![Figure 6. Edited-NEIL1 possesses loss-of-function properties and a compromised DNA-damage repair capability. (A, left) Quantification of colonies formed in OCIMY5 with different NEIL1 status. Each experiment was performed in triplicate, and data were expressed as the mean ± SD of triplicate wells. (Right) Representative images of colonies formed in each cell line on the methylcellulose-based media. Arrows are pointing to the colonies formed. (Bottom left) Cell growth curve of OCIMY5 with different NEIL1 status over the course of 4 days. (Bottom right) Bar chart showing the proportion of OCIMY5 with different NEIL1 status in different cell cycle phases. The percentage of cells are stated on the diagram. (B, left) ROS assay measuring for the positivity of CM-H2DCFDA (ROS marker) in the untreated and melphalan-treated NEIL1-WT and NEIL1-edited OCIMY5. (Right) Enzyme-linked immunosorbent assay quantifying the level of oxidative damage (8-hydroxy-2-deoxyguanosine as the marker) in the untreated and melphalan-treated NEIL1-WT and NEIL1-edited OCIMY5. Relative percentage was calculated by dividing the values of melphalan-treatment over the nontreatment. Both cell lines were treated with 10 μM melphalan for 8 hours before being harvested for the assays. (C) Comet assay of untreated and melphalan-treated (10 μM, 8 hours) cells. Assay was performed in an alkaline condition for specific detection of single-stranded DNA breaks. Tail length was quantified with image J. (Left) Quantification of tail length. (Right) Representative images of each indicated conditions. (D) Relative changes of different cell cycle phases in NEIL1-WT and NEIL1-edited cells on melphalan treatment (10 μM, 8 hours). Relative changes were calculated with this formula: [(% melphalan treated cells−% untreated cells)/% untreated cells]×100. (E) Melphalan (10 μM) was withdrawn from NEIL1-WT and NEIL1-edited cells after 8 hours of treatment and were allowed to recover in complete medium, and their cell viability over the course of 5 days was observed with CTG assay. **P < .05; ***P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/132/12/10.1182_blood-2018-02-832576/4/blood832576f6.png?Expires=1723098141&Signature=jshdzCd0XH-yOq~FiC7TCxfyAJg7~qDIuecbWiNa1nnqiJm5kVh17LyIDQSa5fsBpt-HGSneGz859xDphryyOPbsDwSmFT6D14hJP0EyzWwaERUoq4y9mvFBpwvaZodIxZ~Fy4EIHTTmp-zvdCaVvwWMj56PCFpN~0mEscLE18O-Mj4HLjo2ypqt2BLLd3ADb9FPdb4PmdHEE3gqV3EHaieLR4U3wPFYyOZYgCojForDyrymrXjN~JU1GZLaLqvuFxyVR1~geOdykq-8xDz9zMUb4k28x4ix6T6IvuEM4woSNf8T9eMXeB5nMdmF9Rey81i~CJXmh8uo2qAhbjTVOQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Edited-NEIL1 possesses loss-of-function properties and a compromised DNA-damage repair capability. (A, left) Quantification of colonies formed in OCIMY5 with different NEIL1 status. Each experiment was performed in triplicate, and data were expressed as the mean ± SD of triplicate wells. (Right) Representative images of colonies formed in each cell line on the methylcellulose-based media. Arrows are pointing to the colonies formed. (Bottom left) Cell growth curve of OCIMY5 with different NEIL1 status over the course of 4 days. (Bottom right) Bar chart showing the proportion of OCIMY5 with different NEIL1 status in different cell cycle phases. The percentage of cells are stated on the diagram. (B, left) ROS assay measuring for the positivity of CM-H2DCFDA (ROS marker) in the untreated and melphalan-treated NEIL1-WT and NEIL1-edited OCIMY5. (Right) Enzyme-linked immunosorbent assay quantifying the level of oxidative damage (8-hydroxy-2-deoxyguanosine as the marker) in the untreated and melphalan-treated NEIL1-WT and NEIL1-edited OCIMY5. Relative percentage was calculated by dividing the values of melphalan-treatment over the nontreatment. Both cell lines were treated with 10 μM melphalan for 8 hours before being harvested for the assays. (C) Comet assay of untreated and melphalan-treated (10 μM, 8 hours) cells. Assay was performed in an alkaline condition for specific detection of single-stranded DNA breaks. Tail length was quantified with image J. (Left) Quantification of tail length. (Right) Representative images of each indicated conditions. (D) Relative changes of different cell cycle phases in NEIL1-WT and NEIL1-edited cells on melphalan treatment (10 μM, 8 hours). Relative changes were calculated with this formula: [(% melphalan treated cells−% untreated cells)/% untreated cells]×100. (E) Melphalan (10 μM) was withdrawn from NEIL1-WT and NEIL1-edited cells after 8 hours of treatment and were allowed to recover in complete medium, and their cell viability over the course of 5 days was observed with CTG assay. **P < .05; ***P < .001.