Table 1.
Assessment of cell lines for MSI status by IHC.
Table 2.
Representative cMS mutation analysis in colon cancer cell lines.
Table 3.
Mutant C-terminal peptide sequences encoded for by frameshift mutations in (A) NMD-sensitive and (B) NMD-resistant transcripts.
Figure 1.
Expression of normal BAX protein, but not mutant BAX protein by MSI-High cell lines.
(A) Normal BAX protein (21 kDa, arrow), but no mutant BAX protein (predicted 6.4 kDa) was detected in Western Blot analyses on whole cell lysates of colon cancer cell lines. Cell lines with BAX G8 cMS mutation status: 1. LoVo (−1, +1), 2. SW480 (wt), 3. HCA7 (−1, wt), 4. LS174T (−1), 5. HCT116 (−1, wt), 6. HeLa (wt), 7. LIM1215 (−1). (B) Homozygous −1 base mutation in G8 cMS in LS174T cell line (top) compared to non-mutated cell line, SW1222 (bottom) (reverse DNA sequencing). (C) Normal BAX protein (arrow), but no mutant BAX protein detected by immunoprecipitation: 1. HeLa (wt), 2. LS174T (−1). IgG light chain at 25 kDa.
Figure 2.
Expression of mutant CREBBP and EP300 proteins, arising from NMD-resistant mutant transcripts.
Normal proteins (white arrow) and mutant proteins (black arrow) were detected in Western Blot analyses on whole cell lysates for (A) CREBBP and (B) EP300. (A) CREBBP C5 cMS: 1. SW480 (wt), 2. LoVo (−1, wt), 3. HCT116 (wt), 4. LIM1215 (wt), 5. LS174T (wt). MW (kDa): wt 265, mut 209. (B) EP300 A5 cMS: 1. LoVo (wt), 2. HCT116 (−1, wt), 3. HCA7 (wt), 4. LIM1215 (wt), 5. LS174T (wt). MW (kDa): wt 223, mut 187.
Figure 3.
Validation of mutant CREBBP protein.
Specificity of the mutant protein band was confirmed by gene-specific knockdown in transfectants of siRNA targeting CREBBP. (A) Detection of mutant protein band (black arrow) in whole cell lysates of LoVo cell line, but normal protein only (white arrow) in non-mutated SW480 cell line. (B) Western Blot of LoVo whole cell lysates from siRNA SMARTpool transfectants show decreased expression of normal (white arrow) and mutant (black arrow) CREBBP protein band in CREBBP-targeted lysates (lane 1). Beta-catenin loading control (grey arrow) 92 kDa. (C) Relative intensity of normal (blue) and mutant (red) CREBBP protein bands compared to untransfected cells (PBS). (D) Decreased CREBBP mRNA expression in CREBBP siRNA transfectants (left lane) relative to untransfected cells (right lane).
Figure 4.
Validation of mutant EP300 protein.
Specificity of the mutant protein band confirmed by gene-specific knockdown in transfectants of siRNA targeting EP300. (A) qRT-PCR data to assess siRNAs targeting EP300 (best knockdown using P300-1 and P300-3). (B–C) Decreased mutant EP300 protein detected in Western Blot analyses on immunoprecipitates of EP300 siRNA transfectants relative to untransfected controls (PBS).
Figure 5.
Potential mutant TTK and AIM2 proteins.
(A) Western blot analyses on whole cell lysates identified a potential mutant TTK protein band (black arrow) in multiple mutated cell lines and normal TTK protein (white arrow) only in non-mutated lines: 1. LIM1863 (wt), 2. HCA7 (wt), 3. LIM1899 (−1, wt), 4. HCT116 (−1, wt), 5. LoVo (−1, wt), 6. LIM2537 (−1, wt), 7. LIM2551 (−1, wt). TTK MW (kDa): wt 97, mut 101. (B) Western blot analyses on immunoprecipitates of whole cell lysates identified an AIM2 protein band (black arrow) in all cell lines, including the homozygous mutant LoVo cell line: 1. SW480 (wt), 2. LoVo (−1), 3. HCT116 (−1, wt). 4, LS174T (wt). AIM2 MW (kDa): wt 39.0, mut 40.4.
Table 4.
NMD-R C-terminal peptide sequences from genome-wide search selected for mutation analysis.
Table 5.
HLA-A*0201 SYFPEITHI [26] scores of (A) top ranking NMD-R derived epitopes compared with (B) published MSI-derived epitopes.
Table 6.
Primary antibodies (A) and secondary antibodies (B) used in western blot +/− immunoprecipitation experiments.