Fig 1: Lung transcriptome induced by each oncogenic Kras allele.(A) Volcano plot of the log2 fold change versus p-value of the genes showing differential expression in each allele compared to the wild-type (+/+) Kras allele in the lung. Full-sized plots are provided in Figure 3—figure supplements 2–5. (B, C) Normalized enrichment score and patterns of the indicated Gene Set Enrichment Analysis (GSEA) hallmarks differentially enriched by Kras codon usage (expression) (B) and mutation type (C). Only hallmarks with a false discovery rate (FDR) < 5% are shown. Dot size is adjusted to RAS activity for better visualization. All GSEA hallmarks differentially enriched upon activating KrasLSL alleles with an FDR < 5% are provided in Figure 3—figure supplement 7. (D) STRING analysis of the top ten genes in the differentially enriched GSEA hallmarks identified in RNA-seq analysis of the lungs of Rosa26CreERT2/+;KrasLSL-natG12D/+ versus Rosa26CreERT2/+;KrasLSL-comQ61R/+ mice seven days after tamoxifen injection. Figure 3—source data 1.Differentially expressed genes in each allele from Figure 3—figure supplements 2–6. Figure 3—source data 2.Normalized enrichment scores of the hallmarks identified in Figure 3—figure supplement 1C. Figure 3—source data 3.Ct values from the qRT-PCR analysis in Figure 3—figure supplement 7.
Fig 2: (A) K-Ras gene copy number of BTC cell lines determined by quantitative PCR analysis of genomic DNA. (B) Representative FISH images of K-Ras amplification status in SNU-245 and TFK-1 cells. CEP12 (red), K-Ras (green) and DAPI (blue). (C) Ras activity of cell lines separated according to K-Ras mutation and amplification status. Ras activity was assessed on cell lysates prepared from exponentially growing cells. *P < 0.05. (D) BTC cell lines were separated according to K-Ras mutation and/or amplification status and sensitivity to everolimus at the 5, 10 and 50 nm doses compared using an unpaired t-test.
Fig 3: Conditional KrasLSL alleles with different oncogenic mutations and codon usage.(A) Schematic of generating and activating KrasLSL alleles with the first three coding exons fused and encoded by native (nat) versus common (com) codons with either a G12D or Q61R mutation. (B) PCR genotyping of two independently derived mouse embryonic fibroblast (MEF) cultures (two biological replicates) with the indicated Kras alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) and the unrecombined (KrasLSL*, 389 bp) and recombined (LoxP recombined, 616 bp) KrasLSL allelic products. Gel images were cropped and color inverted for optimal visualization. Full-length gel images are provided in Figure 1—source data 2. (C) Expression levels, determined by immunoblot with an anti-Kras antibody, RAS activity levels, determined by RBD pull-down (RBD-PD) of lysates collected from MEF cells derived from mice with the indicated Kras alleles in the presence of Cre recombinase (CRE). MEF cultures derived from KrasLSL-natG12D/+ mice in the absence of Cre recombinase were used as negative control. MEF cultures were either serum starved overnight (starved) or serum starved overnight followed by serum stimulation for 5 min (stimulated). 20% of the elute from RBD-PD and 30 µg total protein from the total cell lysates were loaded. Tubulin serves as loading control. One of two biological replicates; see Figure 1—figure supplement 3 for the second biological replicate. Full-length gel images are provided in Figure 1—source data 3. Figure 1—source data 1.Full-length gel images of RBD pull downs.Full-length gel images from RAF1-RBD pull downs (top) and whole-cell lysates (bottom) from HEK-HTs ectopically expressing engineered Kras constructs shown in Figure 1—figure supplement 2. Figure 1—source data 2.Full-length gel image of genotyping of mouse embryonic fibroblast (MEF) cultures derived from the KrasLSL alleles in Figure 1B.PCR genotyping of two independently derived MEF cultures with the indicated KrasLSL alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) as well as the unrecombined (KrasLSL, 389 bp) and recombined (LoxP recombined, 616 bp) Kras allelic products. Gel images were color inverted for better visualization. Red box depicts region shown in Figure 1B. Figure 1—source data 3.Full-length gel images of RBD pull-downs in MEFs.Full-length gel images from RBD pull-downs (left) and whole-cell lysates (right) from MEFs derived from KrasLSL alleles with serum starvation, or serum starvation followed by serum stimulation shown in Figure 1C, and same conditions with a second clone of MEF cultures with serial dilutions of 500 µg lysate (left) and 200 µg lysate (right) used for RBD-PD as shown in Figure 1—figure supplement 3. Red box depicts regions shown in Figure 1C and Figure 1—figure supplement 3. Figure 1—source data 4.Full-length gel images of Kras expression and activity using RBD pull-downs from lung tissue.Full-length gel images from RAF1-RBD pull-downs (RBD-PD, top) and whole-cell lysates (bottom) of lungs from mice with KrasLSL alleles seven days after tamoxifen injection as shown in Figure 1—figure supplement 4A. Immunoblots of two separate pull downs from two biological replicates are shown as in Figure 1—figure supplement 4B. Red box depicts the regions shown in Figure 1—figure supplement 4B. Figure 1—source data 5.Ct values from the qRT-PCR analysis in Figure 1—figure supplement 5. Figure 1—source data 6.Sequence of coding exons 1 to 3 of the four KrasLSL alleles.Sequence alignment of the codons in the coding exons 1 to 3 of the indicated KrasLSL alleles in comparison to the murine wild-type sequence (Kras). Top: amino acid sequence. Red nucleotides: optimized codons. Green highlight: G12D mutation. Blue highlight: Q61R mutation.
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