Fig 1. Bilirubin exposure activates ER stress and NFκB pathways in SHSY5Y cells.

SHSY5Y cells were exposed to 50 μM bilirubin for 4h. Whole genome gene expression analysis was subsequently carried out. A) The gene expression pattern was compared to a compendium of 1600 chemical treatments using the cMap; the figure shows the correlation score for the top 10 chemicals, with bilirubin sharing greatest similarly to thapsigargin. B) The heatmap shows the correlation between the top 100 most changed genes in response to bilirubin or thapsigargin (n = 3). Samples and genes were clustered using the Euclidean distance algorithm. Up-regulated genes are shown in red and down-regulated genes in blue. The values represent log₂ fold change compared to control samples. C) Representative images of immunoblots measuring the protein expression levels for ER stress marker proteins and D) NF-ĸB marker proteins (n = 3). E) Densitometry-based quantification of the immunoblots where significant changes in protein expression were detected by ANOVA (*p<0.05, **p<0.01). F) Representative images of morphological changes in the ER caused by application of 50μM bilirubin. The ER was visualised using an antibody for the ER membrane protein, calreticulin. The scale bar represents 10 μm.