Journal: Scientific Reports

Article Title: Identification and functional interpretation of miRNAs affected by rare CNVs in CAKUT

doi: 10.1038/s41598-022-22749-1

Figure Lengend Snippet: The difference in the relative expression of target genes mRNA between MIR484 +/− KO and HEK293 WT. Relative mRNA levels were standardized against GAPDH endogenous control and presented as a scatter plot of 2 −dCt values with standard errors of the mean from three independent replicates. ( A ) Relative levels of MDM2 mRNA , Student's t-test, P = 0.005. ( B ) Relative levels of APAF1 mRNA, Student's t-test, P < 0.0001 ( C ) Relative levels of NOTCH3 mRNA, Student's t-test, P = 0.014. ( D ) Relative levels of FIS1 mRNA, Student's t-test, P = 0.081. ( E ) Relative levels of PKD1 mRNA, Student's t-test, P = 0.742. * denotes a significant difference at P < 0.05; ** denotes a significant difference at P < 0.01; ns denotes a nonsignificant difference in relative target gene mRNA levels.

Article Snippet: Expression levels of the selected hsa-miR-484 target genes were measured by quantitative Real-time PCR on Applied Biosystems Real-Time 7500 system (Applied Biosystems, Inc., Foster City, CA) using TaqMan ® gene expression assay: Hs00947377_m1 (for PKD1 (polycystin-1)), Hs00540450_s1 (for MDM2 (Mouse double minute 2 homolog)), Hs00211420_m1 (for FIS1 (Mitochondrial Fission 1 Protein)), Hs01128537_m1 (for NOTCH3 (Neurogenic locus notch homolog)), Hs00559441_m1 (for APAF1 (Apoptosis protease-activating factor-1)), Hs02786624_g1 (for GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)).

Techniques: Expressing, Control

Journal: Cell Death & Disease

Article Title: Enhanced pro-apoptosis gene signature following the activation of TAp63α in oocytes upon γ irradiation

doi: 10.1038/s41419-022-04659-2

Figure Lengend Snippet: a The six DEGs in this category and the two additional DEGs involved in cell cycle arrest, which are all known from Kenzelmann et al. to be regulated by p53, are indicated . Z -score normalized pseudocounts are represented between −2 (blue) and 2 (red). b Expression profiles of members of the p53 family, Mdm4 , and Bbc3 (PUMA), which do not reach statistical significance in our study but are important for regulating apoptosis. For comparison, expression data for the DEGs Pmaip1 , Cdkn1a , and Mdm2 are shown.

Article Snippet: Mdm2 , Mm01233136_m1 , 4453320.

Techniques: Expressing, Comparison

Journal: Cell Death & Disease

Article Title: Enhanced pro-apoptosis gene signature following the activation of TAp63α in oocytes upon γ irradiation

doi: 10.1038/s41419-022-04659-2

Figure Lengend Snippet:

Article Snippet: Mdm2 , Mm01233136_m1 , 4453320.

Techniques: Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: 3,3′-Diindolylmethane (DIM) decreased MDM2 protein in colon cancer cells. ( A ) HCT-116, ( B ) HT-29 cells, and ( C ) HCT-116 p53 null cells were treated with various doses of DIM for 24 h. Cell proliferation was analyzed by WST-1 assay. ( D ) HCT-116 cells were treated with 40 μM DIM for 24 h then harvested and stained with Propidium iodide for cell cycle analysis. DIM induced cell cycle arrest in the G2 phase where the G2 population increased from 34.9% to 43.0%. ( E ) HCT-116 and HT-29 cells were treated with the indicated concentrations of DIM for 24 h. Cells were harvested, and the lysates were analyzed by Western blotting with the indicated antibodies. All experiments were repeated three times.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques: WST-1 Assay, Staining, Cell Cycle Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: DIM induced proteasome-mediated degradation of MDM2 in HCT-116 cells. HCT-116 cells were pretreated with 40 μM DIM for ( A ) 6 h and ( B ) 24 h before cycloheximide (CHX) (at a final concentration of 10 μg/mL) was added into the culture. Cells were then lysed at the indicated time points after CHX application. ( C , D ) HCT-116 cells were treated with various proteasome or autophagy inhibitors. HCT-116 cells were incubated for ( C ) 6 h and ( D ) 24 h with 40 μM DIM and 10 μM of the indicated inhibitor. Cell lysates were analyzed by Western blotting with the indicated antibodies. ( E ) HCT-116 cells were treated with 40 μM DIM for 24 h. Co-immunoprecipitation was performed using an anti-MDM2 antibody, and the immunoprecipitates were blotted with anti-MDM2 or anti-ubiquitin antibodies.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques: Concentration Assay, Incubation, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: DIM inhibited MDM2 independent of p53. ( A ) Wild-type HCT-116 and p53 knockout HCT-116 cells were treated with various doses of DIM for 24 h. The cell lysates were analyzed by Western blotting with the indicated antibodies. ( B ) Wild-type HCT-116 cells and ( C ) p53 knockout HCT-116 cells were treated with 40 μM of DIM for 6 h. Real-time polymerase chain reaction (PCR) analysis of MDM2 gene expression was conducted as described in Materials and Methods.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques: Knock-Out, Western Blot, Real-time Polymerase Chain Reaction, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: In-silico prediction of MDM2-DIM interaction. ( A ) N terminal structure (p53 binding site) of MDM2. ( B ) Molecular Operating Environment (MOE) software predicted interaction between DIM and the MDM2 N-terminal hydrophobic pocket. ( C ) C-terminal structure (Really Interesting New Gene (RING) domain) of MDM2. ( D ) MOE predicted the binding of DIM to the RING domain grove in MDM2.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques: In Silico, Binding Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: Overexpression of MDM2 reduced the anti-cancer activity of DIM. ( A ) MDM2 overexpressing clones of HCT-116 cells (HCT-116b1 and HCT-116c2) were created and MDM2 expression was confirmed by Western blotting. ( B , C ) Wild-type and MDM2 overexpressing clones of HCT-116 cells were treated with various doses of DIM for 24 h. DIM was added within 24 h of cell seeding to better compare the DIM effect on wild-type and MDM2 overexpression cells. Cell proliferation was assessed by WST-1 assay as described in Materials and Methods. Asterisk represents significance levels (analyzed by t tests) with p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***). All experiments were repeated three times; data shown are mean values + SD. ( D ) Western blotting showed that DIM induced smaller amounts of PUMA and p27 proteins in MDM2 overexpressing cells compared with HCT-116 wild-type cells. The amount of change of protein was noted in numbers compared with the corresponding control group. ( E ) Flow cytometry showed DIM induced a higher level of apoptosis in wild-type HCT-116 cells (total apoptosis population = 24.62%) compared with HCT-116b1 cells (total apoptosis population = 15.80%). Apoptosis was determined by phosphatidylserine (PS) staining with Apopxin™ dye. Necrosis as well as late stage apoptosis were determined by the loss of membrane integrity, detected using DNA Nuclear Green DCS1 dye.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques: Over Expression, Activity Assay, Clone Assay, Expressing, Western Blot, WST-1 Assay, Control, Flow Cytometry, Staining, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: Combination of DIM and MDM2 antagonists.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Chemopreventive Agent 3,3′-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells

