human men1 sirna  (Santa Cruz Biotechnology)

Journal: Journal of Clinical Medicine

Article Title: Identification of New Molecular Biomarkers in Ovarian Cancer Using the Gene Expression Profile

doi: 10.3390/jcm11133888

Figure Lengend Snippet: Sequences of primers used for real-time PCR.

Article Snippet: The Human ATM ELISA Kit (sensitivity: 0.094 ng/mL; LSBio, Seattle, WA, USA), Human BRCA1 ELISA Kit (sensitivity: 0.065 ng/mL; LSBio), BRCA2 ELISA Kit (sensitivity: 0.062 ng/mL; Biomatik, Wilmington, NC, USA), Human KRAS ELISA Kit (sensitivity: 0.115 ng/mL; LSBio, Seattle, WA, USA), C-JUN ELISA Kit (sensitivity: 1.0 ng/mL; MyBioSource, San Diego, CA, USA), Human c-FOS ELISA Kit (sensitivity: 0.188 ng/mL; AssayGenie, Dublin, Ireland), Human NOXA ELISA Kit (sensitivity: 0.078 ng/mL; LSBio, Seattle, WA, USA), Human PUMA ELISA Kit (sensitivity: 0.056 ng/mL; LSBio, Seattle, WA, USA), Human MEN1 ELISA Kit (sensitivity: 0.039 ng/mL; MyBioSource, San Diego, California, USA), Human NOD2 ELISA Kit (sensitivity: 0.062 ng/mL; MyBioSource, San Diego, CA, USA), Human CHEK2 ELISA Kit (sensitivity: 0.062 ng/mL; MyBioSource, San Diego, CA, USA), and Human EGFR ELISA Kit (sensitivity: 0.001 ng/mL; MyBioSource, San Diego, CA, USA) were employed to evaluate the concentrations of ATM, BRCA1, BRCA2, KRAS, c-JUN, c-FOS, NOXA, PUMA, MEN1, NOD2, CHEK2, and EGFR from tissue homogenates, according to the manufacturers’ protocols.

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Identification of New Molecular Biomarkers in Ovarian Cancer Using the Gene Expression Profile

doi: 10.3390/jcm11133888

Figure Lengend Snippet: Summary of mRNA expression analysis of selected genes in patients with ovarian cancer in comparison to the control group.

Article Snippet: The Human ATM ELISA Kit (sensitivity: 0.094 ng/mL; LSBio, Seattle, WA, USA), Human BRCA1 ELISA Kit (sensitivity: 0.065 ng/mL; LSBio), BRCA2 ELISA Kit (sensitivity: 0.062 ng/mL; Biomatik, Wilmington, NC, USA), Human KRAS ELISA Kit (sensitivity: 0.115 ng/mL; LSBio, Seattle, WA, USA), C-JUN ELISA Kit (sensitivity: 1.0 ng/mL; MyBioSource, San Diego, CA, USA), Human c-FOS ELISA Kit (sensitivity: 0.188 ng/mL; AssayGenie, Dublin, Ireland), Human NOXA ELISA Kit (sensitivity: 0.078 ng/mL; LSBio, Seattle, WA, USA), Human PUMA ELISA Kit (sensitivity: 0.056 ng/mL; LSBio, Seattle, WA, USA), Human MEN1 ELISA Kit (sensitivity: 0.039 ng/mL; MyBioSource, San Diego, California, USA), Human NOD2 ELISA Kit (sensitivity: 0.062 ng/mL; MyBioSource, San Diego, CA, USA), Human CHEK2 ELISA Kit (sensitivity: 0.062 ng/mL; MyBioSource, San Diego, CA, USA), and Human EGFR ELISA Kit (sensitivity: 0.001 ng/mL; MyBioSource, San Diego, CA, USA) were employed to evaluate the concentrations of ATM, BRCA1, BRCA2, KRAS, c-JUN, c-FOS, NOXA, PUMA, MEN1, NOD2, CHEK2, and EGFR from tissue homogenates, according to the manufacturers’ protocols.

