Extended Data Figure 6: C18:0 removal does not lead to endoplasmic reticulum stress, and inhibiting UPR does not inhibit mitochondrial fragmentation upon C18:0 removal.

a, C18:0 removal for 24 h does not induce expression of UPR target genes, quantified by quantitative RT–PCR, normalized to RPL19. BiP (also known as HSPA5) is a readout for IRE1 (also known as ERN1) activation, CHOP (also known as DDIT3) is a readout for ATF6 activation, and PERK (also known as EIF2AK3) is a readout of its own activation due to a positive transcriptional feedback loop. Tunicamycin serves as a positive control. y axis is displayed in a logarithmic scale to fit all data points on one graph. The experiment was done in triplicates. b, p-eIF2α, a UPR marker, does not increase upon removal of C18:0 whereas it is induced by tunicamycin, a positive control. See Supplementary Fig. 18 for image of the uncropped full western blot. c, Knocking down mediators of the UPR response does not inhibit mitochondrial fragmentation upon C18:0 removal. HeLa cells were transfected with either control siRNAs or siRNAs targeting UPR mediators as indicated. Left, the mitochondrial fragmentation index; right, representative images. n = 15. d, Inhibiting endoplasmic reticulum stress by means of a chemical chaperone, TUDCA, does not rescue mitochondrial fragmentation upon C18:0 removal. HeLa cells were pre-treated with 500 μg ml⁻¹ TUDCA 30 min before delipidated serum treatment. Left, mitochondrial fragmentation index (n = 15); right, representative images. a, c, d, *P < 0.05, **P < 0.01, two-tailed t-test. Error bars show s.d.