| Autor: |
Escalona, Ruth M., Chu, Simon, Kadife, Elif, Kelly, Jason K., Kannourakis, George, Findlay, Jock K., Ahmed, Nuzhat |
| Rok vydání: |
2023 |
| DOI: |
10.6084/m9.figshare.22606204.v1 |
| Popis: |
Additional file 1: Figure S1. siRNA suppression of TIMP-2 in the OVCAR5 cell line. Diagram showing the location of single siRNA duplexes A, B, C in the TIMP-2 gene. Figure S2. CRISPR/Cas9 editing of the TIMP-2 gene in the OVCAR5 cell line. A Diagram showing the configuration of TIMP-2 CRISPR/Cas9 plasmid. The TIMP-2 linear donor plasmid is ~ 2.74 kb and incorporates the donor GFP and puromycin (under the EF1a promoter). The transfection involves integration of Cas9/gRNA and the linear donor plasmid (GFP and puromycin) genes into cells. The Cas9 targets the TIMP-2 gene in exon1, guided by two gRNA sequences [the gRNA1 (yellow) and gRNA2 (pink)]. The donor genes can be inserted in the cells by transcription in the forward or reverse directions. In both situations, interruption of TIMP-2 expression coinciding with puromycin resistance and GFP expression should occur. (Figure adapted from https://www.origene.com/catalog/gene-expression/knockout-kits-crispr/kn409796/timp2-human-gene-knockout-kit-crispr ). B Puromycin “death” curve in the OVCAR5 cell line. The OVCAR5 cell line was incubated with puromycin concentrations ranging from 0 to 320 µg/mL followed by an MTT assay of OVCAR5 cells after 48 h. The concentration of 3 µg/mL (indicated by red arrow) was used for puromycin selection. Values are mean ± SEM and graph is representative of three experiments done in triplicate. C CRISPR transfected OVCAR5 cells after puromycin selection and GFP sorting. OVCAR5 cells were transfected with CRISPR/Cas9 vectors plus the donor plasmid containing puromycin and GFP genes. After puromycin selection, cells were GFP sorted and visualized under a confocal microscope. After a second GFP sorting, GFP fluorescence was only seen in gRNA2 cells but not in gRNA1 cells. 20× magnification; scale bar 1000 µM. D MTT assay of OVCAR5 TIMP-2 CRISPR/Cas9 transfected and Control cells. After 48 h of incubation the concentration of puromycin that killed 50% of cells was determined (IC50 values). Values are mean ± SEM and the graph is representative of three experiments done in triplicate. Figure S3. Expression of cellular TIMP-2 and corresponding GAPDH by Western blot. Representative full image of a Western blot of TIMP-2 and GAPDH proteins on the cell lysates of parental, CRISPR/Cas9 treated control, gRNA1 and gRNA2 cell lines. Figure S4. Quantification of EdU stained OVCAR5 parental, CRISPR control, and TIMP-2 knocked down gRNA2 and gRNA1 cells. The cells were stained with EdU and propidium iodide (PI) as described in “Methods”. A Flow cytometer representation of percentage of EdU stained cells in S-phase of the cycle for CRISPR transfected cell lines. B Flow cytometer representation of negative controls used to calculate the percentage of EdU stained cells in S-phase of the cycle for the OVCAR5 parental cell line. Red rectangles indicate the areas analysed for EdU positive cells. Table S1. Primers used in the study. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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