Fig 2: Hmgcs2 is expressed in adipose tissue in vivo and in vitro, and adipocytes produce and secrete 3HBA. (a) Schematic diagram of microarray analysis to identify fasting-inducing genes expressed in adipose tissue. The genes upregulated by fasting are sorted in descending order. The following Gene Expression Omnibus DataSet was used for the analysis: GSE46495 (fold-change > 2.0, p < 0.05; 4666 genes). (b) qRT–PCR of Hmgcs2 in epididymal adipose tissue from C57BL/6 J mice after 12 h of feeding and fasting. n = 3. (c–e) qRT–PCR of Hmgcs2 in differentiaed 3T3-L1 adipocytes after 24 h of treatment with 10 mM 3HBA (c), 1 nM insulin (d), and 1 µM dexamethasone (e). n = 3. (f) qRT–PCR of Hmgcs2. n = 3. (g) Intracellular protein of Hmgcs2. n = 1. (h and i) 3HBA concentrations in cell lysate (h) and cell culture supernatant (i) of differentiated and undifferentiated 3T3-L1 adipocytes. n = 3. For measurement of 3HBA, cell lysate was normalized per well of a 6 well plate, and culture supernatant was normalized per 2 mL media for a well of 6 well plate. Here cropped blots were displayed and all full-length blots are included in the supplemental Fig. S2. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. A.U., Arbitrary Unit.

Fig 3: HMGcs2 promoter luciferase(A) Comparison of transcription factors co-transfected with the HMGcs2 promoter luciferase plasmid, n = triplicate wells, each experiment was performed at least 3 times. Mean ± SEM, the means are significantly different by ANOVA, * indicates statistically significant difference from control by Tukey’s post-hoc test.(B) Dose-response of PPARg expression plasmid with the HMGcs2 promoter luciferase plasmid.

Fig 4: Effects of HMGCS2 knockdown and overexpression on PCa cell growth. a LNCaPabl cells were stably infected with a doxycycline-inducible shHMGCS2 vector (ABLshHMGCS2). Following treatment with 1 µM doxycycline, HMGCS2 was effectively downregulated on protein level compared to the mock control as shown by Western analysis. GAPDH was used as loading control. b ABLshHMGCS2 cells were seeded into 96 well plates and incubated in the absence or presence of doxycycline over 5 days. Cell viability was determined with CellTiterGlo viability assay (Promega). Representative images were taken at the end of treatment (magnification 10x). c ABLshHMGCS2 cells were seeded into ULC 96 well plates (Corning) and allowed to form spheroids over 4 days. Then, 1 µM doxycycline (dox) and 5 µM enzalutamide (enza) were added. Cell viability was determined through CellTiterGlo viability assay after 10 days of treatment. Medium was exchanged twice a week. Representative images were taken at day 10 with IncuCyte S3 software. d LNCaP cells were transiently transfected with a HMGCS2 plasmid (LNCaP_HMGCS2). HMGCS2 overexpression was confirmed 72 h afterwards by Western blotting. GAPDH was used as internal control. e LNCaP cells were transiently transfected with a HMGCS2 plasmid and seeded into a 96 well ULC plate (Corning) to allow 3D spheroid formation. After 4 days, 5 µM enzalutamide was added in RPMI with 10% CS_FCS. After 10 days, cell viability was measured via CellTiterGlo assay. Representative images were taken at the end of treatment with IncuCyte S3 software. Data represent the mean plus SEM from at least three independent experiments. (* P < 0.05, ** P < 0.01)

Fig 5: HMGCS2 and AKR1C3 expression is significantly increased in human PCa. a, b Representative staining for HMGCS2 and AKR1C3 in benign (BPH) prostate tissue and PCa. Staining intensity was quantified by determining H-score in the epithelium (ep) (a) and in the stroma (str) (b) as described under methods. c Expression of HMGCS2 and AKR1C3 was correlated with Gleason score (GSC) (GSC = 6: N = 12, GSC = 7: N = 31, GSC = 8: N = 19) and (d) lymph node metastases (N0: N = 41, N1: N = 20). Data are represented as mean + SEM. (* P < 0.05, *** P < 0.001)