Rapamycin rap

Foetal bovine serum (FBS), α‐minimum essential medium (α‐MEM) and Dulbecco's modified eagle's medium (DMEM) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The JetPRIME® transfection reagent was obtained from Polypus‐transfection (Strasbourg, France). The TRAP staining kit, chloroquine (CQ), antibodies against LC3B and p62, and LY294002 were obtained from Sigma Aldrich (St. Louis, MO, USA). RANKL, OPG and macrophage colony‐stimulating factor (M‐CSF) were obtained from R&D systems (Minneapolis, MN, USA). Antibodies against c‐Fos, Src, PI3K (p85), phosphorylated (p)‐Akt (Ser473), Akt, TAK1, Beclin1 and p‐S6 (Ser240/244) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MMP‐9, and CAⅡ were obtained from Abcam (Beverly, MA, USA). EGFP‐pmCherry‐LC3 plasmid was obtained from HedgehogBio Science and Technology Ltd. (Shanghai, China). Beclin1 siRNA plasmid (sc‐29798) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). BCA protein assay kit and rapamycin (Rap), was obtained from Beyotime (Beijing, China). All related reagents were available in our laboratory.

Appressorium development and invasion assay were performed by incubating indicated strains on plastic plates and onion epidermis, respectively, as described in our previous report [23 ]. Briefly, 5 drops (5 µL per drop, 2 × 10⁵ conidia mL⁻¹) of conidial suspensions were placed on a plastic plate and incubated at 28 °C before the conidial morphology was observed, while the appressorium formation was observed under a microscope after 12 h incubation. Moreover, in order to identify the potential signaling pathway by which CgCFEM1 functions, the following treatments at the respective final concentrations were added to the conidial suspensions and analyzed at 24 hpi: 200 nM rapamycin (Rap; Beyotime Biotechnology, Shanghai, China), 10 mM 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cAMP; Sparkjade, Jinan, China) and 10 mM L-glutamine (Macklin, Shanghai, China). All of the relative formation rates were calculated based on the data of three independent replicates, with at least 100 conidia per replicate.