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Figure S3. Live cell imaging of LD growth induced by fusogen. (A) Depicted are live cell images of HepG2 cells either (A1) without any treatment (K-) or (A2) treatment with the DMSO vehicle control. (A3) treatment with a 0.5 mM mixture of oleic acid and palmitic acid for 24 h; (A4) FA and 200 μM propranolol treatment for 3 h. The live cell images were captured at 40x using time-lapse z-stack by phase contrast fluorescence microscopy (Nikon TiE) and further analyzed using the NIS elements software version 4.13. (B) Depicted are live cell images of LD in HepG2 cells. Upon propranolol treatment the smaller LDs (orange arrows) appear to fuse with larger LDs (red arrows). The insets represent actively grown LDs. (B1) Clockwise: r = 9.14 μm, 12.79, 11.34, 7.9, 8.94, 9.04, 10.26, 11.38, 11.09, 10.28, 8.93. (B2) r = 12.41, 11.36, 12.46, 10.88; (B3) r = 16.33, 14.21, 11.11, 17.36; (B4) r = 19.25, 15.88, 19.54). (B5) The fusion events did not result in spherical lipid droplets; hence the volume could not be determined. The figures were captured at 40x using time-lapse phase contrast microscopy (Nikon TiE) and further processed using the NIS elements software version 4.13. The lipid droplets were stained with the blue fluorescent MDP dye (images are not shown). The size bar refers to 10 μm. (PDF 417 kb)

Figure S1. Quantification of intracellular lipid content over time. (A) The plotted graph depicts three treatment conditions, i.e. HepG2 cells without any treatment (K-) or after treatment with the DMSO vehicle control (K+) or after treatment with a 1:1 mixture of the fatty acids OA/PA for 1 h, 2 h, 4 h, 6 h, 24 h, 48 h and 72 h. After 6 h of treatment, the lipid content increased by 25 μg/ml (K+, n = 28; PA/OA, n = 23); after 24 h by 24 μg/ml (K+, n = 15; PA/OA, n = 18), after 48 h by 70 μg/ml (K+, n = 14; PA/OA, n = 17) and after 72 h by 71 μg/ml (K+, n = 7; PA/OA, n = 14) when compared to the DMSO vehicle control (K+), respectively. (T-Test K+ ↔ PA/OA: 1 h, p ≤ 0,00; 2 h, p ≤ 1,87 × 10− 6;4 h, p ≤ 3,5 × 10− 7; 6 h, p ≤ 6,3 × 10− 10; 24 h, p ≤ 2,4 × 10− 7; 48 h, p ≤ 2,6 × 10− 11; 72 h, p ≤ 1,6 × 10− 11). *corresponds to a p-value of p ≤ 0,001. (B) The histogram visualizes the increase in intracellular lipid content over time as compared to the DMSO vehicle control. (PDF 356 kb)

Table S2. List of mechanistically linked steatotic genes. (PDF 185 kb)

Figure S2. Live cell imaging of LD fusion. Live cell images of HuH7 cells treated with 0.5 mM of 1:1 mixture of oleic acid and palmitic acid for 24 h. The smaller LDs (orange arrows) appear to fuse with larger LDs (red arrows). Panel B represents the initiation of fusion events where two LDs (v = 0.86 μm3; v = 2.9 μm3) and LDs (v = 1.49 μm3; v = 6.88 μm3) fuse to form a larger LD (v = 3.2 μm3) and (v = 8.78 μm3), respectively (panel D). The figures were captured at 40x using time-lapse z-stack by phase contrast microscopy (Nikon TiE) and processed using the NIS elements software version 4.13. The lipid droplets were stained with the blue fluorescent MDP dye (images are not shown). The size bar refers to 10 μm. (PDF 192 kb)

Table S1. Patient characteristics of hepatocyte donors. (PDF 170 kb)

Table S3. Compilation of NAFLD responsive genes in in-vitro/in-vivo comparisons. (PDF 96 kb)