Figure 6: Search of Hh transcriptional targets that regulate autophagy.

(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained. (c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control. (d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager. (e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. LC3-II and actin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.