Techniques: Expressing, Comparison, Control

Journal: Journal of Clinical Medicine

Article Title: Identification of New Molecular Biomarkers in Ovarian Cancer Using the Gene Expression Profile

doi: 10.3390/jcm11133888

Figure Lengend Snippet: Analysis of the protein level for ATM, BRCA1, BRCA2, KRAS, c-JUN, c-FOS, NOXA, PUMA, MEN1, NOD2, CHEK2, and EGFR in the tissue homogenates in patients with ovarian cancer in comparison to the control group.

Article Snippet: The Human ATM ELISA Kit (sensitivity: 0.094 ng/mL; LSBio, Seattle, WA, USA), Human BRCA1 ELISA Kit (sensitivity: 0.065 ng/mL; LSBio), BRCA2 ELISA Kit (sensitivity: 0.062 ng/mL; Biomatik, Wilmington, NC, USA), Human KRAS ELISA Kit (sensitivity: 0.115 ng/mL; LSBio, Seattle, WA, USA), C-JUN ELISA Kit (sensitivity: 1.0 ng/mL; MyBioSource, San Diego, CA, USA), Human c-FOS ELISA Kit (sensitivity: 0.188 ng/mL; AssayGenie, Dublin, Ireland), Human NOXA ELISA Kit (sensitivity: 0.078 ng/mL; LSBio, Seattle, WA, USA), Human PUMA ELISA Kit (sensitivity: 0.056 ng/mL; LSBio, Seattle, WA, USA), Human MEN1 ELISA Kit (sensitivity: 0.039 ng/mL; MyBioSource, San Diego, California, USA), Human NOD2 ELISA Kit (sensitivity: 0.062 ng/mL; MyBioSource, San Diego, CA, USA), Human CHEK2 ELISA Kit (sensitivity: 0.062 ng/mL; MyBioSource, San Diego, CA, USA), and Human EGFR ELISA Kit (sensitivity: 0.001 ng/mL; MyBioSource, San Diego, CA, USA) were employed to evaluate the concentrations of ATM, BRCA1, BRCA2, KRAS, c-JUN, c-FOS, NOXA, PUMA, MEN1, NOD2, CHEK2, and EGFR from tissue homogenates, according to the manufacturers’ protocols.

Techniques: Comparison, Control

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Identification of MEN1- modulated genes in breast cancer cells. ( A ) RT-qPCR of MEN1 in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus ( n = 3). ( B ) Quantitative Western immunoassays (WES) of menin expression in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus ( n = 3). ( C ) Venn diagrams of differentially expressed genes (fold change ≥1.5 or ≤0.66) in T47D or MCF-7 cells after sh MEN1 knockdown compared with vehicle controls ( n = 2). ( D ) Pathway annotation analysis of MEN1 -upregulated and MEN1 -downregulated genes in T47D or MCF-7 cells using DAVID including cancer hallmark pathways. ( E ) Schematic illustration of five major metabolic pathways. ( F ) Expression heat maps of oxidative phosphorylation (OXPHOS) and glycolytic genes in both MEN1 knockdown T47D and MCF-7 cells (fold changes relative to vehicle controls). ( G ) Bar charts of the expression levels of representative OXPHOS and glycolytic genes affected by MEN1 knockdown in T47D or MCF-7 cells using RT-qPCR. Data are presented as mean ± S.D. Unpaired two-tailed Student’s t -test was used for statistics. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Quantitative RT-PCR, shRNA, Western Blot, Expressing, Knockdown, Phospho-proteomics, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Identification of menin-associated proteins (MAPs) in breast cancer cells. ( A ) and ( B ) WES of BirA-Menin fusion proteins ( A ) and biotin-labeled proteins ( B ) in total lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after incubating with or without doxycycline and biotin. ( C ) Schematic purification and proteomic identification of MAPs using LC–MS/MS. ( D ) Heatmap of the quantification of 35 MAPs commonly shared in T47D and MCF-7 cells. MAPs further verified by WES immunoassays were indicated by arrows. ( E ) Network analysis of 35 MAPs in MCF-7 cells. The distance between menin and MAPs represented the quantitative ratio of each MAP and menin. MAPs marked in blue were further assayed by WES. ( F ) Nuclear or cytoplasmic lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after streptavidin beads pull-down were detected by WES with antibodies against menin, KMT2A, MED12, WAPL, GATA3, LaminA/C, or GAPDH. FL, full length; SP, spliced form.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Labeling, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Expression correlation relationship of menin/MAPs genes and OXPHOS/glycolytic genes. ( A ) Workflow of the in silico correlation analysis of gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. ( B ) Heatmaps of the expression correlation between MEN1 /selected 4 MAP genes and OXPHOS genes (upper) or glycolytic genes (lower) in normal (N) and tumor (T) samples. The genes are arranged from the highest to the lowest according to gene expression correlation coefficients of MEN1 –OXPHOS genes or MEN1 –glycolytic genes in breast tumors. ( C ) and ( D ) Scatter plots and linear regression analyses of MEN1/selected MAPs expression and mean expression of OXPHOS genes ( C ) or glycolytic genes ( D ) in normal and tumor samples. ( E ) Violin plots (lower panel) shows the average expressions of the genes of OXPHOS complexes I-V and glycolysis in the samples of each of the corresponding 4 groups are shown as violin plots. Based on the median values (where ≥median is “high” and

