Journal: Oncotarget

Article Title: RACK1 promotes lung cancer cell growth via an MCM7/RACK1/Akt signaling complex

doi: 10.18632/oncotarget.17120

Figure Lengend Snippet: ( A ) A549 cells were transfected with siRACK1, siCon, GFP-RACK1, or GFP. MCM7 immunoprecipitates were probed for P-Ser, P-Thr, and MCM7 as indicated. ( B ) A549 cells were transfected with siRACK1 or siCon. MCM7 immunoprecipitates were probed for Cdt1, MCM4, MCM6, and MCM7. ( C ) A549 cells were treated with siRACK1, siCon, GFP-RACK1, or GFP as indicated. The chromatin (Chr) and non-chromatin (Non-Chr) fractions of these cells were purified and immunoblotted with anti-MCM7 antibodies. Antibodies against Histone 3 and GADPH were used as internal controls.

Article Snippet: Antibodies for MCM4, MCM6, Cdt1, p27, E2F1, and Histone 3 were from Proteintech Group.

Techniques: Transfection, Purification

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: A Immunoblot analysis of stable U2OS cell lines left untreated or incubated with DOX to induce expression of WT or mutant forms of GFP‐FAM111A. B DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse‐labeled with EdU, and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image‐based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n > 2,000 cells per condition). See also Appendix Fig S6A . C Cells treated as in (B) were pre‐extracted, fixed, and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC ( n > 2,000 cells per condition). D–F Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Fig S6B–D . G Analysis of FAM111A interactors. U2OS/GFP‐FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP), and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/−DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR < 0.05; s 0 = 1). H U2OS or U2OS/ ΔFAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies. I U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies. J As in (C), except that cells were stained with RFC1 antibody ( n > 2,000 cells per condition). K Quantification of data in (J) for S phase (EdU‐positive) cells (red bars, mean). Data information: Data are representative of at least three (A–F, H–K) and two (G) independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Incubation, Expressing, Mutagenesis, Labeling, Staining, Cytometry, Immunoprecipitation, Mass Spectrometry, Control, Transfection, Plasmid Preparation

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Validation of FAM111A interactors identified by mass spectrometry (Fig G; ). U2OS/GFP‐FAM111A cell lines treated or not with DOX were subjected to GFP immunoprecipitation (IP) followed by immunoblotting with indicated antibodies. U2OS cells transfected with constructs expressing indicated FLAG‐tagged RFC1 fragments were subjected to FLAG IP followed by immunoblotting with indicated antibodies. U2OS cells transfected with non‐targeting control (CTRL) or FAM111A siRNA were pre‐extracted, fixed, and stained with FAM111A antibody. U2OS cells labeled with EdU were pre‐extracted, fixed, and stained with indicated antibodies. Endogenous FAM111A localizes to nucleoli in G1 phase and G2 phase (EdU‐negative) cells and relocates to nuclear foci in S phase (EdU‐positive) cells. U2OS cells transfected with indicated siRNAs were pre‐extracted, fixed, and stained with FAM111A and RFC1 antibodies. Immunoblot analysis of U2OS/GFP‐FAM111A cell lines treated or not with DOX. Scale bars, 10 μm. Data information: Data (A–F) are representative of three independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Biomarker Discovery, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Control, Staining, Labeling

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Overview of heterozygous FAM111A mutations found in patients with gracile bone dysplasia or Kenny–Caffey syndrome. Immunoblot analysis of U2OS cell lines left untreated or incubated with DOX to induce expression of the indicated GFP‐FAM111A alleles. U2OS/GFP‐FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP‐FAM111A WT cells used in Figs and (see Fig A). U2OS/GFP‐FAM111A cell lines treated or not with DOX, pulse‐labeled with EdU, and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. Quantification of data in (C) for S phase (EdU‐positive) cells (red bars, mean (A.U., arbitrary units); n > 2,000 cells per condition). See also Appendix Fig S6J . As in (C), except that cells were pulse‐labeled with EU. Quantification of EU incorporation in cells in (E) (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6L . U2OS/GFP‐FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre‐extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC. As in (G), except that cells were stained with RPB1 antibody. Immunoblot analysis of U2OS/GFP‐FAM111A cell lines treated or not with DOX in the absence or presence of Z‐VAD-FMK. As in (I), using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles. Purified recombinant FLAG‐FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto‐proteolytic activity was analyzed by immunoblotting. GFP IPs from U2OS cell lines expressing GFP‐FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR < 0.05; s 0 = 1). Data information: Data are representative of at least three (B–K) and two (L) independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Incubation, Expressing, Labeling, Staining, Purification, Recombinant, Activity Assay, Mutagenesis, Mass Spectrometry

