Journal: The Journal of investigative dermatology

Article Title: Coexpression of MTH1 and PMS2 Is Associated with Advanced Disease and Disease Progression after Therapy in Melanoma.

doi: 10.1016/j.jid.2021.07.166

Figure Lengend Snippet: Figure 1. Coexpression at the protein level is observed in advanced stages of CMM and after disease progression to therapy. (a) TCGA data show a correlation between PMS2 and MTH1 deletions and amplifications. (b) Altered MTH1 and PMS2 mRNA expression is associated with shorter DFS and OS. (c) TCGA mRNA data shows a correlation between PMS2 and MTH1. (d) MTH1 and PMS2 mRNA correlated in our stage IV cohort and (e) in an independent cohort. (f) Coexpression of MTH1 (green) and PMS2 (red) proteins is shown in stages III/IV (Bar ¼ 50 mm). (g) Survival analysis showing that moderate to high MTH1 expression together with PMS2 expression is associated with shorter PFS. (h) Induced protein expression of MTH1 and PMS2 was observed at progression after MAPKi or ICI therapy (Bar ¼ 50 mm). CMM, cutaneous malignant melanoma; DFS, disease-free survival; ICI, immune checkpoint inhibitor; MAPKi, MAPK pathway inhibitor; OS, overall survival; PFS, progression free survival; TCGA, The Cancer Genome Atlas.

Article Snippet: Membranes were incubated overnight with primary antibodies against MTH1 (1:1,000, Novus Biologicals, Littleton, CO) and PMS2 (1:1,000, Novus Biologicals), followed by secondary antibody incubation with anti-rabbit (1:1,000) or anti-mouse (1:2,000) (Cell Signaling Technology, Danvers, MA).

Techniques: Biomarker Discovery, Expressing

Journal: The Journal of investigative dermatology

Article Title: Coexpression of MTH1 and PMS2 Is Associated with Advanced Disease and Disease Progression after Therapy in Melanoma.

doi: 10.1016/j.jid.2021.07.166

Figure Lengend Snippet: Figure 2. TH579 is an effective treatment option for CMM, including ICI-resistant tumor cells. (a) Induced apoptosis on cosilencing of MTH1 and PMS2 when compared with silencing MTH1 or PMS2 alone (Student’s t-test, n ¼ 3). (b) Downregulation of PMS2 and upregulation of p-H2AX in CMM cells after 24-h treatment with 1 mM TH1579 (n ¼ 2). (c) Reduced cell viability after 72-h treatment with TH1579 on spheroids generated using patient-derived short-term cell lines (ANOVA test, n ¼ 3) and (d) reduction in tumor spheroid sizes (ANOVA test, n ¼ 3). (e) % Reduction in sphere area when comparing treatment with DMSO control (Students t-test, n ¼ 3). (f) Increased apoptosis in patient-derived short-term cell lines after 48-h treatment with 1 mM TH1579. (g) Quantification of (f) (Student’s t-test, n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 3D, three-dimensional; CMM, cutaneous malignant melanoma; h, hour; ICI, immune checkpoint inhibitor; p-H2AX, phosphorylated H2AX; PI, propidium iodide; PRE, pretreatment; PROG, progression; siCombo, Combo small interfering RNA; siControl, control small interfering RNA; siMTH1, MTH1 small interfering RNA; siPMS2, PMS2 small interfering RNA.

Article Snippet: Membranes were incubated overnight with primary antibodies against MTH1 (1:1,000, Novus Biologicals, Littleton, CO) and PMS2 (1:1,000, Novus Biologicals), followed by secondary antibody incubation with anti-rabbit (1:1,000) or anti-mouse (1:2,000) (Cell Signaling Technology, Danvers, MA).

