Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Molecular events for CRBP1 gene in cervical epithelium samples. A: In order to know the gain of copy number of the CRBP1 gene, DNA of healthy cervix and CC samples, were subjected to real time PCR with specific Taqman probes. White bar (healthy cervix samples) represents the mean of the normal cervices (n = 26) without extra copies of CRBP1 gene. Black bars show CC samples with gain of copy number (2-20X); while gray dotted line bars are showing CC samples that do not change in the copies number. Values above the cut-off line (as 1), being assigned as increased gene copy number compared with normal cervical epithelium. CRBP1 Hs01437985_cn probe, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs00894322_cn probe were used as reference; the relative genomic copy number was calculated using the comparative Ct methods [26]. In X-axis represents cervical samples, Y-axis relative copies fold change of CRBP1 gene. B: CRBP1 expression was observed as positive immunostaining result on tissue microarray as mentioned in Methods section The DNAs used for gain of copy number (panel A) were also used for the methylation assay. Methylation result represents the methylation of the CRBP1 promoter. In this case, each healthy or CC sample, correspond to each column for CRBP1 expression and methylation status. Interestingly, in most of the cases, there was an association between the lack of expression of the CRBP1 gene and its methylation status.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunostaining, Microarray, Methylation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: CRBP1 immunodetection in the uterine cervix samples. A: (1) Cytoplasmic CRBP1 expression is present in cells of the basal layer of normal cervical epithelium (healthy tissue); (2) the immunodetection in the transformed cells of a cervical cancer (CC03) tissue harboring gain of CRBP1 gene. (3) CC samples without gain CRBP1 gene showing negative immunostaining (CC16 sample). A kidney tissue section (4) was used as positive control, while a heart tissue section for negative control (5). B: cervical progression spectrum. The tissue section shows a brownish reaction (positive reaction) in the basal cell layer of the “normal” region, in the high-grade lesion, and also in the invasive region. All tissue sections were hematoxylin counterstained, 200X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunodetection, Expressing, Transformation Assay, Immunostaining, Positive Control, Negative Control, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Association between CRBP1 gene gain copy number and its expression in cervical cancer samples

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Immunodetection

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Correlation between CRBP1 expression and clinic pathological variables in cervical cancer

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Expressing, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Immunolocalization of CRBP1 by immunofluorescence in cervical cells. Nuclei were Dapi stained in blue color (A-C). The immunodetection of CRBP1 was observed in green color (D-F). Cytoplasmic immunodetection of CRBP1 in the merge imaging (G-I). 100X original amplification.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Immunofluorescence, Staining, Immunodetection, Imaging, Amplification

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

doi:

Figure Lengend Snippet: Methylation promoter of CRBP1 gene in cervical cancer samples. Example of CRBP1 gene promoter methylation analysis. Lanes: Healthy cervix sample, CC03 and CC06 samples with un-methylated status; lanes CC 10 and CC 16 with methylated status; HeLa cells as un-methylated control (109 bp), or MCF-7 cells as methylated control (99 bp). MW: molecular weight marker of 100 bp.

Article Snippet: Incubation with the monoclonal mouse anti-CRBP1 antibody (ab24090 Abcam) was performed overnight at 4°C, at 1:100 dilution in 1% bovine serum albumin in phosphate buffered saline (PBS).

Techniques: Methylation, Molecular Weight, Marker

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: Bioinformatics analysis of aspartate-β-hydroxylase (ASPH) overexpression in CRC: ( a ) analysis of ASPH mRNA expression in normal mucosa and colorectal cancer (CRC) samples from TCGA database (UALCAN website); ( b ) ASPH promoter methylation status in normal mucosa and CRC samples from TCGA database (UALCAN website; beta-value scale ranges from 0 = unmethylated to 1 = fully methylated); ( c ) analysis of ASPH protein levels by mass spectrometry in normal mucosa and CRC samples from the CPTAC database (UALCAN website; Z-values represent standard deviations from the median across the samples); ( d ) putative copy number alterations of the ASPH gene computed by GISTIC algorithm in TCGA database (c-BioPortal website; shallow deletion = 1− copy, gain = 1+ copy, amplification ≥ 2+). Box plots represent the quartile distribution of data around the median value. Statistics are reported in each graph as the calculated p value.