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, In Silico, Gene Expression

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Bioenergetic dynamics are regulated by menin and MAPs in T47D and MCF-7 cells. ( A ) and ( B ) Glycolytic and OXPHOS ATP productions in T47D ( A ) or MCF-7 ( B ) cells infected with vehicle, sh MEN1 , sh KMT2A , sh MED12 , sh WAPL , or sh GATA3 lentivirus. Statistics represented the difference of glycolytic or OXPHOS ATP production between shRNA knockdown and vehicle controls. ( C ) and ( D ) Bar charts representing mitochondrial functions in the single knockdown of MEN1 , KMT2A , MED12 , WAPL , or GATA3 and their vehicle control in T47D ( C ) or MCF-7 ( D ) cells. ( E ) Schematic summary of mitochondrial dynamics affected by the knockdown of MEN1 or MAPs. ( F , G ) Bar charts represented the glycolytic functions in T47D ( F ) or MCF-7 ( G ) cells subject to gene knockdown by sh MEN1 , sh KMT2A , sh MED12 , sh WAPL , or sh GATA3 lentivirus. ( H ) Schematic summary of glycolytic functions affected by the knockdown of MEN1 or MAPs. Data are presented as mean ± S.D. ( n = 15–20 technical-replicate wells). Statistical significance was performed by an unpaired two-tailed Student’s t -test between treated groups and corresponding controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Infection, shRNA, Knockdown, Control, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: Integrity of the menin–KMT2A complex is required for OXPHOS functions. ( A ) WES of T47D or MCF-7 cells treated with DMSO or 1 μM MI-503 for 3 days (left). Relative protein expression normalized to the average of LaminA/C in WES (right). FL, full length; SP, spliced form. ( B ) Nuclear lysates of T47D or MCF-7 cells treated with DMSO or 1 μM of MI-503 for 3 days were immunoprecipitated with the menin antibody or IgG, and assayed by WES (upper). Relative protein expression in WES (lower). The protein expression in DMSO treated input was normalized as 1. FL, full length; SP, spliced form. ( C ) Glycolytic or OXPHOS ATP production in T47D or MCF-7 cells treated with 1 μM MI-503 for 0, 1, 3, 6, and 72 h, or DMSO control for 72 h. ( D , E ) Bar charts of the Seahorse mitochondrial stress test ( D ) and glycolytic stress test ( E ) on T47D or MCF-7 cells treated with 1 μM MI-503 for 0, 1, 3, 6, and 72 h, or DMSO for 72 h. Data are presented as mean ± S.D. ( n = 10–15 technical-replicate wells). An unpaired two-tailed Student’s t -test was used to determine statistical significance for the difference between MI-503-treated groups and its controls. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, Immunoprecipitation, Control, Two Tailed Test