Techniques: Generated, Derivative Assay, Control, Small Interfering RNA

Journal: Cell

Article Title: Endonucleolytic function of MutLalpha in human mismatch repair.

doi: 10.1016/j.cell.2006.05.039

Figure Lengend Snippet: Figure 5. MutLa Contains a Metal Binding Site in the C-Terminal Portion of PMS2 (A–D) MutLa was incubated in the absence or presence of 20 mM Fe2+ as indicated (see Experimental Procedures). Products were separated on 4%–12% SDS (A and D) or 12% SDS (B and C) gels and visualized by Western blot using a-PMS2 directed against amino acids 9–54 (A), a-PMS2 directed against residues 800–862 (B), a-MLH1 directed against amino acids 633–662 (C), or a-MLH1 directed against the full-length MLH1 polypeptide (D). Arrows indicate the major PMS2 product resulting from radical cleavage. Location of PMS2 peptides produced by single- hit cyanogen bromide cleavage (Grachev et al., 1989) at PMS2 methionine residues 599, 622, 672, 676, and 711 are shown in (B). (E) Human PMS2 residues located between Met-676 and Met-711 were employed for homology search using the BLAST routine available at http:// www.ncbi.nlm.nih.gov/blast. Alignments shown for the five sequences at the bottom were obtained by running the human PMS2 DQHATDEKYNFE motif against indicated polypeptide sequences using MegAlign (DNAStar). Conserved residues are shown in red. Asterisks indicate human PMS2 residues altered in this study. (F) Wild-type MutLa, MutLaE705K, and MutLaD699N were subjected to Fe2+-dependent hydroxyl radical cleavage and analyzed as described in (A). Controls were incubated in the absence of Fe2+ as indicated.

Article Snippet: After transfer to PVDF membranes (GE Healthcare), proteins were visualized by Western analysis using a-PMS2 antibodies directed against PMS2 residues 9–54 (Novus) or residues 800–862 (Santa Cruz Biotechnologies) or a-MLH1 antibodies against the full-length protein (Calbiochem) or MLH1 residues 633–662 (Novus).

Techniques: Binding Assay, Incubation, Western Blot, Produced

Journal: Scientific Reports

Article Title: Mismatch repair proteins play a role in ATR activation upon temozolomide treatment in MGMT -methylated glioblastoma

doi: 10.1038/s41598-022-09614-x

Figure Lengend Snippet: MMR knockdown of one protein affects stability of its heterodimeric partner. ( A ) Western blot showing shRNA MMR knockdown in LN229 cells, in both the MGMT- and MGMT + cell lines. ( B ) Levels of MSH2, MSH6, and MSH3 were assessed in shMSH2, shMSH6, and shMSH3 MGMT- and MGMT + cells. The membrane was stripped and reprobed for MSH6, and then MSH3 after the initial MSH2 exposure. ( C ) Levels of MLH1 and PMS2 were assessed by western blotting in shMLH1 and shPMS2 cells. The membrane was stripped and reprobed for PMS2 after initial MLH1 exposure. Full length blots are shown in the supplemental.

Article Snippet: Other antibodies used include PMS2 (ProteinTech, #66075–1-Ig), MSH3 (BD Biosciences, 611,390), MSH6 (BD Biosciences, 610,918), and GAPDH (ProteinTech, HRP-60004).

Techniques: Knockdown, Western Blot, shRNA, Membrane

Journal: Scientific Reports

Article Title: Mismatch repair proteins play a role in ATR activation upon temozolomide treatment in MGMT -methylated glioblastoma

doi: 10.1038/s41598-022-09614-x

Figure Lengend Snippet: MMR proteins are required for synergy between TMZ and two structurally unique and ATR inhibitors, BAY-1895344 and AZ-20. Synergy assays were performed between TMZ and BAY-1895344 or AZ-20 in the newly created MMR-deficient LN229 isogenic cell lines in triplicate plates before being analyzed by Combenefit ( n = 3). Cells were treated for 6 days with the combination of inhibitors. ( A ) MGMT-cells that are MMR-proficient exhibit exquisite synergy upon treatment with TMZ and BAY-1895344 or AZ-20, but there is a lack of synergy in the MGMT + cells. ( B ) shMSH2, ( C ) shMSH6, ( D ) shMLH1, and ( E ) shPMS2 cells do not display synergy for the combination of TMZ and BAY-1895344 or AZ-20 whereas ( F ) shMSH3 MGMT- cells do show synergy. This indicates that the knockdown of MSH2, MSH6, MLH1, and PMS2 affect synergy between TMZ and ATR inhibitors, suggesting a role for these proteins in ATR signaling.

