Journal: Blood Cancer Journal

Article Title: Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma

doi: 10.1038/s41408-021-00554-9

Figure Lengend Snippet: A Control (sh-C) and APOBEC3G knockdown (sh-A3G) MM.1S and H929 cells were evaluated for the presence of micronuclei, using In Vitro MicroFlow Kit (Litron Labs). Images of micronuclei (I) and bar graphs (II) showing percentage of micronuclei are presented. B MM.1S cells were transduced with control shRNA (sh-C) or that targeting A3G (sh-A3G) and following puromycin selection, cultured for 3 weeks. DNA from these and day 0 (baseline control) cells was extracted and the acquisition of new genomic changes in cultured relative to day 0 cells monitored, using whole genome sequencing (30x). Circos plots showing translocations and other genomic changes (I) and bar graphs showing total number of SNVs and indels (II) are presented. C Control and A3G knockdown MM cells were transfected with a plasmid expressing EGFP. EGFP-positive cells were purified, cultured for additional 72 h, genomic DNA isolated, and EGFP sequences amplified using specific primers. The PCR products were cloned into a plasmid vector to transfect into competent E. coli cells and plated on LB agar. Plasmid DNA from 10 colonies/sample were isolated and sequenced to analyze for mutations. Bar graphs show APOBEC-like mutations.

Article Snippet: To evaluate the impact on genomic instability, control and A3G knockdown cells were analyzed for micronuclei, using a flow cytometry-based Micronucleus Assay (MicroFlow kit, Litron Laboratories, New York, USA) as described by us previously [ ].

Techniques: Control, Knockdown, In Vitro, Transduction, shRNA, Selection, Cell Culture, Sequencing, Transfection, Plasmid Preparation, Expressing, Purification, Isolation, Amplification, Clone Assay

Journal: Blood Cancer Journal

Article Title: Dysregulated APOBEC3G causes DNA damage and promotes genomic instability in multiple myeloma

doi: 10.1038/s41408-021-00554-9

Figure Lengend Snippet: Control and A3G-overexpressing U266 cells (described in Fig. ) were evaluated for genomic instability. A Following selection, the cells were evaluated for the presence of micronuclei, using In Vitro MicroFlow Kit (Litron Labs). Images of micronuclei (I) and bar graphs showing percentage of micronuclei (II) are presented. B Control and A3G-overexpressing cells described above were transfected with a plasmid expressing EGFP. EGFP-positive cells were purified, cultured for additional 72 h, genomic DNA isolated, and EGFP sequences amplified using specific primers. The PCR products were cloned into a plasmid vector to transfect into competent E. coli cells and plated on LB agar. Plasmid DNA from 10 colonies/sample were isolated and sequenced to analyze for mutations. I. Bar graphs show APOBEC-like mutations as well as total (or overall) mutations; II. Percent GFP + cells were analyzed by flow cytometry at different time points and plotted in the graph.

Article Snippet: To evaluate the impact on genomic instability, control and A3G knockdown cells were analyzed for micronuclei, using a flow cytometry-based Micronucleus Assay (MicroFlow kit, Litron Laboratories, New York, USA) as described by us previously [ ].

Techniques: Control, Selection, In Vitro, Transfection, Plasmid Preparation, Expressing, Purification, Cell Culture, Isolation, Amplification, Clone Assay, Flow Cytometry