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. 2021 May 8;12(5):459. doi: 10.1038/s41419-021-03744-2

Fig. 1. Inhibition of the Shh pathway induces autophagy.

Fig. 1

SW1736 (A) and KAT-18 (C) cells were treated with the indicated concentrations of GANT61 for 48 h or with 10 μM GANT61 for the indicated lengths of time. B SW1736 cells were treated with the indicated concentrations of cyclopamine (CP) for 48 h or with 10 μM CP for the indicated lengths of time. LC3, p62, and β-actin were analyzed by western blot. D The effect of bafilomycin and chloroquine on GANT61-induced autophagy. SW1736 cells were incubated in the absence or presence of GANT61 (10 μM) for 24 h. Bafilomycin (Baf) (10 nM) or chloroquine (CQ) (10 μM) was added and incubated for another 8 h. LC3 lipidation, p62, and actin levels were analyzed by western blot. Due to the low level of LC3 I, the ratio of LC3-II to β-actin was used for autophagy analysis in this and the remaining figures. Protein band density was analyzed by using NIH Image-J software. Data presented as the mean ± SD (n = 3) relative to control are shown in bar graphs. *p < 0.05, **p < 0.01, compared to untreated control; ##p < 0.01, compared to GANT61 (A–D). E, F GANT61 induces autolysosome formation. SW1736 cells transiently transfected with an expression vector encoding the GFP-LC3 gene were left untreated or treated with GANT61 (10 μM) for 24 h with or without bafilomycin (10 nM) or CQ (10 μM). After nuclear staining with DAPI, green puncta were visualized under a confocal microscope and statistically analyzed. Bar length: 20 μm. **p < 0.01, compared to untreated controls; ##p < 0.01, compared to GANT61. G SW1736 cells were transfected with control or Gli1 siRNA. After incubation for 48 hr, the cells were harvested and analyzed for Gli1, p62, LC3, and β-Actin by western blot. H SW1736 and KAT-18 cells were transfected with an empty vector or the vector encoding the GLI1 gene. After incubation for 48 h, the cells were harvested and analyzed for Gli1, p62, LC3, and β-Actin by western blot. Relative protein levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. Data are the mean ± SD of three experiments. *p < 0.05, **p < 0.01, compared to the transfection control (E, F).