Journal: Oncotarget

Article Title: SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation

doi: 10.18632/oncotarget.15818

Figure Lengend Snippet: ( A ) Quantitative PCR was performed in HEK293, HEK293/SET, HN12, HN12siSET, HN13, and HN13siSET cells to evaluate whether tumor suppressor genes expression was affected by DNA methylation. BRCA1 , MLH1 , and PTEN were assessed through TaqMan probes and GSTP1 through SybrGreen primers. Graphics represent relative quantification of experiments performed in triplicate through 2 −ΔΔCt method. CDNA from HEK293 was used as calibrator and GAPDH and β-globin primers were used as housekeeping. ( B ) Chromatin immunoprecipitation assay was performed to identify whether SET interacts with GSTP1 and PTEN promoters. Conventional PCR using DNA immunoprecipitated with antibody against SET was performed in triplicate. Ctrl lanes represent samples immunoprecipitated with anti-IgG antibody; INPUT samples consist of total DNA, and SET lanes refer to DNA immunoprecipitated with anti-SET antibody.

Article Snippet: BRCA1 (Hs01556193_m1), MLH1 (Hs00979919_m1) and PTEN (Hs02621230_s1) mRNA levels were assessed by TaqMan assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, DNA Methylation Assay, Quantitative Proteomics, Chromatin Immunoprecipitation, Immunoprecipitation

Journal: Oncotarget

Article Title: SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation

doi: 10.18632/oncotarget.15818

Figure Lengend Snippet: ( A ) Quantitative real time PCR was performed using cDNA from HEK293, HEK293/SET, HN12, HN12siSET, HN13, and HN13siSET cells treated with 100 ng/mL TSA (histone deacetylase inhibitor) or 10 μM 5-aza-2'-deoxicytidine (DNA methyltransferase inhibitor). BRCA1 , MLH1 , and PTEN were assessed through TaqMan probes, and GSTP1 , HIF-1A , NFATC3 , and STAT1 through SybrGreen primers. Graphics represent relative quantification of experiments performed in triplicate, calculated using the 2 −ΔΔCt method. CDNA from HEK293 cells was used as calibrator; GAPDH and β-globin primers were used as housekeeping. *( p < 0.05), **( p < 0.01), ***( p < 0.001) and ns (non-significant). ( B ) Transmission electron microscopy was performed in HEK293 and HEK293/SET cells to analyze chromatin compaction.

Article Snippet: BRCA1 (Hs01556193_m1), MLH1 (Hs00979919_m1) and PTEN (Hs02621230_s1) mRNA levels were assessed by TaqMan assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.

Techniques: Real-time Polymerase Chain Reaction, Histone Deacetylase Assay, Quantitative Proteomics, Transmission Assay, Electron Microscopy

Journal: Cancers

Article Title: Multitarget Stool mRNA Test for Detecting Colorectal Cancer Lesions Including Advanced Adenomas

doi: 10.3390/cancers13061228

Figure Lengend Snippet: List of specific targets tested.

Article Snippet: MLH1 , Hs00179866_m1 , Y , , .

