Fig 2: Cold- and CL316243-dependent upregulation of Sgk2 gene expression is mediated by PGC-1a and NT-PGC-1a. (A) Effect of cold on Sgk2 mRNA and protein levels in BAT. 9-week-old BL6 female mice were housed at 28°C (n=10) or exposed to 4°C for 5h (n=10). (B) Effect of cold on Sgk2 mRNA and protein levels in IWAT. 9-week-old BL6 female mice were housed at 28°C (n=7) or exposed to 4°C for 7days (n=7). (C) Effect of ß3AR stimulation on Sgk2 expression in BAT. 8-week-old BL6 male mice were administered with vehicle (n=8) or a ß3AR agonist CL316243 (n=8) for 5h. (D) Effect of ß3AR stimulation on Sgk2 expression in IWAT. 8-week-old BL6 male mice were administered with vehicle (n=5) or CL316243 (n=5) for 7days. (E) Upregulation of Sgk2 expression by a ßAR agonist or cAMP in brown adipocytes. Differentiated brown adipocytes (7days) were treated with vehicle, isoproterenol (5µM) or dibutyryl cAMP (0.5mM) for 4h (n=4 per group). (F) Upregulation of Sgk2 expression by cAMP in primary beige adipocytes. Differentiated beige adipocytes (7days) were treated with vehicle or dibutyryl cAMP (0.5mM) for 4h (n=5 per group). (G) Enrichment of PGC-1a/NT-PGC-1a on the Sgk2 promoter in cold-activated BAT. The PGC-1a/NT-PGC-1a ChIP-seq peak (Chang et al., 2018) was visualized by the Integrative Genomics Viewer (IGV) v2.3. (H) PCR analysis of PGC-1a/NT-PGC-1a recruitment to the ERRE of the Sgk2 promoter. ChIP was carried out with PGC-1a antibody (Chang et al., 2018) in nuclear extracts of BAT isolated from mice exposed to 4°C for 5h. (I) Decreased induction of Sgk2 expression in Ppargc1a -/- brown adipocytes treated with dibutyryl cAMP (n=5 per group). (J) PGC-1a- and NT-PGC-1a-dependent upregulation of Sgk2 expression in Ppargc1a -/- brown adipocytes (n=4 per group). (K) Schematic presentation of transcriptional regulation of Sgk2 by cold or ß3AR agonist. All data are presented as the mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 determined by Student’s t test.

Fig 3: Sgk2 ablation does not alter βAR-stimulated mitochondrial respiration in brown adipocytes. (A) No change in brown adipogenesis by Sgk2 ablation. Sgk2+/+ (WT) and Sgk2−/− (KO) brown preadipocytes were differentiated into brown adipocytes for 7days and treated with or without isoproterenol for 4h (n=3–4 per group). (B) Effect of Sgk2 ablation on downstream signaling in isoproterenol-stimulated brown adipocytes. (C) Effect of Sgk2 ablation on mitochondrial respiration in differentiated brown adipocytes (7days) in the absence and presence of βAR stimulation. Oxygen consumption rates (OCR) were measured at baseline and after addition of oligomycin and FCCP (n=4 per group). All data are presented as the mean±SEM. **p<0.01, #p<0.0001 determined by Student’s t test.

Fig 4: SGK2 activation induces phosphorylation of downstream substrates in brown adipocytes. (A) Cold-dependent activation of SGK2 signaling in BAT. 9-week old BL6 mice were exposed to 4°C for indicated times. (B) No change in expression of genes involved in brown adipogenesis and thermogenesis by SGK2 activation. Brown preadipocytes were transduced with retrovirus expressing an empty vector or SGK2-S356D, followed by differentiation into brown adipocytes for 7days (n=3 per group). (C) Effect of SGK2 activation on downstream signaling in brown adipocytes starved for serum for 8h. (D) Effect of SGK2 activation on mitochondrial respiration in brown adipocytes. Oxygen consumption rates (OCR) were measured in differentiated brown adipocytes (7days) at baseline and after addition of oligomycin and FCCP (n=5 per group). All data are presented as the mean ± SEM. *p < 0.05 determined by Student’s t test.

Fig 5: SGK2 knockdown facilitates ferroptosis by downregulating GPX4.A Protein levels of ferroptosis-related genes were examined in SGK2 overexpressed or knockdown PC3 cells by western blotting. B Western blotting analysis of GPX4 expression in PCa tissues without lymph node metastasis(N0) and PCa tissues with lymph node metastasis(N1). C, D Immunohistochemistry (IHC) analysis of GPX4 expression difference and H-scores of GPX4 among PCa tissues without lymph node metastasis(N0) and PCa tissues with lymph node metastasis(N1) and corresponding Lymph Node Metastases tissues. Scale bars, 20 µm. E Western blot assay showed the reversion efficiency of overexpressed GPX4 after SGK2 knockdown in PCa. F, G Cell viability of PCa cells with SGK2 knockdown, with/without GPX4 overexpression, were measured after treating with Era (5 µM), Era (5 µM) and Ferr-1(5 µM) for 24 h by CCK-8 assay. H Concentrations of MDA were measured in the indicated group. I Intracellular ROS of the indicated group was stained by DCFH-DA and determined by flow cytometry. Data are presented as representative images or as the mean ± SD of three independent experiments. *P < 0.05.