Bodipy 493 503 dye

The LD accumulation of the test compounds was evaluated according to the studies published by co-author Professor Chia-Hung Yen [1 (link)]. The LD accumulation was detected by BODIPY^® 493/503 dye (Thermo Fisher Scientific). LD accumulation was achieved by treating cells with OA conjugated to BSA. All data were analyzed with GraphPad Prism 6.01 software (La Jolla, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s comparison test was used to compare differences between multiple groups. A p-value < 0.05 was considered statistically significant.

Different experimental groups were designed, and each group of cells was inoculated in 6-well plates at 5 × 10⁵ per well and placed in an incubator (37 °C, 5% CO₂) for 24 h to reach 40–50% confluence. Cells were lysed in 5% Triton X-100 Cell Lysis Buffer. Intracellular neutral lipids were detected using BODIPY 493/503 dye (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. Intracellular triglyceride (TAG) and phospholipid (PL) content was measured using a triglyceride quantitative detection kit (BioAssay Systems, Hayward, CA, USA) and a phospholipid quantitative detection kit (BioAssay Systems), respectively.

To observe PLA accumulation, cells were grown as described in section “Culture Conditions for PLA Production” and washed twice with water. Pellets were then resuspended in water and PLA accumulation was immediately visualized in the cells using BODIPY 493/503 dye (Thermo Fisher Scientific) diluted at 1 μg/mL and imaged using Eclipse Ni-E epifluorescence microscope (Nikon France S.A, Champigny sur Marne, France).

The accumulation of LDs was detected by BODIPY^® 493/503 dye (Thermo Fisher Scientific). LD accumulation was achieved by treating cells with OA conjugated to BSA. Cells were seeded in μClear^® 96-well plates (Greiner Bio-ONE, Frickenhausen, Germany) and loaded with OA with testing drugs or DMSO for the indicated periods. Cells were then fixed with paraformaldehyde and stained with 2 μg/mL Hoechst 33342 and 1 μg/mL BODIPY^® 493/503. Four (in screening) or nine (in further experiments) fields for each well were picked and images for nuclei and LD were acquired and analyzed automatically by an HCS instrument (ImageXpress Micro System, Molecular Devices, Sunnyvale, CA, USA). A granularity analyzing module was used to identify nuclei and LDs. The diameter settings for defining nuclei and LDs are 8–25 and 0.5–2 μm, respectively. For monitoring fatty acid uptake, BODIPY-labelled dodecanoic acid (BODIPY^® FL C12, Thermo Fisher Scientific) conjugated to BSA was used in the LD assay. For the LD accumulation kinetic study, Hoechst 33342 and BODIPY^® 493/503 were added at the same time as OA. Time-lapse images of living cells in each well were acquired once an hour for 24 h by an ImageXpress Micro System automatically.

We seeded C3H10T1/2 cells on coverslips and fixed them with 4% polyformaldehyde for 10 min at RT. Then, we washed the cells with sterile 1× PBS three times. Next, we used 0.2% Triton X-100 in sterile 1× PBS to permeabilize the cells for 10 min at RT. We blocked the cells with 1% BSA in sterile 1× PBS for 1 h at RT and incubated the cells with appropriate primary and secondary antibodies in 1% BSA. To visualize LDs, we added 0.3 μg/ml BODIPY 493/503 dye (Thermo Fisher, D3922) to the cells during incubation with secondary antibodies and then stained cells with DAPI (Sigma). We mounted cell samples on glass slides with Vectashield antifade mounting medium and scanned the samples on a Leica SP5 confocal system, which was mounted on an inverted microscope.

To visualize LDs, treated Huh7 cells were washed twice with sterile PBS and fixed with 4% paraformaldehyde for 15 min at RT. After being washed three times with PBS, the cells were stained with freshly prepared ORO (Sigma, USA) or BODIPY 493/503 dye (Thermo Fisher Scientific, D3922) for 20 min at RT. Then, the staining solution was removed, and the samples were incubated with 4’, 6’- diamidino-2-phenylindole (DAPI, Roche, Switzerland) to stain the nuclei. Finally, the samples were washed with PBS for three times. The stained LDs were observed using confocal microscopy and then photographed.

An LD assay was performed as described in previous research [36 (link)]. The accumulation of LDs was detected with the BODIPY^® 493/503 dye (Thermo Fisher Scientific). LD accumulation resulted from treating cells with bovine serum albumin (BSA)-conjugated oleic acid (OA). Cells were seeded in μClear^® 96-well plates (Greiner Bio-ONE, Frickenhausen, Germany) and loaded with 125 μM of OA with testing compounds at a concentration of 40 μM for 16 h. Cells were then fixed with paraformaldehyde and stained with 2 μg/mL of Hoechst 33342 and 1 μg/mL of BODIPY^® 493/503. Nine fields for each well were picked, and images of nuclei and LD were acquired and analyzed automatically with an HCS instrument (ImageXpress Micro System, Molecular Devices, Sunnyvale, CA, USA). A granularity analyzing module was used to identify nuclei and LDs. The diameter settings for defining nuclei and LDs were 8–25 and 0.5–2 μm, respectively. The average LD counts/cell of BSA-conjugated OA + drug vehicle (DMSO)-treated wells (hereinafter referred to as OA) were used as the standard for 100% of fatty loading.