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. 2024 Jun 6;15:4847. doi: 10.1038/s41467-024-49224-x

Fig. 1. PNPLA3 degrades PUFA-LDs independently of ATGL.

Fig. 1

a ATGL−/− HeLa cells transfected with FLAG-PNPLA3 were treated overnight with (from left to right) 250 μM of OA, LA, α-LA, AA (250 μM), or DHA (125 μM) + EPA (125 μM), and then immunostained with a FLAG antibody. LDs were stained with BODIPY. b Quantification of LD area in (a). c ATGL−/− HeLa cells transfected with FLAG-tagged (from left to right) PNPLA3-WT, PNPLA3-S47A, or PNPLA3-I148M were treated overnight with 250 μM LA and then imaged as in (a). d Quantification of LD area in (c). e ATGL−/− HeLa cells transfected with Myc-tagged PNPLA3 or co-transfected with Flag-G0S2 were treated overnight with 250 μM LA and then imaged as in (a). f Quantification of LD area in (E). g WT control (left) or CGI-58 −/− (right) Huh7 cells transfected with FLAG-PNPLA3 were treated with 250 μM LA overnight and then imaged as in (a). h Quantification of LD area in (g). i PNPLA3−/− primary mouse hepatocytes were isolated and infected with null, PNPLA3 WT or PNPLA3 I148M expressing adenovirus, and loaded with 250 μM OA + 3H-OA (left) or 250 μM LA + 14C-LA (right). After cell lysis, isolated LDs were normalized for total TG content and incubated at 37 °C with 20 μM ATGListatin in a 5% FA-free BSA solution. At the indicated intervals, fractional aliquots were removed from the reaction mixture, and albumin-bound FA was extracted for scintillation counting (n = 4 per condition from a representative experiment). Scale bar in microscopic images = 10 μm. Error bars in (i) represent mean ± SD. (*) indicates statistical significance between [Null] and [PNPLA3 WT], (#) indicates statistical significance between [PNPLA3 WT] and [PNPLA3 I148M], and (+) indicates statistical significance between [Null] and [PNPLA3 I148M]. Box-plot elements: center line, median; box limits, upper and lower quartiles. *p < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t test. Specific p values: b Untransfected+OA vs PNPLA3 + OA: p = 0.488; Untransfected+OA vs Untransfected+LA: p = 0.961; Untransfected+LA vs PNPLA3 + LA: p = 1.22E-08; Untransfected+LA vs PNPLA3+aLA: p = 1.35E-06; Untransfected+LA vs PNPLA3 + AA: p = 2.25E-08; Untransfected+LA vs PNPLA3 + DH/EPA: p = 1.59E-08. d PNPLA3 WT + LA vs PNPLA3 S47A + LA: p = 2.46E-05; PNPLA3 WT + LA vs PNPLA3 I148M + LA: p = 6.43E-04; PNPLA3 S47A + LA vs PNPLA3 I148M + LA: p = 0.237. f p = 9.60E-06. h p = 9.22E-04. i OA loaded LDs: Null vs PNPLA3 WT/2 h: p = 0.0235, 4 h: p = 0.0011, 6 h: p = 0.030; Null vs PNPLA3 I148M/2 h: p = 0.458, 4 h: p = 0.804, 6 h: p = 0.715; PNPLA3 WT vs PNPLA3 I148M/2 h: p = 0.0060, 4 h: p = 0.00051, 6 h: p = 0.0538; LA loaded LDs: Null vs PNPLA3 WT/2 h: p = 3.63E-05, 4 h: p = 8.38E-04, 6 h: p = 3.91E-08; Null vs PNPLA3 I148M/2 h: p = 0.708, 4 h: p = 0.885, 6 h: p = 0.0029; PNPLA3 WT vs PNPLA3 I148M/2 h: p = 2.68E-06, 4 h: p = 6.87E-05, 6 h: p = 6.02E-07.