Fig 1: Glutamine and leucine uptake in KRAS mutant CRC cells associated with the expression of selected AATs: (A, B) l-glutamine and l-leucine uptake levels in KRAS mutant or wt CRC cell lines at 20 min, determined by radiolabeled amino acid transport assay. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical comparisons were computed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Heat map shows mRNA expression levels (fold change) of SLC-amino acid transporter genes in different CRC cell lines from three independent biological replicates (n = 3). (D) Western blot images show the protein expression of AATs (SLC38A2, SLC7A5, and SLC1A5), total-MEK1/2, phospho-MEK1/2, total ribosomal S6, and phospho-ribosomal S6 protein in different CRC cell lines. ß-actin was used as protein loading control. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates.
Fig 2: Overexpression of mutant KRAS induce the expression of AATs, AA uptake, and mTOR activation in KRAS wt cell line HKe3. (A) Western blot images show the expression of AATs (SLC7A5/LAT1, SLC1A5/ASCT2, and SLC38A2/SNAT2), phospho‐MEK1/2, phospho‐S6 ribosomal protein, and KRAS in HCT116‐KRASWT/G13D, Hke3‐KRASWT/G13D−, Hke3‐KRASWT/WT+, and Hke3‐KRASWT/G13D+ cells. KRAS blot show two bands in the lane 3 and 4, as the Hke3‐KRASWT/WT+ and Hke3‐KRASWT/G13D+ cells express HA‐tagged and untagged KRAS. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates. (B, C) l‐glutamine and l‐leucine uptake levels in HCT116 and HKe3 cell lines expressing KRAS wt and KRAS mutation. (D) Proliferation rate of HCT116‐KRASWT/G13D, Hke3‐KRASWT/G13D−, Hke3‐KRASWT/WT+, and Hke3‐KRASWT/G13D+ cells at 24, 48 and 96 h. (E) Bar graph show colony‐forming efficiency of HCT116‐KRASWT/G13D, Hke3‐KRASWT/G13D−, Hke3‐KRASWT/WT+, and Hke3‐KRASWT/G13D+ cells. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical comparisons were computed by one‐way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 3: Knockdown of oncogenic KRAS inhibits the expression of AATs, AA uptake, and mTOR activation in CRC cells. (A) Western blot images show the impact of knockdown of oncogenic KRAS on the protein expression of AATs (SLC7A5/LAT1, SLC1A5/ASCT2, and SLC38A2/SNAT2), KRAS, pan-RAS, total-MEK1/2, phospho-MEK1/2, total-S6, and phospho-S6 ribosomal protein in CRC cell lines, SW480, SW620, and HCT116. ß-actin was used as protein loading control. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates. (B, C) Impact of knockdown of KRAS on the l-glutamine and l-leucine uptake, measured by radioisotope-based membrane transport assays. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical comparisons were computed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 4: Upregulation of AATs in CRC patient samples and their association with oncogenic KRAS mutations: (A–C) Box‐whisker plots show mRNA expression levels of AATs (SLC1A5, SLC7A5, and SLC38A2) in TCGA colon adenocarcinoma patient samples (n = 286) and normal samples (n = 41). Box‐whisker plots represent the interquartile range, middle line indicates the median, and the whiskers indicate minimum/maximum values. (D–F) Correlation of mRNA expression levels of AATs (SLC1A5, SLC7A5, and SLC38A2) in CRC patient samples harboring KRAS wt (n = 317) and KRAS mutations (n = 217), retrieved from gene expression omnibus database (NCBI‐GEO). Error bars represent mean ± SEM; P‐values were calculated by unpaired t‐test.
Fig 5: Knockdown of AATs inhibits AA uptake, mTOR activation, and proliferation in CRC cells: (A, B) Changes in the l-glutamine and l-leucine uptake levels in different CRC cell lines (SW480, SW620, HCT116, and DLD-1) following the transfection of shRNAs against SLC7A5, SLC38A2, and SLC1A5. (C) Impact of knockdowns of SLC1A5, SLC7A5, and SLC38A2 genes on the proliferation of CRC cell lines SW480, SW620, HCT116, and DLD-1 at 24, 48, 72, 96 and 120h. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical comparisons were computed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). (D) Representative western blot images show the changes in the protein expression of AATs, total- and phospho-S6 ribosomal protein in HCT116 and DLD-1 cells after the shRNA transfection. ß-actin was used as protein loading control. All the experiments were performed three times independently, and the blots are representative of three independent replicates.
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