(E) On-target site analysis. h. Cell viability of the treated cells was measured using the MTT assay and the results are shown as the signal from the AzaC-treated sample normalized to that of the untreated sample at each time point. (C,D) inducibility by AzaC. A549 (C) Rabbit polyclonal to ARPM1 and RKO (D) cells were either untreated (No) or treated (n = 4 per group) with the indicated concentrations of AzaC for up to 3 days (D1, D2, D3). The mRNA level from the AzaC-treated sample (AzaC) was normalized to that from the untreated sample (No) and shown as relative mRNA. Data are representative of two independent experiments. *< 0.05, **< 0.01.(TIF) pone.0161899.s002.tif (963K) GUID:?FEB4DFDC-9F65-4359-BCEB-927E6F177342 S3 Fig: Growth curve and cell morphology are not significantly different between original cells and IL-7 eGFP reporter cells. (A, B) A549 and A549#6 cells as well as RKO and RKO#5 cells (104 cells per 12 well, n = 4 per group) were plated (D0) and the number of cells was counted every other day through day 7 (D1, D3, D5, D7). The A549 and A549#6 cell growth curve (A) and RKO and RKO#5 cell growth curve (B) are shown. (C, D) The cells were plated onto six wells (105/well) and photographed at 36 h to observe cell morphology. A549 and A549#6 cell images (C) and RKO and RKO#5 cell images (D) are shown. Scale bar is 50 m.(TIF) pone.0161899.s003.tif (2.1M) GUID:?580796FE-8AF8-450A-8A9A-761135197427 S4 Fig: expression comparison between original cells and reporter cells as well as potential off-target site analysis in reporter cells. (A) expression comparison. A549 and A549#6 cells as well as RKO and RKO#5 cells were either untreated (No) or treated (n = 4 per group) with IFN- (50 ng/mL) alone for 12 h or IFN- (50 ng/mL) for 12 h after treatment with AzaC for 2 days (5 M for A549s, 2 M for RKOs). expression levels were measured by qRT-PCR. The signals from reporter cells (A549#6 and RKO#5) were normalized to those from original cells (A549 and RKO), respectively. (B) Potential off-target site analysis on reporter cells. The genomic DNAs from A549#6 and RKO#5 cells were PCR-amplified using on-target confirmation primers (on-target F/R primers). PCR products were then cloned and 20 independent clones were sequenced and analyzed for potential mutations. Agarose gel image of the PCR products (top). Sequence of on-target and potential off-target site (middle). None of the 20 sequences from A549#6 and RKO#5 contained mutation in the potential off-target site. sgRNA and PAM sites are underlined. Representative chromatograms of the read sequences from clones derived from both cell types (bottom).(TIF) pone.0161899.s004.tif (414K) GUID:?EDAAE4EF-6184-48E0-A3B1-1C8563112EA3 S5 Fig: Cytotoxicity test for ellipticine on A549 and A549#6 cells. A549 (A) and A549#6 (B) cells (104/well) plated on 96-wells were either untreated (No) or treated (n = 3 per group) with the Olcegepant hydrochloride indicated concentration of ellipticine for up to 36 h. Cell viability of the treated cells was measured using the MTT assay and the results are shown as the signal from the ellipticine-treated sample normalized to that from the untreated sample at each time point. Data are representative of two independent experiments. *< 0.05, **< 0.01, ***< 0.001.(TIF) pone.0161899.s005.tif (230K) GUID:?04C0C97B-FE34-4FE4-A041-FA117E1B40A1 S1 Table: DNA primers used for reporter cell establishment. (XLSX) pone.0161899.s006.xlsx (12K) GUID:?811A2DCE-87B9-4CEF-9BB7-1A05C9A39ADF S2 Table: DNA primers used for qRT-PCR analysis. (XLSX) pone.0161899.s007.xlsx (11K) GUID:?EAF28CA2-B8C7-411A-B5AD-078CA9B6E59F Data Availability StatementAll relevant data are within the paper and Olcegepant hydrochloride its Supporting Information files. Abstract Interleukin-7 (IL-7) is a Olcegepant hydrochloride cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report gene transcription based on the eGFP protein.