Fig 2: Macrophage ABHD5 potentiates CRC growth by suppressing SRM in vivo.(a) Immunoblotting assay of SRM in the PMs from WT or TgSRM mice. (b) The 6-week-old WT or TgSRM mice were subcutaneously inoculated with MC-38 cells, and the tumour volume was measured dynamically. The data represent means±s.e.m., (n=8, **P<0.01, Student's t-test). (c,d) Relative mRNA levels of ABHD5 (c) and SRM (d) in macrophages from the WT, TgABHD5, TgSRM or TgABHD5+SRM mice. MC-38 tumour cells were subcutaneously injected into the thighs of 6-week-old C57BL/6 mice. Ten days later, tissue macrophages were isolated. NSM, spleen macrophages from tumour-free mice; TSM, spleen macrophages from MC-38 tumour-bearing mice from MC-38 tumour tissues. The data represent means±s.e.m., (n=6, *P<0.05, **P<0.01, Student's t-test). (e) The relative spermidine levels in the plasma, PMs or TAMs from WT, TgABHD5, TgSRM or TgABHD5+SRM mice that were inoculated with MC-38 tumours subcutaneously for 10 days. The data represent means±s.e.m., (n=3, each sample was pooled from 3 individual samples; *P<0.05, **P<0.01, Student's t-test). (f) The 6-week-old WT, TgABHD5, TgSRM and TgABHD5+SRM mice were subcutaneously injected with MC-38 cells, and the tumour volume was determined dynamically. The data represent means±s.e.m., (n=10, *P<0.05, **P<0.01, Student's t-test). (g) The percentage survival of WT, TgABHD5, TgSRM and TgABHD5+SRM mice bearing MC-38 tumours. The data represent means±s.e.m., (n=10, *P<0.05, Student's t-test). (h) The 6-week-old WT BALB/c mice were subcutaneously injected with CT-26 tumours and treated with the CM from PMs of WT, TgABHD5, TgSRM and TgABHD5+SRM mice. The tumour volume was determined dynamically. The data represent means±s.e.m., (n=10, *P<0.05, **P<0.01, Student's t-test). (i) The percentage survival of subcutaneous CT-26 tumour-bearing BALB/c mice treated with the CM from peritoneal macrophages of WT, TgABHD5, TgSRM and TgABHD5+SRM mice. The tumour volume was measured dynamically. The data represent means±s.e.m., (n=10, *P<0.05, Student's t-test). NS, not significant.

Fig 3: Sspermidine synthase (SRM) promotes the secretion of extracellular vesicles in prostate cancer cells. A, Representative images of LysoTracker staining in control and SRM knockdown 22Rv1 cells. Scale bars, 10 µm. The number of vesicles in 22Rv1 cells was counted. Values represent mean ± SE (n = 3). B, Confocal immunofluorescence micrographs of 22Rv1 cells transfected with SRM siRNA. Cells were stained with anti-CD63 antibody. Scale bars, 10 µm. The number of vesicles in PCa cells was counted. The values represent mean ± SE (n = 5). C, Schematic model of the regulation of extracellular vesicle (EV) secretion in PCa. The novel signaling network of miR-1908 and its target SRM regulating EV secretion in PCa cells. In the normal cells, expression of miR-1908 was high, therefore expression of SRM was suppressed. However, because of downregulation of miR-1908 in PCa cells, expression of SRM was upregulated and led to the acceleration of EV secretion from PCa cells, resulting in the promotion of cancer malignancy. Therefore, targeting SRM in PCa could be a novel therapeutic target for PCa

Fig 4: Macrophage ABHD5 suppresses C/EBP?-mediated SRM expression.(a) A schematic depiction of different mouse SRM promoter regions cloned into the pGL4-basic plasmid. The constructs were designated PS1–PS5. (b) Each of the constructs PS1–PS5 (0.4 µg ml-1) was transfected into the PLKO or ABHD5-KD RAW cells, and then the luciferase activity was assayed. The data represent means±s.e.m., (n=3, **P<0.01, Student's t-test). (c) A diagram of the PS1 construct with a C/EBP?-binding element, which was mutated from 5'-GAGCAA-3' to 5'-GAACTA-3' located at -605/-600. (d) The construct PS1 (PS1-WT; 0.4 µg ml-1) and the C/EBP? element-mutated PS1 (PS1-mut) (0.4 µg ml-1) were transfected into the PLKO or ABHD5-KD RAW cells for 24 h, and then the reporter-gene assay was performed. The data represent means±s.e.m., (n=3, **P<0.01, Student's t-test). (e) The PLKO or ABHD5-KD RAW cells transfected with control siRNA (siNC) (20 nmol ml-1) or C/EBP? specific RNA (siC/EBP?) (20 nmol ml-1) for 24 h were collected for ChIP assay. The interaction between C/EBP? protein and SRM promoter DNA was determined. (f) The construct PS1 (0.4 µg ml-1) and siNC (20 nmol ml-1) or siC/EBP? (20 nmol ml-1) were co-transfected into the PLKO or ABHD5-KD RAW cells for 24 h. Then, the reporter-gene assay was performed. The data represent means±s.e.m., (n=3, *P<0.05, **P<0.01, Student's t-test). (g) SRM mRNA levels of the PLKO or ABHD5-KD RAW cells transfected with siNC (20 nmol ml-1) or siC/EBP? (20 nmol ml-1) for 24 h. The data represent means±s.e.m., (n=3, *P<0.05, **P<0.01, Student's t-test). (h) Immunoblotting assay of SRM and C/EBP? in the PLKO or ABHD5-KD RAW cells transfected with siNC (20 nmol ml-1) or siC/EBP? (20 nmol ml-1) for 24 h. (i) The cell viability of CT-26 treated with different CM from the PLKO or ABHD5-KD RAW cells, which were transfected with siNC (20 nmol ml-1) or siC/EBP? (20 nmol ml-1) for 24 h. The data represent means±s.e.m., (n=5, **P<0.01, Student's t-test). (j) The 6-week-old WT mice were subcutaneously inoculated with CT-26 cells in the thighs and treated with different CM as described in i for 2 weeks. Then, the tumour volumes were measured. The data represent means±s.e.m., (n=6, **P<0.01, Student's t-test). NS, not significant.

Fig 5: Macrophage ABHD5 suppresses ROS-dependent C/EBP? expression.(a) The ROS production in the PLKO or ABHD5-KD RAW cells treated with NAC (20 mM), GSH (10 mM) or vehicle control PBS for 24 h were measured. The data represent means±s.e.m., (n=5, **P<0.01, Student's t-test). (b) The ROS production in the pCDNA3.1 or pCDNA-ABHD5-transfected RAW cells. The data represent means±s.e.m., (n=5, **P<0.01, Student's t-test). (c) ABHD5, C/EBP? and SRM mRNA levels in the RAW cells as described in a. The data represent means±s.e.m., (n=4, **P<0.01, Student's t-test). (d) Immunoblotting assay of C/EBP? and SRM in the RAW cells described in a. (e) The ChIP assay was performed to determine the interaction between C/EBP? protein and SRM promoter in the RAW cells described in a. (f) Supernatant spermidine levels in the RAW cells described in a. The data represent means±s.e.m., (n=4, **P<0.01, Student's t-test). (g) Tumour volumes of the mice that were subcutaneously inoculated with CT-26 cells in the thighs and treated with the CM from the RAW cells described in a for 2 weeks. The data represent means±s.e.m., (n=6, **P<0.01, Student's t-test). NS, not significant.