doi: 10.3390/ijms21134642

Figure Lengend Snippet: DIM enhanced the anti-cancer activity of Nutlin-3a and RG-7388. HCT-116 cells were treated with a single agent or DIM in combination with Nutlin-3a ( A ) or RG-7388 ( B ). Cell proliferation was determined by WST-1 assay. The combination index (CI) was calculated by CompuSyn . ( C ) HCT-116 cells were treated with a single agent of Nutlin-3a (10 μM) or a combination of Nutlin-3a and DIM (40 μM). Western blotting was performed using the indicated antibodies. ( D ) HCT-116 cells were treated with a single agent of RG-7388 (5 μM) or a combination of RG-7388 and DIM (40 μM). Western blotting was performed using the indicated antibodies. ( E , F ) HCT-116 cells were treated for 6 h with a single agent or a combination of DIM (40 μM), Nutlin3a (10 μM), and RG-7388 (5 μM). MDM2 mRNA expression was analyzed as described in Materials and Methods.

Article Snippet: The MDM2 gene expression was measured by real-time PCR using a TaqMan TM gene expression assay (cat. # 4331182-Hs01066931_m1 for MDM2, cat. # 4331182-Hs99999901_s1 for 18s, Applied Biosystems, Foster City, CA, USA).

Techniques: Activity Assay, WST-1 Assay, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Senescence determines the fate of activated rat pancreatic stellate cells

doi: 10.1111/j.1582-4934.2012.01573.x

Figure Lengend Snippet: Gene expression profile of senescent PSC. ( A ) Young PSC were treated for 7 days with doxorubicin (doxo; 25 ng/ml; upper panel), H 2 O 2 (150 μM; middle panel) and staurosporine (stauro; 3 nM; lower panel) to induce senescence. The mRNA expression of α-SMA (SMA), p53, Mdm2, IL-6, CDKN1A/p21 and the housekeeping gene HPRT was analysed by real-time PCR, and relative amounts of target mRNA were determined. ( B ) Upper panel: Untreated young and old PSC were subjected to gene expression analysis as described in ( A ). Lower panel: Time course of α-SMA mRNA expression in untreated PSC. The first time-point refers to young PSC, the following ones to cells of consecutive passages. ( A ) and ( B ): One hundred per cent mRNA expression of each gene corresponds to untreated young PSC. Data of at least six independent experiment were used to calculate mean values and S.E.M. * P < 0.05 versus untreated young PSC, # P < 0.05 versus passage 3. ( C ) IL-6 protein levels in supernatants of young PSC, treated for 7 days as indicated, were measured as described in the Materials and methods section. The results are averaged values (±S.E.M.) of at least five independent experiments. * P < 0.05 versus untreated young PSC.

Article Snippet: Relative quantification of target cDNA levels by real-time PCR was performed in an ABI Prism 7000 sequence detection system using TaqManTM Universal PCR Master Mix and the following Assay-on-DemandTM rat gene-specific fluorescently labelled TaqManTM MGB probes (Applied Biosystems): Rn00561420_m1 (IL-6), Rn01759928_g1 (α-SMA; acta2), Rn00589996_m1 (CDKN1A/p21), Rn03399818_s1 (mdm2), Rn00755717 (p53) and Rn01527840_m1 (HPRT).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction