. Cell lystates were immunoblotted as in Figure 2.
Figure 4.
Structures of NTZ analogues.
Table 1.
Effect of Nitazoxanide analogs (10 µM) on autophagosome induction, EGFP-LC3 and mTORC1 inhibition.
Figure 5.
Effect of NTZ on Mtb proliferation and survival of THP-1 cells.
(A) Differentiated THP-1 cells infected with Mtb H37Rv bearing a luciferase-reporting plasmid were treated with various concentrations of NTZ or TIZ for the indicated times. Intracellular Mtb was measured as luciferase activity at 24, 48 and 72 h. (B) Infected THP-1 cells were treated as in (A) but after 24 h treatment the drugs were removed and cells were incubated with medium without drug. Luciferase activity was measured at 24 h, 48 h (24 h post-wash) and 72 h (48 h post-wash). (C) Differentiated THP-1 cells were exposed to various concentrations of NTZ, TIZ or rapamycin for 4 h. Endogenous LC3 processing (using LC3 antibodies) and mTORC1 activity were determined by immunoblotting as in Figure 2.Viability of THP-1 cells treated with drugs for the indicated times was measured with the MTT assay (D) or by propidium iodide (PI) staining (E, F). PI levels were measured quantitatively by automated microscopy, and viable cells was calculated as the percentage of PI-negative cells. (F) After drug removal at 24 h, THP-1 cell survival was measured by PI at 48 h (24 h post-wash) and 72 h (48 h post-wash).
Figure 6.
Effects of NTZ on Mtb proliferation and viability of PBMC.
Peripheral blood mononuclear cells isolated from healthy human subjects and infected with Mtb H37Rv bearing a luciferase-reporting plasmid were treated with various concentrations of NTZ for the indicated times. Intracellular Mtb was measured as luciferase activity. Data presented are: subject 1withMOI 10 (A), subject 2with MOI 1 (B) and subject 2 with MOI 10 (C). Viability normalized to DMSO-treated controls was measured at 48 and 72 h, with PBMC from subject 2 using the MTT assay (D) or by PI staining (E).(F) Viability, normalized to DMSO treated controls, was measured with PBMC from subject 3 at 72 h.
Figure 7.
Inhibition of NQO1 by NTZ and TSC2-dependent mTORC1 inhibition.
(A) Relative activity of NQO1 as determined by a modified Prochaska microtiter plate bioassay, in which 0.25 µg/ml pure NQO1 enzyme was treated with different concentrations of NTZ, dicoumarol (DIC), and rapamycin (RAPA) for 1 h. (B) MCF-7 cell lysates were treated with various concentrations of NTZ for 1 h, and relative activity of NQO1 was determined by the Prochaska bioassay. (C) MCF-7 cells or (D) TSC2+/+ and TSC2−/−MEFs were treated with NTZ, DIC, and rapamycin for 4 h and subjected to immunoblotting for total and phosophorylated S6K as previously described.(E) Total NQO1 or (F) total and phosphorylated S6K was assessed by immunoblotting in MCF-7 or HEK 293T cells treated with indicated concentrations of drugs for 8 h. Lines in (F) were used to indicate juxtaposition of noncontinguous lanes from the same gel and image exposure.
Figure 8.
Effect of NTZ and TIZ on autophagosome formation in the presence of tuberculosis drugs.
MCF-7 cells expressing EGFP-LC3 were exposed to 3 µM or 10 µM NTZ (A) or TIZ (B), without or with 5 µg/ml ethambutol (EMB), pyrazinamide (PZA), isoniazid (INH), streptomycin (STM) or rifampicin(RMP). Representative images of treated cells are shown in (C). Scale bar, 10 µm.