Journal: EMBO Reports

Article Title: FAM111 protease activity undermines cellular fitness and is amplified by gain‐of‐function mutations in human disease

doi: 10.15252/embr.202050662

Figure Lengend Snippet: Immunoblot analysis of parental U2OS cells (−) or derivative stable cell lines conditionally expressing GFP‐FAM111A WT at different levels. Cells in (A) were treated with DOX for 16 h, pulse‐labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition; A.U., arbitrary units). As in (B), except that cells were treated with DOX for 24 h. Immunoblot analysis of parental U2OS cells (−) or derivative stable cell lines expressing WT or patient‐associated GFP‐FAM111A alleles. Cells in (D) were pulse‐labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition). Quantification of data in (E) (red bars, mean). Representative images of U2OS/GFP‐FAM111A cell lines that were treated or not with DOX for the indicated times, fixed, and co‐stained with PCNA and RPA2 antibodies. Scale bar, 10 μm. Quantification of data in (G) (gray bars, average; n > 2,000 cells per condition). U2OS/GFP‐FAM111A cell lines treated or not with DOX were stained with γ‐H2AX antibody and analyzed for γ‐H2AX signal intensity by QIBC (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6K . As in (I), except that cells were stained with RFC1 antibody, pre‐extracted and fixed, and stained with DAPI. RFC1 signal intensity in S phase cells (gated based on DAPI signal intensity) was analyzed by QIBC (red bars, mean; n > 2,000 cells per condition). See also Appendix Fig S6M . As in (I), except that cells were stained with RPB1 antibody and analyzed for RPB1 signal intensity by QIBC (red bars, mean; n > 2,000 cells per condition). U2OS cell lines conditionally expressing untagged ectopic FAM111A alleles were treated or not with DOX for 24 h, labeled with EdU, fixed, and stained with DAPI. Cells were then subjected to QIBC analysis for quantification of EdU and DAPI signal intensities ( n > 2,000 cells per condition). Immunoblot analysis of stable U2OS/FAM111A cell lines transfected or not with FAM111A siRNA targeting the 3′UTR, and subsequently treated or not with DOX to express ectopic untagged FAM111A alleles. Data information: Data (A–M) are representative of three independent experiments with similar outcomes.

Article Snippet: For immunoprecipitations, cleared lysates were incubated with FLAG agarose (Sigma‐Aldrich), GFP‐Trap Agarose (Chromotek), or anti‐goat RFC1 antibody (2 μg/sample) coupled to Protein G agarose beads (Thermo Fisher Scientific) for 2 h on an end‐over‐end rotator at 4°C, washed in EBC buffer, and treated with Benzonase to minimize chromatin‐mediated interactions.

Techniques: Western Blot, Stable Transfection, Expressing, Labeling, Staining, Transfection

Journal: PNAS Nexus

Article Title: A PITX2–HTR1B pathway regulates the asymmetric development of female gonads in chickens

doi: 10.1093/pnasnexus/pgad202

Figure Lengend Snippet: Identification of HTR1B and DRD4 as downstream target genes of PITX2. A) Combined overlap analysis of genes with left gonad-specific expression in RNA-seq and genes with Pitx2 peaks located within 0–1 kb of TSS in ChIP-seq. B) GO analysis of overlapping genes. C) The expression of HTR1B and DRD4 was confirmed by RNA-seq and RT-qPCR. D) IGV tracks show the Pitx2 binding peaks on HTR1B and DRD4 loci. E) Immunofluorescence staining results of HTR1B and DRD4 in female gonads during development. Note the much stronger HTR1B and DRD4 signals in the left than the right gonad, particularly in the cortex regions (arrowheads). Scale bars: 100 μm. Data are mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were incubated with the HTR1B antibody (Bioss, bs-1125R, 1:500), DRD4 antibody (Boster, A00998-2, 1:500), MCM2 antibody (Abcam, ab4461, 1:200), and βIII-tubulin antibody (R&D Systems, MAB1195-SP, 1:500) overnight at 4°C and the corresponding secondary antibodies conjugated to Alexa Fluor 555 (Invitrogen, 1:500) for 2 h at room temperature.

Techniques: Expressing, RNA Sequencing, ChIP-sequencing, Quantitative RT-PCR, Binding Assay, Immunofluorescence, Staining

Journal: PNAS Nexus

Article Title: A PITX2–HTR1B pathway regulates the asymmetric development of female gonads in chickens

doi: 10.1093/pnasnexus/pgad202

Figure Lengend Snippet: Activation of HTR1B, not DRD4, receptors in the female right gonads increased the expression of ovarian genes and gonadal cell proliferation. A) Schematic diagram showing the method for embryonic gonad injections. The agonists of HTR1B or DRD4 were injected into the right gonads at E4.5 and sample collection at E6.5. Scale bar: 1 mm. B) RT-qPCR showed the up-regulation of ERα and LHX9 upon HTR1B receptor activation by RU24969 (RU). C) The expression of ERα and LHX9 was down-regulated after DRD4 receptor activation by PD168077 (PD). D) Representative image showing MCM2 immunofluorescence staining in gonads after HTR1B receptor activation by RU24969 (RU). The white dashed box indicates the magnified area at the bottom showing single gonads. Scale bars: 100 μm. E) Quantification of MCM2 positive cells in gonads after HTR1B receptor activation by RU24969 (RU). Data are mean ± SEM; ** P < 0.01 after the t test; n = 4 in each group.

