Extended Data Fig. 2: KrasG12D mediates Slc7a5 expression and metabolic reprogramming.
From: The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer
a, RNA in situ hybridization (ISH) images showing Slc7a5 and Slc1a5 expression. Dashed boxes highlight areas shown in high magnification. Scale bars, 200 µm. b, qRT-PCR analysis of indicated genes in mouse intestinal tissues. Transcript levels are shown as delta–delta threshold cycle (ΔΔCT) relative to Gapdh. c, Top, Slc7a5 immunoblotting in intestinal tissue lysates. Representative of three independent experiments. ß-actin as loading control. Bottom, bar graph showing Slc7a5 protein level quantification, normalized to ß-actin. d, Left, ISH images showing Slc7a5 expression in Apcfl/+ and Apcfl/+ KrasG12D/+ tumors. Scale bars, 100 µm. Right, bar graph showing quantification of Slc7a5 positivity calculated as mean percentage (Apcfl/+, n = 5; Apcfl/+ KrasG12D/+, n = 4). e, Slc7a5 substrates in small intestinal tissues as measured by MSI. Boxes depict interquartile range, central line indicates median and whiskers indicate minimum/maximum values (WT, Apcfl/fl, Apcfl/fl KrasG12D/+, n = 5 and KrasG12D/+, n = 4). f, Schematic of genetic crossing to generate Apcfl/+ iKrasG12D mice. g, Brightfield images of Apc iKrasG12D organoid lines in presence or absence of Doxycycline for 48 hours. Representative of four individual wells from three independent lines. Scale bar 100 μm. h, Relative expression of metabolic genes in Apc iKrasG12D organoids from RNAseq reads normalized to the presence of Dox. i, Heatmap shows relative abundance of metabolites (peak area/µg of protein) upon doxycycline withdrawal in Apc iKrasG12D organoid lines using LC-MS. Data represent mean (n = 3 biological replicates in technical triplicates each per group). In a, d images representative of 4 mice per genotype. b, c, h n = 3 per genotype. b, c, d, h data are mean ± s.e.m. P using two-way ANOVA, with Tukey’s test (b), Fisher’s test (h), one-way Mann–Whitney U test (c, d) and one-way ANOVA, with Tukey’s test (e).
