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Journal: iScience
Article Title: KDM5B predicts temozolomide-resistant subclones in glioblastoma
doi: 10.1016/j.isci.2023.108596
Figure Lengend Snippet: KDM5B expression predicts TMZ-triggered subclonal growth (A) Working model. Subclonal dynamics occurs under therapeutic pressure of primary treatment. It results from shifting abundance of treatment-resistant vs. treatment-sensitive cellular hierarchies. Some hierarchies dominate at the naive stage, others appear to arise newly under drug exposure. A bystander pool of cells does not participate in the dynamic course of subclones. (B) Cartoon illustrating the experimental barcoding setup. Naive BN46 and BN118 patient cells were provided with the ClonTracer barcoding construct, exposed to 500 μM TMZ or 0.5% DMSO, and analyzed by NGS at the indicated time points. TMZ-resistant cells, TMZ-r; expanding TMZ-resistant cells, TMZ-rex. (C) NGS-based quantification of detectable barcode identities (BCIs) from (B). Bar charts representing ratios of identifiable vs. undetectable BCIs at the indicated experimental stages. Data points from naive patient samples BN46 and BN118, presented as mean ± SD. (D–H) Note: green-scale, newly arising BCIs under TMZ exposure vs. gray-scale, dominant BCIs at the Naive stage of the experiment. Data from patient sample BN118. (D) Subclonal dynamics from (B), visualized as a stacked plot. Data represent the relative frequencies of the top 50 BCIs, as defined at the TMZ-rex stage, displayed for each stage of the experiments. (E) Subclonal dynamics from (B), visualized as a bubble plot. Data represent relative barcode abundances, barcode identity as specified, at the indicated time points of the experiments. Barcodes of at least 1% relative abundance shown. (F) Subclonal dynamics from (B), visualized as a ranked plot. Data represent individual ranking positions of the top 10 BCIs, as defined at the TMZ-rex stage, displayed for each stage of the experiments. (G) Sorting experiment. Cartoon illustrating cell sorting of KDM5B high barcoded cells at the Naive stage of the experiment. Subclonal dynamics and top TMZ-rex can be predicted; visualized as a slice plot (red boxes). Data represent ranking positions of the top 10 BCIs, as defined at the TMZ-rex stage, color-coded for identity and displayed in relative abundance for each stage of the experiment. Abundance of lower-ranked thousands of bystander identities in white. (H) Subclonal dynamics and top TMZ-rex can be predicted; visualized as a ranked plot (red boxes). Data represent individual ranking positions of the top 10 BCIs, as defined at the TMZ-rex stage, color-coded for identity and displayed as ranking positions at the TMZ-rex stage and for the Naive -sorted KDM5B high cells. Note the similarity of ranking positions among the individual BCIs at both time points. See also Figure S3 and Table S1 .
Article Snippet: Lentivirus was produced in HEK293T cells using the TransIT-TKO® transfection reagent Mirus (Mirus Bio; Madison, Wisconsin, USA), Opti-MEMTM Reduced Serum Medium (Thermo Fisher Scientific) and the following plasmids: 2.4μg ClonTracer Barcoding Library (containing barcodes, RFP and puromycin resistance cassette, Addgene #67267; RRID: Addgene_67267), 2.4μg pCMV delta R8.2 (Addgene #12263; RRID: Addgene_12263), 1.2μg pCMV-VSV-G (Addgene #8454; RRID: Addgene_8454).
Techniques: Expressing, Construct, FACS
Addgene inc is a verified supplier 
Figure S3 and 