Extended Data Fig. 1: PEX2/10/12 downregulation increases iBAs lipolysis without effects on differentiation levels.
From: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis
(a) Quantification of peroxisomes in the proximity to LDs and LDs in proximity to peroxisomes in iBAs at basal state and stimulated state (peroxisome quantification, cell number = 10; LD quantification, cell number = 14 in control and 15 in Iso treatment). LD labelled by LipidTOX Deep Red dye (red) and peroxisomes labelled by EGFP-PTS1 (green). Scale bar, 5 μm. (b) Quantification of peroxisomes in the proximity to LDs in HepG2 cells (Cell number = 15). LD labelled by LipidTOX Deep Red dye (red) and peroxisomes labelled by EGFP-PTS1 (green). Scale bar, 10 μm. (c) Differentiation and screening strategy in iBAs. A pool of three different duplexes was utilized to knock down individual PEX target. (d-e) IBAs were transfected with siRNAs to knock down Pex2/10/12. After 72 h, lipolysis was determined as level of glycerol and NEFA released into starvation medium during 2 hours at basal state and 1 hour at stimulated state induced by 1 μM Iso (N = 5, F = 21.58 in basal state and 15.38 in stimulated state in d; F = 11.81 in basal state and 8.797 in stimulated state in e). (f-h) IBAs were transfected with siRNAs to knock down Pex2/10/12. After 72 h, Pex2/Pex10/Pex12, Pparg2 and Adipoq transcripts were quantified by qPCR (N = 3). (i) IBAs were transfected with siRNAs to knock down Pex2/10/12. After 72 h, levels of adipocyte differentiation were determined via high-content imaging (N = 6, F = 4.51). (j) IBAs were transfected with siRNAs to knock down Pex2/10/12. After 72 h, cells were stained by Oil Red O and the latter was extracted by isopropanol for quantification (N = 3, F = 1.816). (k-l) IBAs were transfected with siRNAs to knock down Pex2. After 72 h, peroxisome mass was analyzed by IB or IF via peroxisomal membrane protein PMP70 (N = 4 in Pex2 and 8 in Nc siRNA, F = 22.61). Scale bar, 40 μm. Repeated 3 times in l. (m) IBAs were transfected with siRNAs to knock down Pex10/12. After 72 h, peroxisome mass was analyzed by IB as indicated. Repeated 3 times. Results are shown as mean ± SEM and analyzed using Student’s two-sided t test (a) and an ANOVA test with Dunnett correction for multiple comparisons between control and other groups (d-k).