Article Snippet: The sections were incubated with the HTR1B antibody (Bioss, bs-1125R, 1:500), DRD4 antibody (Boster, A00998-2, 1:500), MCM2 antibody (Abcam, ab4461, 1:200), and βIII-tubulin antibody (R&D Systems, MAB1195-SP, 1:500) overnight at 4°C and the corresponding secondary antibodies conjugated to Alexa Fluor 555 (Invitrogen, 1:500) for 2 h at room temperature.

Techniques: Activation Assay, Expressing, Injection, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Revista da Associação Médica Brasileira

Article Title: Expression of gamma-aminobutyric acid type A receptor subunit π and minichromosome maintenance-3 in breast cancer patients treated with neoadjuvant chemotherapy: a target molecule or a proliferation marker?

doi: 10.1590/1806-9282.20250507

Figure Lengend Snippet: Gamma-aminobutyric acid type A receptor subunit π and minichromosome maintenance-3 staining examples [(A) minichromosome maintenance-3 Strong nuclear staining (B) minichromosome maintenance-3 Weak nuclear staining (C) gamma-aminobutyric acid type A receptor subunit π Strong cytoplasmic staining (D) gamma-aminobutyric acid type A receptor subunit π Weak cytoplasmic staining].

Article Snippet: Ventana), GABRP (1:1000; ab26055; Abcam, Cambridge, UK), MCM3 (1:1000; PA1651; Boster Bio), HER-2 (anti-HER-2/neu; 4B5.

Techniques: Staining

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Expression patterns of MCM3 in pan-cancer. (A) Differences in MCM3 between tumor and normal tissues based on TCGA and GETx data. (B) Comparison of protein levels based on CPTAC data. (C) Clinical correlation analysis based on GEPIA database. (D) The genomic alteration of MCM3 in pan-cancer. (E) Immunofluorescence results showed the localization of MCM3 in cell lines. **** p < 0.0001.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Expressing, Comparison, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Prognostic value of MCM3 in pan-cancer. (A) The heatmap shows results of univariate Cox regression analysis. (B) Forest plot of MCM3 expression and OS across cancers. (C) Forest plot of MCM3 expression and PFI across cancers. (D) Forest plot of MCM3 expression and DSS across cancers. (E) Forest plot of MCM3 expression and DFI across cancers.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Relationship between MCM expression and immune-related features in pan-cancer. (A) MCM3 expression and tumor microenvironment relate parameters. (B) MCM3 expression and immune cell infiltration. (C) MCM3 expression and immune checkpoints. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Immunotherapy and drug sensitivity analysis. (A) Relationship between MCM3 expression and TMB, MSI, and neoantigens. (B–E) Prognostic significance of MCM3 and proportion of immunotherapy response between high- and low-MCM3 groups in four cohorts receiving ICB therapy. (F) Drug sensitivity analysis of MCM3 based on CTPR and GDSC data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Expressing, Immunopeptidomics

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Single-cell analysis of MCM3. (A) Correlation between MCM3 and 14 biological functions. (B) The top three functions in BRCA, LUAD, MEL and glioma. (C) Datasets of single-cell expression of MCM3 from TISCH website. (D) Distribution of MCM3 among cell types in the GSE111360 and GSE140228 datasets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Single-cell Analysis, Expressing

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Correlation between MCM3 expression and clinical features and prognosis of LGG. (A, B) Relationship between MCM3 and clinical features in TCGA and CGGA cohorts. (C, D) Evaluation of the ability of MCM3 expression to predict prognosis in TCGA and CGGA cohorts. ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Construction of a prognostic nomogram and analysis of MCM3 related functions in LGG. (A) Univariate/multivariate Cox analysis was performed based on TCGA cohort. (B) Establishment of a prognostic nomogram based on multivariate analysis results. (C–F) Nomogram model evaluation, including timeROC, calibration, DCA and KM curves. (G) GO and KEGG analyses based on differentially expressed genes in TCGA. (H) An interaction network between GO and KEGG. (I) The GSEA analysis in TCGA cohort. (J) The waterfall map shows the top 10 genes with the highest mutation probability. (K, L) TMB and TIDE score were compared between the two groups. ** p < 0.01, **** p < 0.0001.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: The prognostic and immunological role of MCM3 in pan-cancer and validation of prognosis in a clinical lower-grade glioma cohort

doi: 10.3389/fphar.2024.1390615

Figure Lengend Snippet: Validation of the association between MCM3 and clinical features and prognosis of LGG. (A) MCM3 protein expression in normal and tumor tissues from HPA database. (B) Representative plots of negative and positive immunohistochemical results. (C, D) Relationship between MCM3 expression and tumor size and grade. (E, F) Survival curves for OS and PFS in clinical cohort. (G, H) Univariate and multivariate Cox regression analysis for OS and PFS.

Article Snippet: The MCM3 antibody was purchased from Boster Biological Technology Company (article number: BA2186 ).

Techniques: Biomarker Discovery, Expressing, Immunohistochemical staining