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. 2017 Oct 24;61(11):e00969-17. doi: 10.1128/AAC.00969-17

FIG 4.

FIG 4

(A) Effects of the various drugs on induction of autophagy in THP-1 macrophages. THP-1 cells were differentiated with PMA and treated with 25 μM either compound 1, compound 10, compound 2, or PA-824 for 24 h. The clarified lysates from these drug-treated macrophages were prepared and subjected to immunoblot analysis as described in Materials and Methods. The data presented in this panel are representative of those from an experiment performed in triplicate. (B) Statistical analysis of replicate immunoblots shown in panel A showing the normalized fold intensity of proteins in macrophages exposed to 25 μM either compound 1, compound 10, compound 2, or PA-824. The values depicted in this panel are means ± SEs from three independent experiments. Statistically significant differences were observed for the indicated groups and were determined by a paired (two-tailed) t test. *, P < 0.05; **, P < 0.01. (C) Dose dependence of 5NP on expression of Beclin-1 and Atg-3 in THP-1 macrophages. THP-1 macrophages were differentiated and treated with various concentrations of 5NP (3.125 μM to 25 μM, as indicated above the gels) for 24 h. As described in Materials and Methods, expression of Beclin-1 and Atg-3 was measured by immunoblot analysis. The blots shown in this panel are representative of those from three independent experiments. (D) Pretreatment with 5NP or its structural analogs increases LC3 punctum formation in THP-1 macrophages. THP-1 macrophages were differentiated and treated with 25 μM either compound 1 or compound 10 for 24 h. As described in Materials and Methods, LC3 punctum formation was visualized by staining with anti-LC3 using confocal microscopy. Bars, 10 μm. (E) Detailed quantitative analysis of LC3 punctum formation in THP-1 macrophages treated with 25 μM either compound 1 or compound 10 for 24 h. The data shown in this panel are the mean number of LC3 puncta ± SE obtained from random fields of the panels shown in panel D. Significant differences were observed for the indicated groups and were determined by a paired (two-tailed) t test. **, P < 0.05.