Extended Data Fig. 1: Generation of a reporter cell line to assess loss of unlimited proliferative capacity.
From: Disruption of the MSL complex inhibits tumour maintenance by exacerbating chromosomal instability
a, Schematics of the experimental system used for the screen. b, Diagram illustrating the design of the DCN-driven fluorescent reporter. Arrows: approximate location of primers used in c, ORF: open reading frame. c, Amplicons detecting the correctly integrated IRES-GFP cassette within the DCN 3’UTR. Edited: TDF reporter line; unedited: unedited parental cells. Schematics at the top show the sequence junctions probed. MW: molecular weight. d, Quantification of DCN upregulation in SSEA1− non-tumorigenic tumor cells as assessed by microarray analysis16. Values are average ± SEM from n = 3 biologically independent tumors. P-value from one-tailed Student’s t-test. e, Flow cytometry analysis of untreated or quisinostat-treated, differentiated TDF reporter cells. f, Flow cytometry analysis of a tumor containing the DCN-GFP reporter and of a control tumor induced by unedited TDF cells. Gates used for sorting cells used in h. g, Quantification of DCN upregulation in SSEA1− non-tumorigenic tumor cells, as assessed by flow cytometry (FACS) detecting expression of the DCN-GFP reporter. Values are average ± SEM from n = 5 biologically independent tumors. P-value from one-tailed Student’s t-test. h, Transplantation assays for secondary tumor formation. Quantification of tumors induced by injection of 1,000 SSEA1+ or GFP+ sorted cells. N = 4 independent injections per condition. P-value from one-sided Fisher’s exact test. i, Quantification of DCN upregulation in HRAS-KO tumors as assessed by flow cytometry detecting expression of the DCN-GFP reporter. Individual values from two biologically independent samples. P-value from one-tailed Student’s t-test. j, Clonogenic assays showing loss of unlimited proliferative capacity by HRAS-KO TDF cells. Individual values from 2 biologically independent samples. P-value from one-tailed Student’s t-test. Scale bar: 100 µm. k, Growth kinetics of tumors induced by wild-type (WT) or HRAS-KO TDF cells. Values are average ± SEM from n = 4 biologically independent tumors. P-value from one-tailed Student’s t-test at the last time point. l, Growth kinetics of WT or HRAS-KO TDF cells as assessed by MTS assays. Values normalized to initial plating show the average ± SEM from n = 3 biologically independent samples. P-value from one-tailed Student’s t-test at the last time point. Source data are provided.