Techniques: TaqMan Assay

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Schematic of the structure of the genetically modified Trp53 allele. The coding sequence for the triple-FLAG tag was inserted after the start codon of the Trp53 gene. ( B ) PCR analysis showing correct insertion of the coding sequences for the triple-FLAG tag into the genetically modified Trp53 allele. A band of 225 bp indicates the presence of a wt (unmodified) allele. A band of 291 bp indicates a FLAG-Trp53 allele. Each lane represents DNA taken from an individual mouse (#32, 38, 40, 44, 45) followed by DNA from a control wt mouse, control DNA from a FLAG-Trp53 KI/+ mouse, and a water-only control. ( C ) Table with observed and expected genotype distribution from inter-crosses of heterozygous FLAG-Trp53 KI/+ mice. ( D ) Table with observed and expected sex distribution from all matings that could give rise to FLAG-Trp53 KI/+ or FLAG-Trp53 KI/KI mice. Frequencies compared by Chi-square test p = 0.0262. ( E ) Kaplan–Meier survival curve showing overall survival of wt, FLAG-Trp53 KI/+ , and FLAG-Trp53 KI/KI mice. Differences in animal survival were compared using the Log-rank (Mantle–Cox) test, * p values ≤0.05. Mice were censored if harvested when healthy for use in experiments. ( F ) Thymocytes from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice were treated for 48 h in vitro with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 1 µg/mL ionomycin or exposed to one dose of 1.25 Gy γ-radiation and then kept for 48 h in culture. Cell viability was measured at various time points by flow cytometry after staining with fluorochrome-conjugated annexin-V and DAPI. The percentages of live, i.e. annexin-V/DAPI double-negative cells, are plotted. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. Data were presented as mean ± SD. n = 4 mice of each genotype. ( G ) Mouse dermal fibroblasts (MDFs) were derived from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice and treated for 72 h with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide or 250 nM taxol. Cellular senescence was measured as a percentage of fluorescein-di-β- d -galactopyranoside (FDG) positive cells by flow cytometry. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. Data were presented as mean ± SD. n = 4 mice of each genotype. ( H ) RNA was extracted from thymocytes and MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice after they had been treated for 6 h with DMSO (vehicle control) or 10 µM nutlin-3a in vitro. qRT-PCR was performed to determine the mRNA levels of the TRP53 target genes Bax, Mdm2, Mlh1, Pmaip1/Noxa, Cdkn1a/p21, Bbc3/Puma , and the Trp53 mRNA levels. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. The data were plotted as fold-change of drug-treated compared to DMSO-treated cells. Data were presented as mean ± SD. n = 4 mice of each genotype. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. All statistical tests were non-significant (i.e. had a p value >0.05). .

Article Snippet: Mlh1 , Mm00503449_m1 , Thermo Fisher Scientific.

Techniques: Genetically Modified, Sequencing, FLAG-tag, Control, In Vitro, Flow Cytometry, Staining, Derivative Assay, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Next-generation sequencing results for the inserted sequences encoding the triple-FLAG tag in the F1 generation of FLAG-Trp53 KI/+ mice. Each line represents the reads from 1 independent F1 mouse. A black dot indicates a matching base in the sequencing reads compared to the reference sequence. ( B ) Representative histology from aged wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice at the time of harvest. No tumour samples showed TRP53 staining by immunohistochemistry, indicating that they did not have mutant TRP53 driving their malignancy. Scale bar = 100 um. ( C ) RNA was extracted from thymocytes and MDFs from wt, FLAG-Trp53 KI/+ and FLAG-Trp53 KI/KI mice that had been treated for 6 h with DMSO (vehicle control) or 1.25 Gy γ-radiation in vitro. qRT-PCR analysis was performed to determine the mRNA levels of the TRP53 target genes Bax, Mdm2, Mlh1, Pmaip1/Noxa, Cdkn1a/p21, Bbc3/Puma and the Trp53 mRNA levels. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. The data were plotted as fold-change compared to DMSO-treated samples. Data were presented as mean ± SD. n = 4 mice of each genotype and treatment. p values were calculated using a two-way ANOVA using Dunnett’s correction for multiple tests. All statistical tests showed that differences were not significant (had a p value >0.05).

Article Snippet: Mlh1 , Mm00503449_m1 , Thermo Fisher Scientific.

Techniques: Next-Generation Sequencing, FLAG-tag, Sequencing, Staining, Immunohistochemistry, Mutagenesis, Control, In Vitro, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: TaqMan TM probes for qRT-PCR.

Article Snippet: Mlh1 , Mm00503449_m1 , Thermo Fisher Scientific.

Techniques:

Journal: Frontiers in Oncology

Article Title: The MSH2 c.793-1G>A variant disrupts normal splicing and is associated with Lynch syndrome

doi: 10.3389/fonc.2023.1131011

Figure Lengend Snippet: The deficiency of MMR was found in the pedigree. H&E and IHC staining was conducted on the serial paraffin embedding tumor tissue sections of III-14, III-15, and III-18. The results indicate III-14: intestinal mucous adenocarcinoma, MLH1+, MSH2−, MSH6−, and PMS+; III-15: poorly differentiated adenocarcinoma, MLH1+, MSH2−, MSH6+, and PMS+; III-18: moderately differentiated adenocarcinoma, MLH1+, MSH2−, MSH6−, and PMS+.

Article Snippet: MMR protein expression was tested by IHC using MLH1 polyclonal antibody clones (Proteintech Group, #11697-1-AP), MSH2 polyclonal antibody (Proteintech Group, #15520-1-AP), MSH6 polyclonal antibody (Proteintech Group, #18120-1-AP), and PMS2 polyclonal antibody (Affinity Group, #DF4351).

Techniques: Immunohistochemistry