Fig. 6: VPS13D and TSG101 are required for efficient FAs transfer from LDs to mitochondria. | Nature Communications

Fig. 6: VPS13D and TSG101 are required for efficient FAs transfer from LDs to mitochondria.

From: An ESCRT-dependent step in fatty acid transfer from lipid droplets to mitochondria through VPS13D−TSG101 interactions

Fig. 6

a–h Confocal image of HEK293 cells in Red C12 pulse-chase assays after treatments with scrambled (a), VPS13D (b), TSG101 (c), CHMP4B (d), CHMP6 (e), CHMP1B (f), IST1 (g), or ALIX siRNAs (h). i, j Quantification of size (i) and number (j) of Red C12-labeled LDs after 5 h of starvation. Scrambled (n = 177 from 10 cells), VPS13D (n = 488 from 11 cells), TSG101 (n = 589 from 15 cells), CHMP4B (n = 418 from 12 cells), CHMP1B (n = 475 from 12 cells), CHMP6 (n = 322 from 13 cells), IST1 (n = 284 from 10 cells) or ALIX (n = 257 from 10 cells) siRNAs treated cells were analyzed. k Relative rate of Red C12 uptake to mitochondria upon 5 h of starvation was quantified by measurement of C12 fluorescence inside mitochondria. Scrambled (30 cells), TSG101 (14 cells), CHMP4B (12 cells), CHMP1B (12 cells), CHMP6 (12 cells), IST1 (11 cells) or ALIX (15 cells) siRNAs treated cells were analyzed. A.U., arbitrary unit. l Quantification of Red C12 fluorescence in whole cell areas. Scrambled (10 cells), VPS13D (11 cells), TSG101 (15 cells), CHMP4B (12 cells), CHMP1B (12 cells), CHMP6 (12 cells), IST1 (10 cells) or ALIX (11 cells) siRNAs treated cells were analyzed. A.U., arbitrary unit. m Seahorse FAO assays to examine the transport and oxidation of endogenous FAs in scrambled, Eto (40 μM) pretreated, VPS13D or TSG101 siRNAs treated HepG2 cells. The p values were labeled in front of the corresponding data points for Eto-pretreated (red), VPS13D siRNA (green), and TSG101 siRNAs (magenta) treated groups, compared with the scrambled siRNA treated group (blue). Oligo: oligomycin; FCCP: Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; RTN: rotenone; AA: Antimycin. Four independent assays were performed with similar results in (i-l), and at least three biological replicates were performed with similar results in m. The statistic analysis was performed with two-tailed unpaired student’s t-test, and data are means ± SD in i–m. Scale bar, 10 μm in whole cell image and 2 μm in insets in a–h.

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