Extended Data Fig. 7: Interplay between SLC7A5, essential amino acids, and protein synthesis.
From: The amino acid transporter SLC7A5 is required for efficient growth of KRAS-mutant colorectal cancer
a, qRT-PCR analysis of indicated genes in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl intestinal organoids. Transcript levels were normalized to Gapdh (n = 3 per group). Data are mean ± s.e.m. P = 2 x 10-6 using two-way ANOVA with Sidak’s multiple comparisons test. b, Immunoblotting of Slc7a5 in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl intestinal organoids. Each lane corresponds to organoids from a biologically independent mouse. Representative of two independent experiments. HSP90 was used as a loading control. c, Measurement of 35S-methionine uptake in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl intestinal crypt cultures. Uptake of 35S was normalized to total protein. Data are mean ± s.e.m. (n = 3 biological replicates in technical duplicates per group). P = 0.5, one-way Mann–Whitney U test. d, Representative ISH for pS6 and p4EBP1 in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl mice (n = 3 biological replicates). Scale bar, 100 μm. e, Ribosome run-off rate measured in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl intestinal organoids. The run-off rate was measured by addition of translation elongation inhibitor Harringtonine for 0 or 180 s, and increase in sub-polysomes relative to polysomes was calculated. The shift between two timepoints’ S:P ratio, represents elongation speed. Data are mean ± s.e.m. (n = 3 biological replicates per group). P = 0.42 using one-way Mann–Whitney U test. f, Representative polysome profiles of intestinal epithelial cells showing RNA distribution in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl mice (n = 3 biological replicates per group). g, Quantification of protein synthesis 35S- methionine in Apcfl/fl KrasG12D/+ Slc7a5+/+ and Apcfl/fl KrasG12D/+ Slc7a5fl/fl intestinal organoids 16 h after depletion of leucine/isoleucine. Data are mean ± s.e.m. (n = 3 biological replicates per group). *P = 0.02 using one-way Mann–Whitney U test.
