Fig. 6: Supplementing 18:3 fatty acid triggers CI-instability in HEK293T cells. | Nature Communications

Fig. 6: Supplementing 18:3 fatty acid triggers CI-instability in HEK293T cells.

From: Kingdom-specific lipid unsaturation calibrates sequence evolution in membrane arm subunits of eukaryotic respiratory complexes

Fig. 6

a Top: Schematic representing workflow. Bottom: Bar-graph showing abundance of different CL-species in mitochondria of Hs-A1-EGFP cells treated with BSA, linoleic and linolenic acid. *– CLs with multiple isomeric species; 68:2| (16:0)2(18:1)2 / (16:0)1(16:1)1(18:0)1(18:1)1; 68:5| (16:1)2(18:1)1(18:2)1/ (16:0)1(16:1)1(18:2)2; 72:5| (18:1)3(18:2)1/ (18:0)1(18:1)1(18:2)2; 68:6| (16:1)2(18:1)1(18:3)1/ (16:0)1(16:1)1(18:2)1(18:3)1; 70:6| (16:1)1(18:1)2(18:3)1/ (16:0)1(18:0)1(18:3)2; 70:7| (16:1)1(18:1)1(18:2)1(18:3)1/ (16:1)1(18:0)1(18:3)2; 72:7| (18:1)2(18:2)1(18:3)1/ (18:0)1(18:1)1(18:3)2; 72:10| (18:2)2(18:3)2/ (18:1)1(18:3)3; 68:7| (16:1)2(18:2)1(18:3)1/ (16:0)1(16:1)1(18:3)2. Diamond: Data points and outliers. CLs with at least 3 data points have only been plotted. n = 6 biological replicates. Error bar: 1 SE from mean. Calculations in Source Data. b. Left: Schematic representing BSA-conjugated lipid supplementation assays. Right: Native-PAGE processed for in-gel fluorescence (IGF). Densitometry of SCs provided. Diamond: Data points. n = 3 biological replicates. Error bar: 1 SD from mean. c Immunoblot showing cleaved product of Ch-A1-EGFP upon supplementation with control BSA or BSA conjugated 18:2 and 18:3 fatty acids. Hsp60 as loading control. *Cleaved bands. Densitometry of cleaved bands normalized by Hsp60 provided. Diamond: Data points. n = 4 biological replicates. Error bar: 1 SD from mean. d Western blots (WB) using anti-Ndufs1, anti-Sdha and other mitochondria-specific (Glud1+Glud2, Hsp60) antibodies. WB sequentially probed with Ndufs1, Glud1+Glud2, Hsp60 and Sdha. <<< Ndufs1 SC; << Ndufs1 HC; < Ndufs1 SubC; $Glud1+Glud2; #Hsp60; *Sdha HC; **Sdha SubC. Diamond: Data points. Densitometry analysis for Ndufs1-SCs normalized to Glud1+Glud2 from 3 biological replicates provided. Error bar: 1 SD from mean. c–d arb. units: Arbitrary unit. Native-PAGE processed for in-gel CI-activity assay. Densitometry of activity bands (bracketed) provided. n = 3 biological replicates. Error bar: 1 SD from mean. (b & e) SC- supercomplex; HC- holocomplex; SubC- subcomplex. All uncropped gels and blots in Source Data. b–e P values from two-sided, paired Student’s t-test with 95% confidence interval without any corrections.

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