Plasmid extraction kit

Monoclonal antibodies against Bcl-2, Bax, P53, P21, Mcl-1, and GAPDH were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against CytC, Fas, FasL, and TRAIL were purchased from Abcam (Cambridge, MA, USA). Mouse anti-γ-H2AX antibody was from Millipore (Billerica, MA, USA). DMEM medium and FBS were purchased from Gibco BRL (Grand Island, NY, USA). FITC annexin V Apopotosis Detection Kit was from BD Biosciences (San Jose, CA, USA). Caspase-Glo 3/7, 8, and 9 assay kits and Plasmid extraction kit were purchased from Promega (Madison, WI, USA). N-acetyl-L-cysteine (NAC) and Reactive Oxygen Species Assay Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine^TM 2000, Dichloro-dihydro-fluorescein diacetate (DCFH-DA), MitoSOX^TM Red, MitoTracker Deep Red FM, MitoTracker Green FM and Hoechst 33342 were from Invitrogen (Carlsbad, CA, USA). Trypan Blue Staining Cell Viability Assay Kit was from Beyotime Institute of Biotechnology (Jiangsu, China). Tetramethylrhodamine methyl ester (TMRM) was from Life Technologies (Carlsbad, CA, USA). Z-LEHD-FMK, Z-IETD-FMK, Z-DEVD-FMK were from Calbiochem (San Diego, CA, USA). Colorimetric TUNEL Apoptosis Assay Kit was from Beyotime (Shanghai, China). Mitochondria/Cytosol Fractionation Kit was from BioVision (San Francisco, CA, USA). DL-2000 DNA Marker was from TaKaRa Biotechnology (Dalian, China).

Bioinformatics software (http://www.targetscan.org) was used to predict the targeting relationship between miR-103a and BDNF and the binding sites between miR-103a and BDNF 3′UTR. The sequence of BDNF 3′UTR promoter containing miR-103a binding site was synthesized, and the BDNF 3′UTR wild-type (WT) plasmid was constructed. On the basis of this plasmid, the BDNF 3′UTR mutant (MUT) plasmid was constructed at the mutation binding site. According to the methods of the plasmid extraction kit (Promega, Madison, Wisconsin USA), the cells in the logarithmic growth were inoculated into 96-well plates and transfected with Lipofectamine 2000 at about 70% cell confluence. BDNF-WT and BDNF-MUT were mixed with mimics NC and miR-103a mimics (Shanghai GenePharma Co., Ltd (Shanghai, China)) respectively, and then co-transfected into 293T cells. After 48 h of transfection, the cells were collected and lysed. The luciferase activity was detected by luciferase detection kit (BioVision, San Francisco, CA, USA) and Glomax 20/20 luminometer (Promega, Madison, Wisconsin USA). The experiment was repeated three times.

Bioinformatics software (http://www.targetscan.org) was used to predict the targeting relationship between miR-27a and SFRP1 and the binding sites between miR-27a and SFRP1 3′UTR. The sequence of SFRP1 3′UTR promoter containing miR-27a binding site was synthesized, and the SFRP1 3′UTR wild-type (WT) plasmid was constructed. On the basis of this plasmid, the SFRP1 3′UTR mutant (MUT) plasmid was constructed at the mutation binding site. According to the methods of the plasmid extraction kit (Promega, Madison, Wisconsin USA), the cells in the logarithmic growth were inoculated into 96-well plates and transfected with Lipofectamine 2000 at the cell density of about 70%. The plasmids of SFRP1-3′UTR-WT and SFRP1-3′UTR-MUT were mixed with mimics NC and miR-27a mimics (Shanghai GenePharma Co., Ltd, Shanghai, China), respectively, and then co-transfected into 293T cells. The cells were collected and lysed after 48 h of transfection. The luciferase activity was detected by luciferase detection kit (BioVision, San Francisco, CA, USA).

Bioinformatics software http://www.targetscan.org was used to predict the targeting relationship between miR-92a and TCF21 and the binding sites of miR-92a to TCF21 3′UTR. TCF21 3′UTR promoter sequence containing miR-92a binding site was synthesized and TCF21 3′UTR wild type plasmid (TCF21-WT) was constructed. On the basis of this plasmid, the mutant type plasmid TCF21 3′UTR (TCF21-MUT) was constructed. The procedure was carried out according to the instructions of plasmid extraction kit (Promega, Madison, Wisconsin, USA). The cells in the logarithmic growth were inoculated into the 96-well plate and transfected with Lipofectamine 2000 when the cell density was about 70%. TCF21-WT and TCF21-MUT were mixed with mimics NC and miR-92a mimics (Shanghai GenePharma Co., Ltd (Shanghai, China)) respectively, and then co-transfected into 293T cells. After 48 h of transfection, luciferase activity was collected and lysed. The luciferase activity was detected by a Glomax20/20 luminometer (Promega, Madison, Wisconsin, USA) with a luciferase detection kit (BioVision, San Francisco, CA, USA). The experiment was repeated three times.

The ORFs of the AMPs (Table S1), codon-optimized for Lactococcus lactis, were cloned into the pTKR plasmid (Fig. S1), an L. lactis/E. coli shuttle vector developed in the Kearney Lab from the L. lactis plasmid, pT1NX (38 (link)) by adding an E. coli ori site and kanR gene for propagation and selection of E. coli clones. This plasmid includes the P1 acid-inducible promoter and the usp45 signal peptide for transport out of the cell. The ORFs were amplified from a gBlock (IDT) by PCR (1 minute melting at 95°C; 35 cycles of 15 seconds melting at 95°C, 15 seconds annealing at T_m + 3°C, 30 seconds extension at 72°C; and 5 minutes elongation at 72°C) using respective primers and pasted into the pTKR plasmid using restriction enzyme cutsites post agarose gel purification. The recombinant plasmid was electroporated into E. coli 10β cells and plated out on LB agar (Thermo Scientific) plate with kanamycin (MP Biomedicals) to pick successfully cloned colonies. Post-colony-PCR screening, cloned colonies were picked and propagated in LB liquid media with kanamycin (25 µg/mL; Thermo Scientific). The pTKR plamids with AMPs cloned were extracted from the pelleted liquid culture using the Plasmid Extraction Kit (Promega) and electroporated into L. lactis MG1363 (LMBP 3019) cells, followed by erythromycin selection on GM17 plates.