Journal: Cancers

Article Title: Menin and Menin-Associated Proteins Coregulate Cancer Energy Metabolism

doi: 10.3390/cancers12092715

Figure Lengend Snippet: MEN1 and OXPHOS expression are increased in breast circulating tumor cells (CTCs). ( A ) t-SNE profile plots and cell clustering of 93 CTCs from 5 breast cancer patients based on the single cell RT-qPCR expression profiling of 11 OXPHOS genes ( NDUFA7 , NDUFA11 , NDUFA13 , NDUFB7 , NDUFS7 , NDUFS8 , NDUFV1 , SDHA , SDHB , SDHC , and SDHD ). ( B ) Violin plots of MEN1 or selected MAPs expression, mean expression of 7 glycolytic genes ( ALDOA , ALDOC , ENO1 , PFKL , PFKP , PGK1 , and TPI1 ) or mean expression of 11 OXPHOS genes (aforementioned) in the five cell clusters. Statistical significance among clusters was carried out using the Duncan multi-range test. ( C ) Mean expression of 7 glycolytic genes and 11 OXPHOS genes in these 93 breast CTCs or in the TCGA primary breast cancer cohort. ( D ) Glycolytic and OXPHOS ATP productions of T47D or MCF-7 cells after circulation ( n = 6–10 technical replicates). ( E , F ) Mitochondrial ( E ) and glycolytic ( F ) functions of T47D or MCF-7 cells after circulation ( n = 5–9 technical-replicate wells). Statistics represented the difference between no circulating control and each treatment. Data are presented as mean ± S.D. An unpaired two-tailed Student’s t -test was used for statistical significance determination. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1.

Techniques: Expressing, Quantitative RT-PCR, Control, Two Tailed Test

Journal: Endocrinology

Article Title: Menin Associates With the Mitotic Spindle and Is Important for Cell Division

doi: 10.1210/en.2019-00274

Figure Lengend Snippet: Cell cycle subcellular localization of menin and MLL1-N. Immunofluorescence microscopy of HeLa cells stained for DNA, α-tubulin (α-Tub) and either (A) menin or (B) MLL1-N. Note that menin localizes to the mitotic spindle poles and mitotic spindle during early mitosis and to intercellular bridge microtubules during cytokinesis, similar to MLL1-N. Scale bars, 5 μm.

Article Snippet: HeLa [CCL2; RRID:CVCL_0030 ( 22 ); ATCC] cell line growth and small interfering RNA (siRNA) treatments with OriGene control nontargeting siRNA (SR30004) and siRNA targeting MEN1 (SR302867A and SR302867B) were used as described previously ( 23 , 24 ).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: Endocrinology

Article Title: Menin Associates With the Mitotic Spindle and Is Important for Cell Division

doi: 10.1210/en.2019-00274

Figure Lengend Snippet: Validation of menin’s localization to the mitotic spindle. (A) Immunoblot analysis showing that siMEN deplete menin protein levels compared with siCont. (B) Immunofluorescence microscopy of HeLa cells treated with siCont or siMEN for 48 h and stained for DNA, α-tubulin (α-Tub), and menin. Note that menin’s localization to the mitotic spindle is depleted in siMEN-treated cells. (C, D) Immunofluorescence microscopy of HeLa cells transfected with the overexpressed GFP-tagged version of menin (GFP-menin) and stained for (C) DNA, α-Tub, and MLL1-N, or (D) MLL1-C. Scale bars, 5 μm. Cont, control; MEN, menin; MW, molecular weight (kDa).

Article Snippet: HeLa [CCL2; RRID:CVCL_0030 ( 22 ); ATCC] cell line growth and small interfering RNA (siRNA) treatments with OriGene control nontargeting siRNA (SR30004) and siRNA targeting MEN1 (SR302867A and SR302867B) were used as described previously ( 23 , 24 ).

