Fig. 9: Evaluation of the role of the H-motif and phosphorylation in the LD binding of HSL in 3T3-L1 adipocytes.

a Subcellular localization of human HSL and its variants in 3T3-L1 adipocytes. Wild-type HSL, HSL with the H-motif (residues 489-538) deleted, and full-length HSL carrying the phosphorylation site mutations (S552A/S649A/S650A), each with an EGFP fused to its C-terminus, were stably expressed in 3T3-L1 preadipocytes. The 3T3-L1 preadipocytes were then induced to differentiate into mature 3T3-L1 adipocytes using dexamethasone, IBMX, and insulin (see Methods). The 3T3-L1 adipocytes were treated with 1 mM IBMX and 10 µM forskolin or the same volume of DMSO control, then the adipocytes were imaged as described in Fig. 3e. b Quantification of the EGFP signal of human HSL and its variants on lipid droplets in 3T3-L1 adipocytes (a total of 15 cells were analyzed from three independent measurements). c Upon treatment with forskolin or DMSO, the protein expression and phosphorylation levels of human HSL and its variants in 3T3-L1 adipocytes were analyzed using Western blot. The data in a and c, are the results of a representative experiment out of three independent experiments. The data in b represent the mean ± SD of three independent measurements. The data in b were analyzed using the unpaired t-test in Prism to calculate the two-tailed P-values. Source data are provided as a Source Data_Fig. 9 file.