Article Snippet: Other antibodies used include PMS2 (ProteinTech, #66075–1-Ig), MSH3 (BD Biosciences, 611,390), MSH6 (BD Biosciences, 610,918), and GAPDH (ProteinTech, HRP-60004).

Techniques: Knockdown

Journal: Scientific Reports

Article Title: Mismatch repair proteins play a role in ATR activation upon temozolomide treatment in MGMT -methylated glioblastoma

doi: 10.1038/s41598-022-09614-x

Figure Lengend Snippet: MMR is likely implicated in the entire ATR/CHK1 signaling axis. Synergy assays were performed between TMZ and AZD7762 in the newly created MMR-deficient LN229 isogenic cell lines in triplicate plates before being analyzed by Combenefit. Cells were treated for 6 days with the combination of inhibitors. ( A ) MGMT- cells that are MMR-proficient and ( B ) shMSH3 MGMT- cells exhibit some synergy upon treatment with TMZ and AZD7762. ( C ) shMSH2, ( D ) shMSH6, ( E ) shMLH1, and ( F ) shPMS2 MGMT- cells do not display synergy for the combination of TMZ and AZD7762. This indicates that the knockdown of MSH2, MSH6, MLH1, and PMS2 adversely affects synergy between TMZ and AZD7762, suggesting a role for these proteins in ATR-CHK1 signaling. ( G ) Western blot of MGMT- and shMSH2 MGMT- cells showing the kinetics of ATR activation through pCHK1 levels after 12.5 µM of TMZ treatment for the time course indicated. From the densitometry analysis of this blot, there is a robust increase in pCHK1 levels over time in the MGMT- MMR-proficient cells, but not in the shMSH2 cells, suggesting that MSH2 is required for ATR activation and signaling to CHK1. The densitometry analysis is based upon the pCHK1 levels from this representative western blot. Full length blots are shown in the supplemental.

Article Snippet: Other antibodies used include PMS2 (ProteinTech, #66075–1-Ig), MSH3 (BD Biosciences, 611,390), MSH6 (BD Biosciences, 610,918), and GAPDH (ProteinTech, HRP-60004).

Techniques: Knockdown, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: Mismatch repair proteins play a role in ATR activation upon temozolomide treatment in MGMT -methylated glioblastoma

doi: 10.1038/s41598-022-09614-x

Figure Lengend Snippet: MMR is required for ATR-activated G 2 -M arrest. Cells were treated with 12.5 µM of TMZ for the timepoints indicated before being harvested for propidium-iodide staining in a 96-well plate format. Data was acquired on a CytoFLEX Flow Cytometer and analyzed with FlowJo software. Data are presented as mean ± SEM ( n = 3). ( A ) TMZ causes G 2 /M arrest as seen in the MMR-proficient cells over 48 h of treatment, which is a sign of increased ATR activation. However, the ( B ) shMSH2, ( C ) shMSH6, ( D ) shMLH1, and ( E ) shPMS2 cells are not arrested in G 2 /M and rather cycle normally in G 1 as the untreated condition, even after 48 h of TMZ treatment. ( F ) shMSH3 cells are arrested in G 2 /M after 48 h of temozolomide treatment like the wild-type LN229 MGMT- cells. This suggests that MSH2, MSH6, MLH1, and PMS2 are required for proper ATR signaling and cell cycling compared to MSH3. Statistical testing was performed using GraphPad PRISM. Where indicated, **** p < 0.0001 comparing the 48 h timepoint of MGMT- G 2 /M with the 48 h timepoint of the other cell lines in a one-way ANOVA with multiple comparisons.

Article Snippet: Other antibodies used include PMS2 (ProteinTech, #66075–1-Ig), MSH3 (BD Biosciences, 611,390), MSH6 (BD Biosciences, 610,918), and GAPDH (ProteinTech, HRP-60004).

Techniques: Staining, Flow Cytometry, Software, Activation Assay