Fig. 4: Metformin reduces LDs accumulation and lysosomal enlargement in olanzapine-treated cells.

A HepG2 cells were treated with 5 mM metformin (Met) alone or with 5 μM U18666A, 25 μM risperidone (RIS), olanzapine (OLA) in the presence or absence of 5 mM metformin for 24 h and processed. Total protein extracts were run on 10% SDS-polyacrylamide gels and probed with anti AMPK, phospho-AMPK (p-AMPK) and actin (ACTB) Abs. The quantification of AMPK phosphorylation levels and the uncropped gels are reported in Supplementary Fig. 5A and B. B HepG2 cells were treated as described above. Total RNA was used to analyse SREBF1 and SREBF2 expression levels by Real Time PCR. Data are expressed as fold increase over levels of untreated HepG2 cells (unt) (one-way ANOVA followed by Tukey’s multiple comparison test, n = 3 experiments; *vs. unt cells). C HepG2 cells were treated with metformin alone or with U18666A, risperidone, olanzapine in the presence or absence of metformin, and with the SREBP2 positive control lovastatin, and the SREBP1 positive control T09 for 24 h. Cytosolic (C) and nuclear (N) extracts were prepared, loaded on 6% and 10% gels and probed with anti SREBP1 and SREBP2, the nuclear marker Nibrin and the cytosolic marker α-Tubulin. The 60 kDa nuclear active SREBP1 and SREBP2 levels were quantified, normalised on Nibrin levels and reported in the graphs. The cytosolic cleaved SREBP2 levels were quantified, normalised on α-Tubulin levels and reported in the graph (one-way ANOVA followed by Sidak’s; n = 3 experiments; *vs. unt cells). Uncropped gels are in Supplementary Fig. 7. D HepG2 cells treated with the indicated compounds were fixed and incubated with a solution of Oil-Red-O 0.2% w/v in 40% v/v isopropanol for 30 min and counterstained with DAPI. Scale bar =10 µm. E The percentage of cell area covered by lipid droplets (n = 45 cells) was quantified and reported in the graph. F The lipid droplets fluorescence (n = 65 cells) was quantified and expressed as fold increase over levels of untreated cells (one-way ANOVA followed by Dunnett’s multiple comparison test; *vs. unt cells). Scale bar =10 µm. G HepG2 treated cells were fixed and incubated with 100 µg/ml Filipin III (blue) and anti LAMP1 Ab (green). Scale bar =10 µm. H Lysosomal number (n = 45 cells) and lysosomal diameter (n = 400 vesicles), and I the percentage of cell area covered by Filipin III accumuli (n = 45 cells) and the diameter of Filipin III positive accumuli (n = 400 vesicles), were quantified and reported in the graphs (one-way ANOVA followed by Sidak’s multiple comparison test; *vs. unt cells).