Fig 2: Alterations of protein S-glutathionylation modification in macrophages under oxidative conditions.a Schematic of the enrichment and quantitative analysis for SSG-modified Cys-peptides. b Enrichment of protein SSG and LC–MS/MS in BMDMs exposed to H2O2 (200 µM) for 0, 15 min. Gene ontology annotations for biological process of proteins with significant S-glutathionylation alterations found to be increased or decreased in any stage by >1.2-fold compared to control. The slice for metabolic process is slightly displaced from the rest of the pie chart. c Alterations in S-glutathionylation sites under exposure to H2O2 [shown as fold changes of protein S-glutathionylation (H2O2 treated/control). Gray, identified protein sites; blue, metabolism-related differential proteins; orange, FABP5]. d mRNA levels of Ptbp1, Otub1, Ctsz, Gapdh, and Fabp5 in BMDMs as evaluated by qPCR, n = 3 biologically independent samples over 3 independent experiments, data are shown as mean ± SEM and analyzed with a 95% confidence interval, P < 0.0001, one-way ANOVA is followed by Tukey’s post-hoc test. Source data are provided as a Source Data file.

Fig 3: FABP5 promotes cell growth via ERRα signaling pathway in PCa cells(A) PCa cells were transfected with control siRNA (20 nM) or siFABP5 (20 nM) and mRNA levels of various factors were determined by qPCR. Results are means ± S.D. for three independent experiments. (B) PNT2 cells were transfected with pCI-neo (e.v.), pCI-neo/FABP5 (FABP5) or pCI-neo/NES-FABP5 (NES-FABP5) and mRNA levels of various factors were measured by qPCR. Results are means ± S.D. for three independent experiments. (C) Schematic of the ATP5B promoter region and luciferase constructs with the wild-type (WT) sequence or mutant deleted for the ERRE (ΔERRE). (D) The luciferase reporter vector driven by the ATP5B promoter (WT) or ΔERRE mutant was co-transfected with hRluc/TK into PC-3 and DU-145 cells, and treated with control siRNA (20 nM) or siFABP5 (20 nM). Luciferase activities were measured and normalized against Renilla activities. Results are means ± S.D. for three independent experiments. (E) The WT or ΔERRE mutant reporter vector was co-transfected with hRluc/TK into PNT2 cells transfected with pCI-neo (e.v.), pCI-neo/FABP5 or NES-FABP5. Luciferase activities were measured and normalized against Renilla activities. Results are means ± S.D. for three independent experiments. *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett's test (A, B, D and E), N.S. = Not significant.

Fig 4: Schematic model for the FABP5-mediated transcriptional controlling network of mitochondrial functions in PCa cells(A) FABP5-ERRα crosstalk is required for mitochondrial functions and cell proliferation in PCa cells. FABP5 activated mRNA expression of more than one metabolic gene via ERRα signaling in PCa cells. (B) FABP5 knockdown induced high energy stress (increase of AMP/ATP ratio) in PCa cells. AMPK was activated by increased AMP/ATP ratio. Moreover, autophagy, apoptosis and cell cycle arrest were induced in PCa cells transfected with siRNA against FABP5 via the AMPK-FOXO3A signaling pathway. Thus, we considered that metabolic genes regulated by FABP5-ERRα played an essential role for energy homeostasis and viability in PCa cells. These results suggested that FABP5 may be a molecular target for the antitumorigenic treatments for PCa.

Fig 5: FABP5 knockdown induces starvation stress and activation of the AMPK-FOXO3A signaling pathway in PCa cell lines(A) Cell cycle analysis of control siRNA (20 nM) or siFABP5 (20 nM) transfected PCa cells by flow cytometry. Percentages of cells at each phase are indicated. (B) Apoptosis was measured by cleaved caspase 3 (active form) level by flow cytometry. (C) Amounts of AMP+ADP and ATP were measured by the microplate reader using firefly luciferase in control siRNA (20 nM) or siFABP5 (20 nM) transfected PCa cells. Data are presented as the means ± SD from four independent experiments. (D) Relative amount of ATP was measured using firefly luciferase in pCI-neo (e.v.), pCI-neo/FABP5 or pCI-neo/NES-FABP5-transfected PNT2 cells. Results are mean ± S.D. for twenty independent experiments. (E) Western blot analysis of AMPK and phosphorylated AMPK in prostate cancer cells transfected with control siRNA (20 nM) or siFABP5 (20 nM). Results shown are representative of three independent experiments. (F) Western blot analysis of FOXO3A and proteins encoded by its target genes in PCa cells transfected with control siRNA (20 nM) or siFABP5 (20 nM). Results shown are representative of three independent experiments. **P < 0.01, two-way ANOVA followed by Tukey's test (A), one-way ANOVA followed by Dunnett's test (B, C and D), N.S. = Not significant.