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Over Expression, Expressing, Methylation, Mass Spectrometry, Amplification

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: Heatmaps of mRNA-clusters significantly correlated with ASPH expression (red = high, green = low), selected in : ( a ) Immune signature ( n = 222); ( b ) Notch signature ( n = 203); ( c ) Invasive signature ( n = 203). Each cluster is ordered according to Pearson’s r coefficients of each marker against ASPH, from higher (left) to lower (right). The maximum r coefficient was 0.330 (SNW1), and the lowest was -0.304 (LFNG); thus, no marker showed a widespread correlation with ASPH among analyzed samples. Microarray Z -scores for the selected markers were extracted from the COAD Firehouse legacy database (TCGA) using the cBioPortal web interface.

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Expressing, Marker, Microarray

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: ASPH expression in the tumor invasive margin (IM) correlates with the pattern of local invasion and patients’ overall survival: invasive front type (I = infiltrative, E = expansive), tumor budding (1 = 0–4 buds, 2 = 5–9 buds, 3 ≥ 10 buds) and overall survival (L = low, H = high ASPH levels dichotomized by the median value) show a strong relation only with ASPH levels scored in CRC IM. Center of the tumor (CT)-scored ASPH shows a statistically significant relation only with tumor budding, while ASPH expressed next to tumor lumen (LM) does not show any relation with these parameters. Box plots represent the quartile distribution of ASPH H-score quantifications in CRC samples around the median value. Statistics are reported in each graph as the calculated p value (Invasive front: Student t -test; Budding: one-way ANOVA; Overall survival: Kaplan–Meier analysis).

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Expressing

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: ASPH silencing or activity inhibition decreases cell invasion and proliferation in a panel of CRC cell lines. Specific ASPH siRNAs visibly downregulate ASPH expression after 18 h ( a ). Concomitantly, cell invasion ( b ) and proliferation ( c ) are significantly inhibited. All these effects are associated with Notch1 inhibition ( d ). One representative experiment out of three is reported. β-Actin was utilized as a loading control. Means ± SEM are reported. *** p < 0.001. DLD1 and SW480 cell lines were exposed to increasing concentration of MO-I-1144. For the invasion test ( e ), filters were recovered and counted after 18 h. Representative images of filters from 20 µM treated cells are shown in ( f ). Cell growth was evaluated after 96 h by the crystal violet assay ( g ). One representative experiment (conducted on 10 replicates) out of three is reported. Mean ± SEM are reported. ** p < 0.01, *** p < 0.001.

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Activity Assay, Inhibition, Expressing, Control, Concentration Assay, Crystal Violet Assay

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet:

Article Snippet: Mouse α-MYH7 monoclonal , Santa Cruz Biotech , Cat# Sc-53090; RRID: AB_2147279.

Techniques: Recombinant, Membrane, Transfection, SYBR Green Assay, DNA Methylation Assay, Mutagenesis, CRISPR, Software, Functional Assay, Microarray, In Vivo, Comparison, Next-Generation Sequencing

Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

Journal: Cancer cell

Article Title: Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation, Stemness, and Braf V600E -Induced Tumorigenesis

doi: 10.1016/j.ccell.2019.01.005

Figure Lengend Snippet: (A) DNA methylation accumulation determined by bisulfite pyrosequencing of selected CGI regions in BrafEV1 and 3 organoids cultured for 2 or 12–14 months and BrafCA-IND1–3 organoids cultured for 5 months. The suffix “m” in BrafEV1–12m and BrafEV3–14m indicates the duration in months for which the organoids were cultured. Whiskers indicate mean (cross bar) ± SD.

