Fig. 4: Biogenesis of LDs participates in endosomal trafficking.

A, B Control or ACSL3-knockdown A549 cells were transiently transfected with GFP-Rab5a (A) or GFP-Rab7a (B), and then treated with Nile Red (1 μM) for 30 min, followed by confocal imaging. The number of Rab5-positive early endosomes (EEs) or Rab7-positive late endosomes (LEs) was quantified by ImageJ (n = 20 for both WT and Sg-ACSL3 in (A, B); p = 0.0001 for (A), and p < 0.0238 for (B)). C, D A549 cells were treated with DMSO or TC (5 μM) overnight. The cells were then fixed with 4% PFA, immunostained with antibodies against Rab5a (C) or Rab7a (D), and co-stained with Bodipy 493/503 (1 μM) and DAPI (1 μg/mL) for 30 min. The number of EEs and LEs was quantified by ImageJ (For C, n = 18 for both WT and TC, p = 0.0103; for D, n = 16 and 17 for WT and TC, respectively, and p = 0.0013). E Control or ACSL3-knockdown A549 cells treated with DMSO or AP1 (1 μM) were incubated with LDL-488 on ice for 1 h, and then released into the warm medium for the indicated times, and the fluorescence of LDL was calculated by ImageJ (n = 30 for all groups; at 180 min, compared to WT, p < 0.0001 for both AP1 and Sg-ACSL3, while compared to AP1, p = 0.0731 for ACSL3). Images were captured by a Zeiss 880 microscope with a 63x objective lens. The scale bar is 5 μm. The graphs represented data from three independent experiments. The statistical significance of differences was determined by using the unpaired two-tailed student’s t test, and data quantifications were expressed as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001; ns: no significance.