Fig 1: BCL-XL contributes to MAPK activation to induce HMGA2 expression. a Venn diagram of repartition of protein expression variations in iTraq experiment. Applied cut-offs on log2 Fold Change are mentioned for each condition. RAS-induced proteins are those for which the log2 Fold Change is higher than 0.28 in MCF10A KRASV12 sh-Ctl compared to MCF10A Lxsn sh-Ctl. BCL-XL-dpdt proteins are those for which the Log2 fold change is lower than -0.18 in sh-BCL-XL compared to sh-Ctl in either EGF-treated MCF10A Lxsn cells (right) or MCF10A KRASV12 cells (left). b–d Western blot showing KRAS, BCL-XL and/or p-ERK expressions in b MCF10A Lxsn and KRASV12 cells, c MCF10A KRASV12 cells 72 h after sh-BCL-XL and d MCF10A Lxsn cells 72 h after sh-BCL-XL (in presence of 20 ng ml-1 EGF in MCF10A Lxsn media). e Correlations between BCL-XL protein expression and KRAS protein expression (left) or p338RAF protein expression (right) in basal subtype tumour samples. Quantified expression of BCL-XL, KRAS and p338RAF from RPPA data were examined for correlation using Pearson’s (Pear.) analysis. The results shown here are based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/. f Box & Whiskers representation of KRASV12 vs. Lxsn log2 Fold Change protein expression (Tukey representation, unpaired t-test with equal SD). Black box represents log2FC for all proteins, dark blue box represents KRAS vs. Lxsn log2FC of BCL-XL-dependent proteins (as defined above), light blue box represents KRAS vs. Lxsn log2FC of BCL-XL-independent proteins (with sh-BCL-XL vs. sh-Ctl log2FC <-0.18 or >0.18 in KRASV12 background). g qPCR analysis of HMGA2 mRNA in MCF10A Lxsn and KRASV12 cell lines infected with sh-BCL-XL during 72 h (in presence of 20 ng ml-1 EGF in MCF10A Lxsn media). Mean and SEM of 3 independent experiments are represented as relative quantity of mRNA normalised to the mean of RPLP0, RPS18 and GAPDH relative expression (two-tailed unpaired t-test). Western blot analysis showing HMGA2 expression in MCF10A Lxsn cell line infected with sh-BCL-XL during 72 h
Fig 2: Prediction and design of amino acid substitutions in each amino acid position of KRpep-2d. The complex of ribbon diagram of K-Ras(G12D)GDP and stick model of KRpep-2d (PDB ID: 5XCO) are shown in the center. Purple amino acids are Cys5 and Cys15. Orange amino acids indicate pharmacophore sequence (Pro6–Val14). Cyan amino acids present Arg1–4,16–19 residues. Chemical structures of representative amino acid examples that were predicted to improve biochemical functions of KRpep-2d in Leu7, Ile9, and Tyr11 are listed. In the upper left side box, Arg residues at both termini of the KRpep-2d were excluded for clarity. The figure was made using PyMOL.
Fig 3: MD simulations of KS-58, KS-58(monocyclic), and KS-58(Ser6Dap). (A) Structural comparison of K-Ras(G12D)GDP-binding mode and K-Ras(G12D)GTP-binding mode of KS-58 (stable snap shots) with K-Ras(G12D)GDP (PDB ID: 4EPR), K-Ras(G12D)GDP/KRpep-2d (PDB ID: 5XCO), K-Ras(G12D)GppNHp (PDB ID: 5USJ), and K-Ras(G12D)GppNHp/KD2 (PDB ID: 6WGN). The magnesium ions are presented as large spheres. Dash lines indicate hydrogen bonds. Arg residues at both termini of the KRpep-2d were excluded for clarity. (B) Lipid membrane accessibility of peptides in MD simulations. Line graph shows moving positions of underlined atoms of Asp8 (Ca–CH2–COOH, red) and Nle3/Anon5 (Ca–CH2–CH2–CH2–CH3, Ca–CH2–CH2–CH2–CH2–CH2–CH2–CH3, green), respectively. KS-58: left panel, KS-58(monocyclic): center panel, KS-58(Ser6Dap): right panel.
Fig 4: Screening of KRpep-2d derivatives. (A) K-Ras(G12D)-binding activity of Biotin-KRpep-2d in ELISA (n = 4, ± SEM). (B) Competitive inhibition activities of KRpep-2d and KS-36 against the interaction of plate-coated K-Ras(G12D) and Biotin-KRpep-2d (100 nM) (n = 4, ± SEM). (C) Amino acid structures introduced into KRpep-2d derivatives and their K-Ras(G12D)-binding activities. Positions of the substituted amino acids in KRpep-2d and introduced amino acids are listed. K-Ras(G12D)-binding activities are listed as fold-value compared to the parental KRpep-2d set as 1.0 (n = 4, ± SEM). 2d-amide is an amide bond (Dap5–Asp15) cyclized KRpep-2d, and 2d-nc is main chain cyclized KRpep-2d.
Fig 5: BCL-XL-dependent RAS target genes expression are sensitive to low dose of RAF inhibitor. a qPCR analysis of BCL2L1, HMGA2, FOSL1, BCL2L11 and CCND1 mRNA in MCF10A KRASV12 cells treated with increasing doses of RAF inhibitor (L779450) during 24 h. Mean and SEM of 3 independent experiments are represented as relative quantity of mRNA normalised to the mean of RPLP0, RPS18 and ACTB relative expression. Insert: western blot showing phosphorylation of ERK and total ERK levels under the same conditions. b qPCR of BCL2L1, HMGA2, FOSL1, BCL2L11 and CCND1 mRNA in MCF10A Lxsn cells grown in the presence of EGF treated with increasing doses of RAF inhibitor (L779450) during 24 h. Mean and SEM of 3 independent experiments are represented as relative quantity of mRNA normalised to the mean of RPLP0, RPS18 and GAPDH relative expression Insert: western blot showing phosphorylation of ERK and total ERK levels under the same conditions. c qPCR of FOSL1 and CCND1 mRNA in EGF-treated MCF10A Lxsn and in MCF10A KRASV12 cell lines infected with sh-BCL-XL during 72 h. Mean and SEM of 3 independent experiments are represented as relative quantity of mRNA normalised to the mean of ACTB, HPRT1 and GAPDH relative expression (two-tailed unpaired t-test)
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