Fig 2: Quantitative characterization of KRas in two human breast cancer cell lines.(a,b) Single-molecule western blot analyses of KRas expression levels. eGFP-labelled KRas was used as the probe Ras. The expression level of the wild-type KRas in the MCF-7 line was measured to be 17.3±1.3 nM, whereas that of the G13D mutant KRas in the MDA-MB-231 line was measured to be 5.5±0.3 nM per 1 mg ml−1 extracts. Error bars denote s.d (n=200). (c) kbind as a function of KRas pull down. Note the same kbind value shared by the wild-type and mutant KRas in the single-molecule kinetics region (the common flat line). The threshold for kbind was measured to be at 1.2 pulled down KRas per μm2 for the mutant KRas from MDA-MB-231, which gave an estimated active fraction of 50%. This value was one order of magnitude higher than 6% of the wild-type KRas from MCF-7, which had the threshold at 10 KRas pulled down per μm2. Error bars denote s.e. (n>150).

Fig 3: RAS dose-dependency in non-liver contexts.a,b, tSNE projection of tdTom+ cells from the pancreas model coloured by sample-of-origin (a) and cluster (b). Schematic in panel a was created using BioRender (https://biorender.com). c,d, Distribution of expression levels at single-cell level for the indicated genes (c) and gene signature (d) in endogenous KrasG12D-driven pancreatic tumour model (PRT mice). Values for preneoplastic “Early” and “PanIN” were divided into two based on the clustering in Extended Data Fig. 1b, as indicated by the colour of the box/violin. Cells from the Cdkn2a/p16 positive Cluster 11 were designated as “OIS”, whilst all the other “Early” and “PanIN” cells were designated as “non-OIS”. PDAC, pancreatic ductal adenocarcinoma. n values indicate number of cells. e, tSNE projections coloured by indicated genes-of-interest. f, Expression of KRAS or HRAS in TCGA samples of the indicated tumour types, separated by RAS mutation status. wt, wild-type; mt, mutant. n values indicate number of patients. g,h, Upregulation of KRAS in human pancreatic (g) and lung (h) cancer cells, compared to normal epithelial cells, in public scRNA-seq datasets. Ductal cell clusters were identified using KRT19 expression, acinar cell clusters by CPA1 and CPA2 (e). KRAS expression in lung epithelial cells of human lung adenocarcinoma samples, comparing between adjacent normal and tumour cells from different disease stages (f). Lung epithelial cell subset is based on annotation by the original authors. n values indicate number of cells. All boxplot centre line indicates median, box limits indicate first- and third-quartiles and whiskers indicate largest values within 1.5 * interquartile range.

Fig 4: Validation of gene signatures in two TIC branches.(a-c) Representative serially sectioned IHC images of indicated tumours for the indicated proteins. All undifferentiated (DS 4) tumours (n = 5 mice) were CK19-positive (a). Dichotomous expression of either Notch1 or Dlk1 are shown even in the same tumour (b). 5 out of 6 Notch1-positive tumours were Tgfβ1-positive (c). Scale bars = 100 µm. Arrows indicate Dlk1-positive cells within the Notch1/Nestin-tumour. Of note, the Dlk1-positive cells tend to be well-differentiated and express low level of NRAS. (d) Random walk plots for geneset enrichment analysis for the indicated genesets against ranked genes between poorly- and well-differentiated HCC based on human liver cancer cell lines. (e) Kaplan-Meier analysis for the indicated gene signatures in TCGA-LIHC (Liver hepatocellular carcinoma) dataset. n values indicate number of genes in signature. HR = Hazard ratio for top quartile vs. bottom quartile, p = Log-rank p-value.

Fig 5: PIASγ is SUMO E3 ligase for RAS proteins(A) HEK293T cells were co-transfected with plasmid constructs expressing Flag-tagged proteins of the PIAS family, Flag-KRAS and/or HA-SUMO3. Equal amounts of protein lysates from various treatments were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with the lysate inputs, were immunoblotted with the anti-Flag or the anti-HA antibody. (B) HEK293T cells were co-transfected with individual Flag-tagged PIAS expression constructs and GFP-HRAS expression construct. Equal amounts of protein lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were immunoblotted with an anti-GFP antibody or anti-Flag antibody. (C) HEK293T cells were co-transfected with plasmid constructs expressing Flag-PIASγ or vector) and UBC9. Equal amounts of protein lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted with antibodies to Flag and RAS. Endogenous RAS and IgGs (light and heavy chains) are indicated. (D) HEK293T cells were transfected with plasmid constructs expressing HA-SUMO3, Flag-KRAS, PIASγ, and/or UBC9 for 24 h after which cells were collected and lysed. Equal amounts of cell lysates were immunoprecipitated with the Flag antibody. Immunoprecipitates, along with lysate inputs, were blotted with antibodies to HA, Flag, PIASγ, and/or UBC9. (E) HEK293T cells were co-transfected with Flag-KRAS and/or HA-SUMO3 expression constructs, and siRNAs to either PIASγ (siPIAS) or luciferase (siLuc) for 24 h as indicated, after which cells were collected and lysed. Equal amounts of cell lysates were immunoprecipitated with the Flag antibody. Flag immunoprecipitates were blotted with antibodies to HA and Flag, respectively. Corresponding cell lysates were also blotted with antibodies to HA, Flag, PIASγ, total ERKs, and p-ERKs, respectively.