Techniques: Biomarker Discovery, Western Blot, Immunofluorescence, Microscopy, Staining, Transfection, Control, Molecular Weight

Journal: Endocrinology

Article Title: Menin Associates With the Mitotic Spindle and Is Important for Cell Division

doi: 10.1210/en.2019-00274

Figure Lengend Snippet: Depletion of menin leads to cell division defects. (A) Immunofluorescence microscopy of HeLa cells treated with siCont or siMEN for 48 h and stained for DNA and α-tubulin (α-Tub). Note that siMEN cells show multiple aberrancies, including multipolar spindles and unaligned chromosomes in metaphase, lagging chromosomes in anaphase, multipolar cytokinesis, and multinucleated interphase cells. Arrows point to uncongressed chromosomes in a metaphase cell (panel with four arrows) and lagging chromosomes in a telophase cell (panel with one arrow). Scale bar, 5 μm. (B) Quantification of the percentage of mitotic cells with defective spindles, uncongressed chromosomes, and cytokinetic defects and interphase cells with more than one nucleus (multinucleated). Data represent the mean ± SD of three independent experiments, 100 cells counted for each. **P < 0.001. (C) Live-cell time-lapse microscopy snapshots of HCT116-GFP-H2B cells treated with siCont or siMEN (42). Representative cell division defects are shown, including cytokinetic arrest, multipolar cell division with cell death, and regression of a dividing cell into a binucleated cell. Time is in minutes. (D) The percentage of cells undergoing normal cell division, dying during cell division, undergoing aberrant cytokinesis and failing cytokinesis, and regressing to a binucleated state were quantified for siCont- or siMEN-treated cells. Data represent the mean ± SD of three independent experiments, 50 cells counted for each. **P < 0.001; ***P < 0.0001. (E) HeLa cells were treated with siCont or siMEN for 24 h, synchronized in G1/S with thymidine (Thy), and released into the cell cycle. Cells were harvested at the indicated time after release and extracts were immunoblotted for menin, cyclin B1 (CycB1), and α-Tub. Line graph shows relative cyclin B1 levels over time (in hours) normalized to α-Tub.

Article Snippet: HeLa [CCL2; RRID:CVCL_0030 ( 22 ); ATCC] cell line growth and small interfering RNA (siRNA) treatments with OriGene control nontargeting siRNA (SR30004) and siRNA targeting MEN1 (SR302867A and SR302867B) were used as described previously ( 23 , 24 ).

Techniques: Immunofluorescence, Microscopy, Staining, Time-lapse Microscopy

Journal: Endocrinology

Article Title: Menin Associates With the Mitotic Spindle and Is Important for Cell Division

doi: 10.1210/en.2019-00274

Figure Lengend Snippet: Pharmacological inhibition of the menin-MLL1 interaction with MI-2 leads to cell division defects. (A) Immunofluorescence microscopy of HeLa cells treated with DMSO or MI-2 (10 μM) for 2 h before mitotic entry and stained for DNA and α-tubulin (α-Tub). Note that MI-2–treated cells show multiple aberrancies, including multipolar spindles and unaligned chromosomes in metaphase, lagging chromosomes in anaphase, multipolar cytokinesis, and multinucleated interphase cells. The arrow points to lagging chromosomes in a telophase cell. Scale bar, 5 μm. (B) Quantification of the percentage of mitotic cells with defective spindles, uncongressed chromosomes, and cytokinetic defects and interphase cells with more than one nucleus (multinucleated). Data represent the mean ± SD of three independent experiments, 100 cells counted for each. *P < 0.01; ***P < 0.0001. (C) Live-cell time-lapse microscopy snapshots of HCT116-GFP-H2B cells treated with DMSO or MI-2 (42). Representative cell division defects are shown, including multipolar cytokinesis and regression of dividing cells into binucleated cells. Time is in minutes. (D) The percentage of cells undergoing normal cell division, dying during cell division, undergoing defective divisions and failing cytokinesis, and regressing to a binucleated state were quantified for DMSO- or MI-2–treated cells. Data represent the mean ± SD of three independent experiments, 50 cells counted for each. **P < 0.001; ***P < 0.0001. (E) HeLa cells were synchronized in G1/S with thymidine (Thy) and released into the cell cycle in the presence of either DMSO or MI-2. Cells were harvested at the indicated time after release and extracts were immunoblotted for menin, cyclin B1 (CycB1), and α-Tub. Line graph shows relative cyclin B1 levels over time (in hours) normalized to α-Tub.

