Embedded vaginal tissue samples were used for analysis of macrophages infiltration as previously described [27]. Anti-CD68 (ab31630, dilution 1:200;Abcam, Cambridge) Anti-CD163 (ab119996, dilution 1:250, Abcam,(CKPV)2 Inhibits ML-281 Candida PD 168393 web albicans VaginitisFigure 1. (CKPV)2’s inhibits Candida albicans SA-40 colonies formation. The inhibitory rate of different concentrations of (CKPV)2 against Candida albicans SA-40 in vitro. Candida albicans were incubated with PBS (vehicle control) or indicated concentration of (CKPV)2 (10211 M, 3610210 M, 10210 M, 361029 M, 1029 M, 361028 M, 1028 M, 361027 M, 1027 M, 361026 M, 1026 M, 361025 M, 1025 M and 1024 M) at 30uC for 2 h, afterwards, Candida albicans were transferred to Sabouraud medium and cultured at 30uC for 48 h. The inhibitory ratio( ) = the CFUs of Candida albicans treated with(CKPV)2/the CFUs of Candida albicans treated with PBS 6100. Experiments in this figure were repeated at least three times and similar results were obtained. doi:10.1371/journal.pone.0056004.gCambridge) were applied to label CD68 and CD163 as makers for M1 and M2 macrophages separately. The immune-fluorescence images were captured on a LEICA DMI3000 B confocal microscope, using 106 and 406 objective.Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). 48 hours after transfection, the mRNA level of MC1R in transfected cells was detected to test RNAi efficiency [15,31].Candida Albicans Phagocytosis by Activated Peritoneal MacrophagesThe macrophages were seeded into 24-well plates (26105/well) for 4 h and incubated with PBS, LPS (5 ng/ml)/IFN-c(10 ng/ml), a-MSH (1026 M) or indicated concentration of (CKPV)2 respectively for 24 h. Heat-inactivated Candida albicans were washed twice with PBS, centrifuged at 1000 rpm for 5 min and stained with Giemsa dye reagent (Jiancheng Technology Co. Ltd, Nanjing, China). The suspension containing 26107 Candida albicans was added to each well containing macrophages. The plates then were carefully incubated at 37uC 22948146 for 1 h. After extensively washes, the number of cells engulfed Giemsa dye stained- Candida albicans was recorded under the microscope [28].cAMP AssayThe primary cultured macrophages were seeded in 24 well plates in DMEM supplemented with 10 FCS and incubated at 37uC for 2 hours for adhesion. Thirty min after indicated treatment/s [32,33], the cells were lysed with 0.1 M HCl for 20 min. The cAMP levels were determined 18325633 with mouse cyclic adenosine monophosphate (cAMP) ELISA Kit (Abcam, UK) based on the manufacturer’s protocol.TNF-a Cytotoxicity AssayThe supernatant (100 ml/well) of the macrophages after indicated treatment/s was added to L929 cells for 20 h [34,35]. MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide) salt (0.25 mg/ml) was added to each well. Afterwards, L929 cells were further incubated in CO2 incubator for 4 hours at 37uC, 100 ml of DMSO was then added to dissolve formazan crystals and the absorbance of each well was observed by a plate reader at a test wavelength of 570 nm. The OD value was normalized to untreated vehicle control group.MC1R Exogenously Expression in COS-7 CellsMouse MC1R cDNA extracted from B16-F10 cells (ATCC, Maryland, USA) was sub-cloned into Amp+ enhancer and promoter reporter vector PRL14.4 to yield PRL14.4-MC1R. Then the MC1R gene was transferred into Phage transfer vector pGEM-T4. The recombinant plasmid was transfected into COS-7 cells as previously described [29,30].Arginase Activity AssayTriton X-10.Embedded vaginal tissue samples were used for analysis of macrophages infiltration as previously described [27]. Anti-CD68 (ab31630, dilution 1:200;Abcam, Cambridge) Anti-CD163 (ab119996, dilution 1:250, Abcam,(CKPV)2 Inhibits Candida albicans VaginitisFigure 1. (CKPV)2’s inhibits Candida albicans SA-40 colonies formation. The inhibitory rate of different concentrations of (CKPV)2 against Candida albicans SA-40 in vitro. Candida albicans were incubated with PBS (vehicle control) or indicated concentration of (CKPV)2 (10211 M, 3610210 M, 10210 M, 361029 M, 1029 M, 361028 M, 1028 M, 361027 M, 1027 M, 361026 M, 1026 M, 361025 M, 1025 M and 1024 M) at 30uC for 2 h, afterwards, Candida albicans were transferred to Sabouraud medium and cultured at 30uC for 48 h. The inhibitory ratio( ) = the CFUs of Candida albicans treated with(CKPV)2/the CFUs of Candida albicans treated with PBS 6100. Experiments in this figure were repeated at least three times and similar results were obtained. doi:10.1371/journal.pone.0056004.gCambridge) were applied to label CD68 and CD163 as makers for M1 and M2 macrophages separately. The immune-fluorescence images were captured on a LEICA DMI3000 B confocal microscope, using 106 and 406 objective.Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). 48 hours after transfection, the mRNA level of MC1R in transfected cells was detected to test RNAi efficiency [15,31].Candida Albicans Phagocytosis by Activated Peritoneal MacrophagesThe macrophages were seeded into 24-well plates (26105/well) for 4 h and incubated with PBS, LPS (5 ng/ml)/IFN-c(10 ng/ml), a-MSH (1026 M) or indicated concentration of (CKPV)2 respectively for 24 h. Heat-inactivated Candida albicans were washed twice with PBS, centrifuged at 1000 rpm for 5 min and stained with Giemsa dye reagent (Jiancheng Technology Co. Ltd, Nanjing, China). The suspension containing 26107 Candida albicans was added to each well containing macrophages. The plates then were carefully incubated at 37uC 22948146 for 1 h. After extensively washes, the number of cells engulfed Giemsa dye stained- Candida albicans was recorded under the microscope [28].cAMP AssayThe primary cultured macrophages were seeded in 24 well plates in DMEM supplemented with 10 FCS and incubated at 37uC for 2 hours for adhesion. Thirty min after indicated treatment/s [32,33], the cells were lysed with 0.1 M HCl for 20 min. The cAMP levels were determined 18325633 with mouse cyclic adenosine monophosphate (cAMP) ELISA Kit (Abcam, UK) based on the manufacturer’s protocol.TNF-a Cytotoxicity AssayThe supernatant (100 ml/well) of the macrophages after indicated treatment/s was added to L929 cells for 20 h [34,35]. MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide) salt (0.25 mg/ml) was added to each well. Afterwards, L929 cells were further incubated in CO2 incubator for 4 hours at 37uC, 100 ml of DMSO was then added to dissolve formazan crystals and the absorbance of each well was observed by a plate reader at a test wavelength of 570 nm. The OD value was normalized to untreated vehicle control group.MC1R Exogenously Expression in COS-7 CellsMouse MC1R cDNA extracted from B16-F10 cells (ATCC, Maryland, USA) was sub-cloned into Amp+ enhancer and promoter reporter vector PRL14.4 to yield PRL14.4-MC1R. Then the MC1R gene was transferred into Phage transfer vector pGEM-T4. The recombinant plasmid was transfected into COS-7 cells as previously described [29,30].Arginase Activity AssayTriton X-10.
