Fig. 2: Golgi targeting features of CyMA precursors and the role of enzymes in futile cycle creation and disruption.

a CLSM and colocalization of GALNT2-RFP HeLa cells treated with 1a (1 μM, 10 min) (n = 6). b CLSM of HeLa treated with 1a (500 nM) at indicated times. c Golgi fluorescence intensity in HeLa pretreated with endocytosis inhibitors and treated with 1a (500 nM) (n = 6). d CLSM and Golgi fluorescence in HeLa cells pretreated with vehicle, ML211, or DC661, then treated with 1a (1 μM, 1 h) (n = 6). e Golgi fluorescence in HeLa transfected with indicated TE siRNA and treated with 1a (5 μM) (n = 5). f HRMS of 1a and its palmitoylated form. g CLSM and “Golgi-specificity” analysis of HeLa cells pretreated with vehicle or DHHC inhibitor (2-BP) for 30 min and treated with 1a (2 μM, 20 min). h CMC determination of peptide-SH form and peptide-SPalm form of 1a. i TEM of peptide-SH form and peptide-SPalm form of 1a. j CLSM and the Golgi fluorescence intensity of HeLa pretreated with vehicle, Triacsin C (10 or 20 μM), Triacsin C (20 μM) + palmitoyl-CoA (50 μM) for 30 min, and incubated with 1a (500 nM) (n = 10). k Golgi fluorescence in HeLa pretreated with 1a (1 μM, 30 min), switched to media with DC661 or 2-BP (n = 6). l FRAP and mobile/immobile fraction analysis of Golgi after 1a (2 μM, 30 min) or C6-NBD-Ceramide (10 μM, 1 h) treatment (n = 10). m Golgi fluorescence in CES-negative (HeLa, KPCA-C) and CES-positive (HepG2, RAW264.7) cells treated with 1a (2 μM) (n = 6). n Golgi fluorescence intensity in CES-positive cells (HepG2, RAW264.7) pretreated with vehicle, Nevadensin, or Loperamide, and treated with 1a (1 μM) (n = 6). o Schematic of CyMA assembly/disassembly dynamics. Scale bar = 20 μm. Data are mean ± s.d. (n-values as indicated in the panel). Statistical significance was determined by a two-tailed Student’s t test. Reproducibility and statistical details are provided in the Methods. Source data are provided as a Source Data file.