Article Snippet: HeLa [CCL2; RRID:CVCL_0030 ( 22 ); ATCC] cell line growth and small interfering RNA (siRNA) treatments with OriGene control nontargeting siRNA (SR30004) and siRNA targeting MEN1 (SR302867A and SR302867B) were used as described previously ( 23 , 24 ).

Techniques: Inhibition, Immunofluorescence, Microscopy, Staining, Time-lapse Microscopy

Journal: Endocrinology

Article Title: Menin Associates With the Mitotic Spindle and Is Important for Cell Division

doi: 10.1210/en.2019-00274

Figure Lengend Snippet: Depletion of menin or inhibition of menin-MLL1 interaction does not perturb Kif2A localization. (A) Immunofluorescence microscopy of HeLa cells treated with siCont or siMEN for 48 h and stained for DNA and α-tubulin (α-Tub). Note that Kif2A remains localized to the mitotic spindle in siMEN-treated cells. Scale bar, 5 μm. (B, C) Immunofluorescence microscopy of HeLa cells treated with DMSO or MI-2 (10 μM) for 2 h before mitotic entry and stained for (B) DNA, α-Tub, and Kif2A, or (C) menin. Note that Kif2A remains localized to the mitotic spindle in MI-2–treated cells, whereas menin localization to the mitotic spindle decreases. Scale bar, 5 μm.

Article Snippet: HeLa [CCL2; RRID:CVCL_0030 ( 22 ); ATCC] cell line growth and small interfering RNA (siRNA) treatments with OriGene control nontargeting siRNA (SR30004) and siRNA targeting MEN1 (SR302867A and SR302867B) were used as described previously ( 23 , 24 ).

Techniques: Inhibition, Immunofluorescence, Microscopy, Staining

Journal: The American Journal of Pathology

Article Title: miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation

doi: 10.1016/j.ajpath.2016.10.021

Figure Lengend Snippet: Menin is down-regulated in CCA. A and B: By real-time PCR and immunoblots, menin expression is decreased in CCA cell lines compared to H69. Significance is shown versus H69 cells. C: Flow cytometry analysis demonstrated a decrease in menin protein expression in Mz-ChA-1 cells compared to H69 cells. D: By real-time PCR, menin expression decreased in advanced-stage human CCA tissue biopsy specimens compared with normal control. Data are expressed as means ± SEM performed in triplicate (A–D). n = 3 independent samples (A and B). ∗P < 0.05 versus H69 or human control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Modulation of Menin Expression, Proliferation, and Angiogenesis in Vitro Mz-ChA-1 cells were targeted for the transient knockdown of menin expression using a human MEN1 siRNA (Santa Cruz Biotechnology) (sc-35922) along with siRNA Transfection Reagent (Santa Cruz Biotechnology) (sc-29528), according to the vendor's instructions.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Flow Cytometry, Control

