Fig. 3: Biogenesis of LDs is essential for AP1-C-induced enlarged intermediate endosomes.

A A549 cells were treated with DMSO, Oleic acid (OA) (200 μM), or Triacsin C (TC) (5 μM) overnight and then co-stained with Bodipy 493/503 (1 μM) and AP1-C (10 μM) for 1 h, followed by confocal imaging. The fluorescence intensity of cells was quantified by ImageJ (n = 30 for all columns; compared to the control group, for the Bodipy 493/503 group, p < 0.0001 for both OA and TC; for the AP1-C group, p < 0.0001 for both OA and TC). B–E A549 cells were transiently co-transfected with GFP-Rab5a and mc-Rab7a, treated with DMSO or TC (5 μM) overnight, and subsequently treated with DMSO or AP1-C (10 μM) for an additional 6 h before confocal imaging (B). The colocalization (MCC) of Rab5a and Rab7a was quantified using ImageJ (C) (p < 0.0001), the diameter of vacuoles (diameter > 2 µm) was calculated using ZEN Black software (D) (p = 0.0007), and the fluorescence intensity was quantified using ImageJ (E) (p < 0.0001). Sample sizes were n = 8 for the control group and n = 7 for the TC-treated group in (C–E). F Knockdown efficiency of ACSL3 in A549 cells was analyzed by immunoblotting analysis (p < 0.0001). G–J Control or ACSL3-knockdown cells were transiently transfected with GFP-Rab7a, and then treated with DMSO or AP1-C (10 μM) for 6 h, followed by treatment with Nile Red (1 μM) for 30 min (G). Images were finally captured by a Zeiss 880 microscope with a 63x objective lens. The scale bar is 5 μm. The number of GFP-Rab7a-positive vacuoles (diameter >2 µm) in scramble-Sg-expressing cells or ACSL3-Sg-expressing cells was quantified using ZEN black software (H) (n = 17 and 11 for Scramble-Sg and Sg-ACSL3, respectively; p < 0.0001). The fluorescence intensity of AP1-C in scramble-Sg-expressing cells or ACSL3-Sg-expressing cells (I) (n = 17 and 11 for Scramble-Sg and Sg-ACSL3, respectively; p < 0.0001), as well as the number, size, and area of Nile Red-positive LDs in scramble-Sg-expressing cells treated with or without AP1-C (J) were calculated using ImageJ (n = 13 and 17 for control and AP1-C 6 h, respectively; p = 0.0067 for number of LDs, p = 0.0008 for size of LDs, and p < 0.0001for % area of LDs). 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.