Article Snippet: EZ DNA Methylation Lightning Kit , Zymo , Cat#D5030.

Techniques: DNA Methylation Assay, Cell Culture

Journal: Cancer cell

Article Title: Aging-like Spontaneous Epigenetic Silencing Facilitates Wnt Activation, Stemness, and Braf V600E -Induced Tumorigenesis

doi: 10.1016/j.ccell.2019.01.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EZ DNA Methylation Lightning Kit , Zymo , Cat#D5030.

Techniques: Recombinant, Transfection, Methylation, Clone Assay, Microarray, DNA Methylation Assay, Knock-Out, CRISPR, Expressing, Plasmid Preparation, Software

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 2 DACH1 deletion PCa enhances AR signaling. A Interrogation of human PCa gene expression data [26], showing candidate genetic drivers ERG, ETV1/ETV4/FLI1, SPOP, FOXA1, and unknown. Samples with DACH1 homozygous (deep) genetic deletions (29/333) are shown as an additional subtype. The AR score (the average of the AR target gene expression) refers to a group of AR-responsive genes [26], and together with the expression Z-score of the AR target genes, are shown as colorimetric scales. The AR score-based gene names are shown. The androgen receptor (AR) activity, inferred by the induction of AR target genes, was increased in DACH1 homozygous (‘deep’) deletion PCa compared with normal (P = 2 × 10−5 by t-test) and ERG mutation groups (P = 0.003 by t-test). B AR mRNA and AR protein levels, shown for each DACH1 deletion sample, were not significantly different. C The iCluster [29], mRNA cluster, and SCNA (somatic copy-number alteration), and DNA methylation status are shown for the PCa classified by the corresponding gene deletion subtypes. D DACH1 homozygous deletions were enriched for iCluster 2 and 3 [29], mRNA cluster 2 (P = 0.0003 by Fisher exact test, SCNA (“more” somatic copy-number alteration, P = 0.0004 by Fisher exact test), but not for DNA methylation.

Article Snippet: The following antibodies were used: rabbit polyclonal DACH1 antibody (Proteintech 10914-1-AP), rabbit polyclonal Ku80 antibody (Invitrogen PA517454), mouse monoclonal Ku70 antibody (Fisher MS329P), mouse monoclonal FLAG antibody (Sigma F3165), and rabbit polyclonal GAPDH antibody (sc-25778).

Techniques: Gene Expression, Targeted Gene Expression, Expressing, Activity Assay, Mutagenesis, DNA Methylation Assay

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 3 Prostate-specific Dach1 gene deletion promotes prostate hyperplasia and dysplasia in OncoMice (15 weeks). A Schematic representation of transgenes integrated into mice. B Representative immunohistochemistry for Dach1, with data quantitated as mean ± standard error of the mean (SEM) for N = 20 (4 separate mice, with 5 views per mouse, in each group). C Blinded quantitative histology grading of prostate of multigenic mice at 15 weeks. Data are shown as mean ± SEM for N = 15 (5 separate mice, with 3 prostate areas [anterior, ventral, lateral] per mouse) in each group). H&E staining demonstrates the presence of a focal atypical intraductal proliferation in Dach1−/−prostate, compatible with prostatic intraepithelial neoplasia (PIN). Representative immunohistochemistry with results shown as mean ± SEM for Ki-67 (n = 20, 4 separate mice for each genotype, 5 views per mouse) (D), Beclin 1 (n = 9, 3 separate mice for each genotype, 3 views per mouse) (E); and AR (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 12 for Dach1fl/flmice, 3 separate mice, 2 views for one mouse and 5 views for other two mice) (F). Scale bars, 50 μm. A Student’s t test was performed for all comparisons.

Article Snippet: The following antibodies were used: rabbit polyclonal DACH1 antibody (Proteintech 10914-1-AP), rabbit polyclonal Ku80 antibody (Invitrogen PA517454), mouse monoclonal Ku70 antibody (Fisher MS329P), mouse monoclonal FLAG antibody (Sigma F3165), and rabbit polyclonal GAPDH antibody (sc-25778).

