Figure 2.

Psoralidin-induced autophagy in CTPE cells. (upper left panel) Whole-cell lysates were prepared for western blot analysis to determine the expression levels of LC3IB and IIB, Plac8, LAMP1, Beclin, Atg-3, Atg-5, Atg-7, Atg-12 and Atg16L. β-actin was used as a loading control. (lower left panel) Total RNA was extracted and qRT-PCR performed to measure differences between the steady-state levels of expression of LC3B, Lamp1 and Plac8 from Pso-treated and -untreated CTPE cells. (upper right panel) Confocal microscopy was used with CTPE cells immunostained with Plac8, LAMP1 and LC3B antibodies. Autophagosome formation is marked by the presence of cytoplasmic puncta. (lower right panel) Localisation of Plac8 and LC3B; Cells treated in presence or absence of Pso and immunostained for Plac8 detection (green- Alexfluor 488) to visualise the overlap with LC3B (Red-alexafluor 594) which is shown as yellow. Student’s t-test was used to calculate statistical significance between vehicle control and treatment at each concentration. *P<0.05 and ***P<0.001. Densitometric analysis for western blots from two or three experiments are given in Supplementary Information.