Ampicillin

PCR amplified regions of genes encoding mCherry, Superfolder GFP, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from F. prausnitzii and the 23S rRNA gene of S. mutans (for primer sequences see table S1) were cloned blunt-end via the EcoRV restriction site into the pGEM 5Zf(+) vector (Promega, Germany) and the resulting plasmids were transformed in E. coli DH5α. Positive clones were selected via blue/white screening on LB agar plates containing 100 μg/ml ampicillin (Sigma, Germany). The clones were cultivated overnight in LB containing 100 μg/ml ampicillin and the plasmids were isolated using the Plasmid Mini Kit (Qiagen, Germany). Cloned plasmids were verified by sequencing.

The S. flexneri serotype 3b strain SFL1520 (also known as strain K-1770) was clinically isolated and kindly provided by K. A. Talukder from the International Centre for Diarrhoeal Disease Research, Bangladesh. The strain SFL1520 was grown aerobically (180 rpm) at 37 °C in Luria Bertani broth. The antibiotic susceptibility pattern of SFL1520 was determined using the disk diffusion method (Kirby-Bauer) (Cavalieri et al. 2005 ). We tested SFL1520 for resistance against a range of modern antibiotics (Oxoid, United Kingdom), including ampicillin (10 µg), cefoxitin (30 µg), chloramphenicol (30 µg), erythromycin (30 µg), kanamycin (30 µg), nitrofurantoin (300 µg), penicillin (1 U), tetracycline (30 µg), streptomycin (10 µg), and trimethoprim/sulfamethoxazole (1.25/23.75 µg).
The bacterial DNA was extracted using the Genome Tip 100/G (Qiagen) according to the manufacturer’s instructions. The Rapid Sequencing Kit SQK-RAD004 (Oxford Nanopore Technologies) was used for the library preparation and subjected to the MinION Flow cell (R9.4, Oxford Nanopore Technologies) for sequencing. The Nextera XT DNA library preparation kit (Illumina) was used for MiSeq v3 300-bp paired-end sequencing.

pcDNA4/HisMaxB mammalian expression vector was purchased from Invitrogen (#V864-20(43-0078F), USA). pcDNA4/HisMaxB-YAP1 (#18978) and pcDNA4/HisMaxB-YAP-S127A (#18988) were obtained from Addgene (Cambridge, MA, UK). In brief, prepared plasmids were amplified using Plasmid Plus Midi Kit (Qiagen, Hilden, Germany) and then selected by ampicillin (Sigma, MO, USA). Purified plasmid DNA was transfected using 4D-nucleofector X Kit for mammalian fibroblasts and Amaxa 4D-nucleofector (Lonza, Walkersville, MD, USA), following as manufacturer’s instructions. Zeocin selection reagent (#R250-01) (100 μg/ml) was used to select transfected cells. The transfection results were confirmed by DNA sequencing analysis (Bioneer, Co., Daejeon, Korea). In brief, DNA was extracted from WT-YAP-transfected and YAPS127A-transfected hTERT-hNOFs by using DNA Mini Kit (Qiagen, Hilden, Germany), and thereby RT-PCR was performed as indicated in RT-PCR method above. The following primer sequences to detect the mutation of phosphorylation site (serine at 127) in YAP; 5’- GTCATGAACCCCAAGACG -3’ (forward) and 5’- GGCAGAGGTACATCATCAGG-3’ (reverse). After electrophoresis, the exact DNA fragment was cut out with a scalpel and then DNA sequencing analysis was identified from Gel DNA extraction (iNtRON, Korea). Genomic identities were confirmed using BLASTN (http://www.ncbi.nlm.nih.gov/BLAST).