Fig. 3. ZNF189 regulates hormone-stimulated lipolysis.

(A and B) Representative Western blots of ZNF189, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cytosolic), and Lamin A/C (nuclear) in cytosolic (Cyt) and nuclear (Nuc) adipocyte fractions (A) and nuclear fractions from adipocytes electroporated with siRNAs targeting ZNF189 (siZ) or nontargeting control (siC) (B). (C to E) Gene expression (C), adiponectin secretion (D), and glycerol levels (E) of siC and siZ adipocytes. Glycerol was measured in conditioned media following a 3-hour incubation under basal or stimulated conditions using either isoprenaline or ANP. (F) Overview of the ANP and isoprenaline signaling pathways. NPR, natriuretic peptide receptor; βAR, beta-adrenergic receptor; Gαs, Gs alpha subunit, AC = adenylate cyclase, PKG/PKA, protein kinase G and A; DG, diglyceride; MG, monoglyceride. (G) Representative Western blots for HSL phosphorylated at serine-660 (activating phospho-site; pHSL^Ser660), total HSL (HSL), and GAPDH from basal, isoprenaline- and ANP-treated siC and siZ cells. (H) Principal components analysis of 307 lipid species measured by shotgun lipidomics of basal and ANP-stimulated siC and siZ adipocytes. Confidence ellipses display 95% confidence intervals. (I) Total triglycerides for the samples in (H) presented as fold change versus same siRNA basal companion. (J and K) Individual triglyceride species comparing siC versus siZ adipocytes. Volcano plots for ANP responsiveness of all triglycerides are shown in (J), and lipid abundance and ANP responsiveness are highlighted for the species with altered responsiveness following ZNF189 depletion in (K). In (J), adjusted P reflects FDR correction on multiple comparisons analyses performed using limma. Bar charts are presented as means ± SEM. In (C) to (E) and (I), replicates are highlighted by dots. For targeted analyses, data are based on at least three independent experiments. ***P < 0.001 for Welch’s t test [(C) and (I)] or Tukey’s multiple comparisons test (E).