Journal: The American Journal of Pathology

Article Title: miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation

doi: 10.1016/j.ajpath.2016.10.021

Figure Lengend Snippet: Increased menin expression decreases proliferation. Mz-ChA-1 cells overexpressing menin with pCMV6-MEN1 vector exhibit a decrease in Ki-67 proliferative marker expression. A–C: Increased menin expression in pCMV6-MEN1 Mz-ChA-1 cells by real-time PCR (A) and flow cytometry (B) decreased Ki-67 proliferative marker expression by real-time PCR (C). D: Decreased cell migration as measured by wound healing assay. E: Decreased cell invasion as measured by Boyden chamber assay in pCMV6-MEN1 Mz-ChA-1 cells. Data are expressed as means ± SEM performed in triplicate (A–E). ∗P < 0.05 versus Mz-ChA-1 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Modulation of Menin Expression, Proliferation, and Angiogenesis in Vitro Mz-ChA-1 cells were targeted for the transient knockdown of menin expression using a human MEN1 siRNA (Santa Cruz Biotechnology) (sc-35922) along with siRNA Transfection Reagent (Santa Cruz Biotechnology) (sc-29528), according to the vendor's instructions.

Techniques: Expressing, Plasmid Preparation, Marker, Real-time Polymerase Chain Reaction, Flow Cytometry, Migration, Wound Healing Assay, Boyden Chamber Assay, Control

Journal: The American Journal of Pathology

Article Title: miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation

doi: 10.1016/j.ajpath.2016.10.021

Figure Lengend Snippet: Menin expression negatively regulates angiogenesis. A: By real-time PCR, Mz-ChA-1 MEN1 knockout cells increased expression of angiogenic factors compared to Mz-ChA-1 control cells. B: By real-time PCR, pCMV6-MEN1 Mz-ChA-1 cells decreased expression of angiogenic factors compared to Mz-ChA-1 control cells. Data are expressed as means ± SEM performed in triplicate (A and B). ∗P < 0.05 versus Mz-ChA-1 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Modulation of Menin Expression, Proliferation, and Angiogenesis in Vitro Mz-ChA-1 cells were targeted for the transient knockdown of menin expression using a human MEN1 siRNA (Santa Cruz Biotechnology) (sc-35922) along with siRNA Transfection Reagent (Santa Cruz Biotechnology) (sc-29528), according to the vendor's instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Knock-Out, Control

Journal: The American Journal of Pathology

Article Title: miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation

doi: 10.1016/j.ajpath.2016.10.021

Figure Lengend Snippet: miR-24 negatively regulates menin. A: Real-time PCR evaluation of miR-24 expression in CCA and H69 cell lines demonstrates increased levels in CCA lines compared to H69 cells. B: Luciferase luminescence shows decreased menin expression with miR-24 mimic treatment. C: Left panel: miRNA-sequence data demonstrate increased expression of miR-24 in human CCA tumors compared with matched normal tissue. Right panel: Statistical significance of increased miR-24 expression is validated with an unpaired t-test. Data are expressed as means ± SEM (A–C). n = 3 (A and B); n = 9 (C). ∗P < 0.05 versus normal matched human control.

Article Snippet: Modulation of Menin Expression, Proliferation, and Angiogenesis in Vitro Mz-ChA-1 cells were targeted for the transient knockdown of menin expression using a human MEN1 siRNA (Santa Cruz Biotechnology) (sc-35922) along with siRNA Transfection Reagent (Santa Cruz Biotechnology) (sc-29528), according to the vendor's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Luciferase, Sequencing, Control

Journal: The American Journal of Pathology

Article Title: miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation

doi: 10.1016/j.ajpath.2016.10.021

Figure Lengend Snippet: miR-24 drives proliferation. A: Real-time PCR confirmed knockdown of miR-24 in Mz-ChA-1 cells by hairpin inhibitor. miR-24 knockdown increased menin expression via fluorescence-activated cell sorting (B) and decreased expression of angiogenic factors via real-time PCR (C). Data are expressed as means ± SEM performed in triplicate unless otherwise stated (A–C). ∗P < 0.05 versus Mz-ChA-1 control cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Modulation of Menin Expression, Proliferation, and Angiogenesis in Vitro Mz-ChA-1 cells were targeted for the transient knockdown of menin expression using a human MEN1 siRNA (Santa Cruz Biotechnology) (sc-35922) along with siRNA Transfection Reagent (Santa Cruz Biotechnology) (sc-29528), according to the vendor's instructions.

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Expressing, Fluorescence, FACS, Control