Techniques: Immunohistochemistry, Staining

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 4 Prostate-specific Dach1 gene deletion in TRAMP mice induces PIN lesions with increased TGFβ activity. Genome-wide expression analysis of TRAMP Dach1+/+ vs. Dach1−/−PIN lesions was analyzed for enrichment of known targets of upstream regulators using Ingenuity Pathway Analysis (IPA) and represented as (A) barplot was calculated by IPA activation Z-score labeled and as (B) bubble plot with size of the bubbles proportional to –log10 p values. C IHC was conducted for SMAD activation using SMAD2P, quantitated and shown as (D) mean ± SEM (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 10 for Dach1fl/flmice, 2 separate mice, 5 views per mouse). E–G Western blot of either PCa cell lines for the presence of DACH1 (E, F) or (G) TGFβ-treated (10 ng/ml for 24 h) PC3 cells illustrating induction of nuclear vimentin and cytoplasmic cyclin D1. Protein loading controls are β-tubulin (a marker of cytoplasmic proteins) and Lamin B1 (a marker for nuclear protein enrichment). H Microarray-based gene expression analysis of PC3 cells stably expressing DACH1, showing restraint of genes mediating TGFβ signaling (shown with blue arrows), including reduction of TGFB2 and TGFBR2 [33].

Article Snippet: The following antibodies were used: rabbit polyclonal DACH1 antibody (Proteintech 10914-1-AP), rabbit polyclonal Ku80 antibody (Invitrogen PA517454), mouse monoclonal Ku70 antibody (Fisher MS329P), mouse monoclonal FLAG antibody (Sigma F3165), and rabbit polyclonal GAPDH antibody (sc-25778).

Techniques: Activity Assay, Genome Wide, Expressing, Activation Assay, Labeling, Western Blot, Marker, Protein Enrichment, Microarray, Gene Expression, Stable Transfection

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 6 DACH1 facilitates the recruitment of, and co-accumulates with, Ku70/Ku80 proteins at sites of DNA damage. A Co-accumulation of Ku-70/Ku-80 at laser micro irradiation-induced DSBs sites in Dach1+/+ 3T3 cells. B, C 24 h after transfection, the accumulation of DACH1 and Ku70/Ku80 in Dach1−/−3T3 cells transfected with EGFP or EGFP-tagged DACH1 and red fluorescent protein (RFP)-tagged Ku70 or RFP-tagged Ku80 expression vectors were treated with laser micro-irradiation (403 nm) to induce DSBs. Time is shown after micro-irradiation. Accumulation of the transfected proteins was indicated by EGFP (green) or RFP (red) fluorescence at laser-irradiated sites. Co-accumulation was visualized in yellow merged images. Time is shown in minutes and -fold increase in foci intensity is shown as mean ± SEM for N = 5 separate cells.

Article Snippet: The following antibodies were used: rabbit polyclonal DACH1 antibody (Proteintech 10914-1-AP), rabbit polyclonal Ku80 antibody (Invitrogen PA517454), mouse monoclonal Ku70 antibody (Fisher MS329P), mouse monoclonal FLAG antibody (Sigma F3165), and rabbit polyclonal GAPDH antibody (sc-25778).

Techniques: Irradiation, Transfection, Expressing

Journal: Cell Reports Medicine

Article Title: Arachidonic acid released by PIK3CA mutant tumor cells triggers malignant transformation of colonic epithelium by inducing chromatin remodeling

doi: 10.1016/j.xcrm.2024.101510

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibody anti-FBW7 , Proteintech , Cat# 28424-1-AP; RRID: AB_2881138.

Techniques: Microarray, Recombinant, Cloning, Mutagenesis, Immunohistochemistry, Protein Purification, In Vitro, Sequencing, Methylation