Dulbecco s modi ed eagle medium dmem

For protein quantify, BCA protein quantitation reagent kit (Beyotime, P0010S, Shanghai, China) was purchased from Beyotime Institute of Biotechnology, China. For western blotting, the rst antibodies against β-actin (PA1-46296, Invitrogen), HA Tag Antibody (26183, Invitrogen), Flag Tag Antibody (PA1-984B, Invitrogen), Her2 Antibody (MA5-13105, Invitrogen), Granzyme B Antibody (MA1-80734, Invitrogen), Perforin Antibody (14-9392-82, Invitrogen) and TNF-α Antibody (ARC3012, Invitrogen) and goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody conjugated HRP (G-21234, Invitrogen) were purchased from Thermo Fisher Scienti c, Inc. The ECL western-blotting substrate kit (32106, Pierce) and ELISA kits for the cell cultured supernatant immune factors: TNF-α (BMS607-3, Invitrogen) and IFN-γ (BMS606, Invitrogen) were purchased from Thermo Fisher Scienti c (China) Co. Ltd., Beijing, China.
Human breast cancer cell lines SK-BR-3 and mouse breast cancer 4T1 cells were all from the American Type Culture Collection (ATCC; USA). Chinese hamster ovary (CHO) cells were obtained from the BeNa Culture Collection (Beijing, China). SK-BR-3, 4T1 and CHO cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; Beyotime, Shanghai, China) and 100µg/ml penicillin-chain Mycin (Beyotime, Shanghai, China).

To test the binding of recombinant Semaphorins to MGE-derived interneurons, 100 µl dissociated neurons were plated with a density of 80.000 cells/100 µl culture media onto Laminin/PLL HNO3-glass coverslip, (19.5 µg/ml Laminin (Sigma-Aldrich) and 5 µg/ml Poly-L-Lysine (PLL, Invitrogen)). Warm FBS-containing culture medium was used for 2 h (Dulbecco's Modi ed Eagle Medium (DMEM, Invitrogen) supplemented with 10% FBS, 10000 U/ml penicillin, 10000 µg/ml streptomycin, 0.065% D-glucose and 0.4 mM Lglutamine). Next, the culture medium was replaced for neurobasal serum-free culture medium (37°C).
Cells were incubated for a total of 46 h and exposed for 2 h to control or Sema3C-AP tenfold conditioned media diluted in freshly warmed FBS-free neurobasal medium (37°C). Control conditioned medium or Sema3C-AP conditioned media were obtained as already described [22, 23] . The dissociated cells were xed for 15 min with 4% PFA/PBS and further immunostaining were performed.

CPT (purity 98%, HPLC, Xian Yuxuan Biotechnology Co., Ltd.), RPMI 1640, Dulbecco's Modi ed Eagle Medium (DMEM), fetal bovine serum (FBS), Opti MEM medium, trypsin-EDTA and penicillin/streptomycin were purchased from Gibco (Grand Island, NY, USA). KO143, was obtained from MCE (Newark, NJ, USA). Non-denaturing non-reducing protein Beyotime Biotechnology (Shanghai, China). Mitoxantrone was brought from Meilunbio (Dalian, Liaoning, China). Rhodamine123 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin was obtained from Bairui Biotechnology (Nanjing, China). Goat Anti-Rabbit IgG H&L FITC (Abcam, Cambridge, UK). MTS, BSA were provided by Biosharp (Hefei, Anhui, China). RIPA, PMSF were given by Dingguo Biotechnology (Beijing, China).

Two HCC cell lines, including SNU-387 and Huh7, and immortalized human hepatocytes (THLE-2) were purchased from YaJi Biological (Shanghai, China). SNU-387 cells were cultured in Roswell Park Memorial Institute (RPMI-1640; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS). Huh7 cells were kept in Dulbecco's Modi ed Eagle Medium (DMEM; Gibco) supplemented with 10% FBS. THLE-2 cells were maintained in Bronchial Epithelial Cell Growth Medium (BEGM; Gibco) containing 10% FBS. All cells were maintained in a thermostatic incubator at a constant temperature of 37℃ with moist air and 5% CO 2 .