Fig 2: Inhibition of autophagy enhances GANT61-induced apoptosis.A SW1736 cells were treated with the indicated concentrations of GANT61 for 48 h or with 10 µM GANT61 for the indicated lengths of time. B, D SW1736 cells were incubated in the absence or presence of GANT61 (10 µM) minus or plus 5Z (1 or 5 µM) (B) or CQ (5 or 10 µM) (D) for 24 h. Cell lysates were prepared and analyzed for PARP, caspase-8 (C8), cleaved caspase-8 (CC8), caspase-3 (C3), and cleaved caspase-3 (CC3) by western blot. C-PARP cleaved PARP. C, E SW1736 cells were incubated in the absence or presence of GANT61 (10 µM) minus or plus 5Z (1 or 5 µM) (C) or CQ (5 or 10 µM) (E) for 24 h. Single-cell suspensions were stained for propidium iodide (PI) and annexin V followed by flow cytometry. Data in bar graphs are the mean ± SD of three independent experiments. F, G SW1736 cells were transfected with scrambled or TAK1 siRNA (F) or Beclin-1 siRNA (2.5 nmole each) (G). After incubation for 24 h, the cells were left untreated or treated with GANT61 for 24 h. Cell lysates were analyzed for p62, LC3, Beclin-1, TAK1, PARP, C8, CC8, C3, CC3, and ß-Actin by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05; **p < 0.01, compared to the untreated control; #p < 0.05; ##p < 0.01, compared to GANT61 (A–E).

Fig 3: 5Z suppresses IAV-induced TAK1 phosphorylation and restores the levels of epithelial junction proteins in vivo.a 5Z inhibits H5N1 virus-induced downregulation of intercellular junction proteins. C57BL/6 mice were first treated with vehicle or 5Z (2 mg/kg body weight) 12 hr before virus infection. Mice were left uninfected or infected with H5N1 viruses (1000 pfu/mouse, 4 mice/group) and then treated daily with the same dose of vehicle or 5Z. Mice were sacrificed 48 hr after virus infection. One portion of lung tissue was analyzed for the levels of TAK1, Snail, and junction proteins by Western blot with the indicated antibodies. The density of the bands from 8 mice per group was analyzed using NIH Image-J software and normalized by the arbitrary units of ß-actin. *p < 0.05, **p < 0.01. b 5Z does not affect H5N1 virus replication. c 5Z blocks H5N1 virus-damaged intercellular junction structure. The sections of the paraffin-embedded lung tissue blocks were stained with antibodies against occludin and claudin-1 as described in Methods. d, e 5Z improves the pathological changes in the lungs of H5N1 virus-infected mice. Sections of paraffin-embedded lung tissue blocks were stained with H & E d and graded based on the filtration of inflammatory cells as described in Methods. Data represent the mean ± SD from three sections per mouse, 4 mice per group e. *p < 0.05, **p < 0.01. f, g Female C57BL/6 mice (6-8 weeks old, 10 mice/group) were mock-infected with PBS or infected intranasally with H5N1 viruses (1000 pfu/mouse) and then treated daily with the vehicle or 5Z (2 mg/kg body weight) for 7 days. Mice were weighed and monitored for survival for 16 days. Percent of bodyweight changes f and percent survival g were plotted. *p < 0.05, compared to the untreated controls. h Schematic model of H5N1-induced disruption of intercellular junctions. H5N1 virus activates TAK1 through the Toll-like receptor 3 (TRL3), leading to the activation of p38 and ERK kinases. These kinases induce the expression of Itch, which functions as a ubiquitin ligase to induce occludin ubiquitination and degradation of occludin. Whether Itch mediates the degradation of intercellular junction proteins remains to be investigated. In contrast, H5N1 viruses activate the PI-3 kinase pathway but do not activate p38 and JNK in alveolar epithelial cells. Activation of the Gli1 and Snail transcription factors by H5N1 viruses suppresses the expression of intercellular junction proteins. H5N1 virus infection may also downregulate the levels of intercellular junction proteins in part by inducing the proteasomal degradation of these proteins.

Fig 4: Inhibition of autophagy potentiates the antiproliferative effect of GANT61.SW1736 cells seeded in 96-well plates (2000 cells/well) were incubated with the indicated concentrations of GANT61 minus or plus CQ (10 µM) (A) for 72 h or 5Z (2 or 5 µM) (B) for 48 h. Cell viability was measured by using a CellTiter-Glo kit. Data are the mean ± SD of the triplicate from one representative of three independent experiments with similar results. **p < 0.01, compared to the untreated control; ##p < 0.01, compared to the counterparts in SW1736 left untreated or treated with the same concentrations of GANT61. C The mechanisms of Shh pathway inhibition-induced autophagy. Shh binding to Ptch releases the restriction of Smo activity. Activated Smo disrupts the interaction between Sufu and Gli1. Inhibition of the Shh pathway by the Smo inhibitor cyclopamine or by the Gli1 inhibitor GANT61 activates TAK1, which then activates JNK and AMPK. JNK promotes autophagy by phosphorylating Bcl-2 and dissociating it from Beclin-1. Free Beclin-1 becomes available for the initiation of autophagy. AMPK phosphorylates ULK1 and activates it. ULK1 is involved in the assembly of the pre-initiation complex and the early formation of autophagosomes. Activation of the autophagic pathway by inhibition of the Shh pathway attenuates apoptosis by multiple pathways including the activation of NF-kB and JNK by TAK1. Autophagy itself appears to also suppress apoptosis, albeit its underlying mechanism remains unknown. In addition, it is also not clear how Gli1 suppression leads to TAK1 activation.

Fig 5: TAK1 mediates GANT61-induced autophagy.A SW1736 cells were incubated in the absence or presence of GANT61 (10 µM) minus or plus 5Z (1 µM) for 48 h. Alternatively, SW1736 cells were transfected with scrambled or TAK1 siRNA (2.5 nmole each). After incubation for 4 h, the cells were left untreated or treated with GANT61 (10 µM) for 48 h. Cell lysates were prepared and analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05; **p < 0.01, compared to the untreated control; #p < 0.05; ##p < 0.01, compared to GANT61-treated SW1736 cells. B, C Inhibition of GANT61-induced autolysosome formation by 5Z. SW1736 cells transiently transfected with the expression vector encoding the GFP-LC3 gene were incubated in the absence or presence of GANT61 (10 µM) minus or plus 5Z (1 µM) for 48 h. After nuclear staining with DAPI, the cells were examined under a confocal microscope for the presence of autophagosomes (B). The number of autophagosome puncta per cells was statistically analyzed (C). Bar length: 20 µm. **p < 0.01, compared to untreated control; ##p < 0.01, compared to GANT61.