Fig 1: Msh3 overexpression cell line shows increased expansion frequency and increased MutSβ formation with no effect on contraction frequency. (A) Representative immunoblot showing Msh3 protein expression in Msh3 add-back cell line (Msh31.7X) in comparison to Msh3+/+and Msh3−/− cell lines. (B) Quantitative analysis of Msh3 protein expression normalised to actin and relative to Msh3+/+cell line. Msh3 add-back cell line has an average Msh3 expression level of 1.7 times wild type expression. n = 9 for Msh3 protein expression in all cell lines, n = 7 for Msh2 and Msh6 protein levels in Msh3+/+ cell lines, n = 6 for Msh2 and Msh6 protein levels in Msh3−/− cell line and n = 5 for Msh2 and Msh6 protein levels in Msh31.7xcell line. *P = 2.23 × 10−20 and **P = 1.87 × 10−6, both compared to Msh3 level in Msh3+/+cells. ***P = 0.032 compared to Msh6 level in Msh3+/+cells. (C) Expansion frequencies for Msh3−/−, Msh3+/+ and Msh31.7Xcell lines. n = 4 for Msh3−/−and n = 3 for Msh3+/+and Msh31.7X. *P = 0.029 compared to wild type. (D) Contraction frequencies of Msh3−/−, Msh3+/+and Msh31.7Xcell lines, n = 3. (E) Representative immunoblot for co-immunoprecipitation of Msh3 and Msh2 and investigation of MutSβ formation in Msh3−/−, Msh3+/+ and Msh32.9X cell lines. (F) Quantitative analysis of immunoprecipitated Msh3 and Msh2 protein abundance, n = 4 for all samples. *P = 5.44 × 10−13 for Msh3 and P = 4.30 × 10−11 for Msh2 compared to wild type; and **P = 1.66 × 10−4 for Msh3 and P = 4.96 × 10−3 for Msh2 compared to wild type. For panels B, C, D and F error bars denote ±SEM.
Fig 2: The key gene MSH3 has an important impact on the prognosis of patients with RCC. (A) Expression of five model genes in the TCGA-KIRC dataset. (B) Correlation graph between MSH3 and the other four genes. (C) Model gene-related thermograms that vary with MSH3 expression. (D) Expression of MSH3 in the TCGA-KIRC dataset. (E) A survival analysis curve that reflects the relationship between MSH3 expression levels and OS in patients with RCC. (F) ROC curve of MSH3 predicting 5-year survival probability of RCC patients. (G–K) Box diagram showing the correlation between MSH3 and clinical characteristics of RCC patients. The symbol * indicates p<0.05 and *** indicates p<0.001.
Fig 3: Msh3E976A ATPase Walker B mutant shows a decreased expansion frequency compared to Msh3+/+ wild type. (A) Schematic showing Walker B motif consensus sequence and the E976A mutation, ‘h’ denotes any hydrophobic amino acid. (B) Representative immunoblot showing Msh protein expression in Msh3+/+ and Msh3E976Acell lines. Msh3E976A cells show Msh3 expression at near wild type levels. (C) Representative immunoblot of co-immunoprecipitation of Msh3 and Msh2 and MutSβ formation in Msh3+/+and Msh3E976A cell lines. (D) Expansion frequencies for Msh3E976A cell line in comparison to Msh3+/+, n = 3, *P = 0.037. (E) Contraction frequencies of Msh3E976Aand Msh3+/+ cell lines, n = 5 for Msh3+/+and n = 4 for Msh3E976A. (F) Representative ATPase analysis of MutSβ and MutSβ-Msh3E976A (MutSβ EA) in the absence or presence of mismatched DNA containing a two-nucleotide insertion/deletion mispair. Pi, [32P]phosphate. (G) Average ATP hydrolysis activity, n = 3. *P = 0.028 compared to wild type MutSβ assayed without DNA (unshaded bars); **P = 0.001 compared to wild type MutSβ assayed with DNA (shaded bars). For panels D, E and G, error bars denote ±SEM.
Fig 4: Loss of Msh3 causes a defect in CTG repeat expansions but not contractions. (A) Schematic showing predicted CRISPR/Cas9 cut site in exon 2 of human Msh3 gene. Sequencing results show in-frame nine base pair deletion with corresponding loss of codons for amino acids 116–118. (B) Representative immunoblot with an antibody against Msh3 residues 136–349, showing loss of Msh3 expression in Msh3−/−cells in comparison to Msh3+/+ wild type cell line. Expression of Msh2 and Msh6 was retained. (C) Quantitative analysis of Msh protein expression in Msh3+/+ and Msh3−/−cell line, normalised to actin and relative to Msh3+/+. n = 9 for Msh3 levels; n = 7 for Msh3+/+Msh2 and Msh6 protein levels; and n = 6 for Msh3−/−Msh2 and Msh6 protein levels. *P = 2.23 × 10−20. (D) Growth curve analysis of Msh3+/+and Msh3−/−cell lines, n = 3. (E) Expansion frequencies of Msh3+/+and Msh3−/− cell lines, n = 3 for Msh3+/+and n = 4 for Msh3−/−, *P = 0.0019. (F) Contraction frequencies of Msh3+/+and Msh3−/− cell lines, n = 3. For panels C-F, error bars denote ±SEM.
Fig 5: Msh3 polymorphic cell lines show no significant difference in expansion frequency in comparison to Msh3+/+ cell line. (A) Schematic showing polymorphic changes to Msh3 sequence. (B) Representative immunoblot showing Msh protein expression in Msh3T363Iand Msh3T1045Acell lines at comparable expression levels to Msh3+/+wild type cells. (C) Representative immunoblot of co-immunoprecipitated Msh3 and Msh2 showing MutSβ complex formation in Msh3T363Iand Msh3T1045A cell lines alongside Msh3+/+cells. (D) Expansion frequencies for polymorphic Msh3 cell lines assayed in parallel to Msh3+/+. Error bars denote ±SEM, n = 3.
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