N terminal p3xflag cmv plasmid

Invasion assay was measured by counting the THLE-2 invaded cells after overexpression with empty N-Terminal p3XFLAG-CMV plasmid (Sigma-Aldrich, Saint Louis, MO, USA) or with plasmid containing BRAF WT and BRAF V600E. Corning BioCoat™ Matrigel Invasion Chamber (Corning, NY, USA) with 8 μm pores inserts was used to perform the analysis according to manufacturer instructions. After 24 h from transfections THLE-2 cells were seeded in the number of 0.5 × 10⁵ in serum-free LHC-8 medium and incubated for 24 h under standard conditions. Non-invasive cells from the upper surface of the membrane were removed with cotton swabs. The invading cells were fixed and stained using Differential Quick III Stain Kit (Polysciences, Inc., Warrington, PA, USA), and photographed under the light microscope (Nikon Eclipse E200, Tokyo, Japan). The number of cells was calculated using ImageJ software and presented as a bar chart.

RNA was extracted from the THLE-2 cells after 48 h from cell transfection with N-Terminal p3XFLAG-CMV plasmid (Sigma-Aldrich, Saint Louis, MO, USA) and with plasmid containing BRAF WT and BRAF V600E using RNA extraction kit NucleoSpin RNA (Macherey-Nagel, Düren, Germany). Changes in gene expression were quantified using DNB-SEQ 30M PE100 reads sequencing by BGI Genomics (Shenzhen, China). Fastq files were aligned to reference genome GRCh38 using STAR aligner [77 (link)]. Read counts were created using GENCODE v34 and featureCounts [78 (link)]. Differentially expressed transcripts were determined using DESeq2 [79 (link)] using a false discovery rate (FDR) of 0.1.

The influence of selected BRAF mutation on cell proliferation and growth was investigated using Cell Proliferation Reagent WST-1 (Roche, Mannheim, Germany). THLE-2 cells were seeded on a 24-well plate at a concentration of 5 × 10⁴ cells/well. After 24 h cells were transfected with N-Terminal p3XFLAG-CMV plasmid (Sigma-Aldrich, Saint Louis, MO, USA) or with containing the following BRAF: WT, D594A, V600E, L537M, and E648G. Cells were incubated for 48 h under standard condition. Next, the absorbance was measured with accordance to manufacturer instructions using Synergy LX multi-mode reader (BioTek, Winooski, VT, USA).