Journal: Cancer research

Article Title: Inhibition of miR-193a expression by Max and RXR? activates K-Ras and PLAU to mediate distinct aspects of cellular transformation

doi: 10.1158/0008-5472.CAN-11-0425

Figure Lengend Snippet: MiR-193a targets PLAU and K-Ras. A, Relative expression levels of PLAU and miR-193a during cellular transformation of ER-Src cells at the indicated times points after treatment with tamoxifen. B, Relative expression levels of K-Ras and miR-193a during cellular transformation of ER-Src cells at the indicated times points after treatment with tamoxifen. C, Sequence complementarity (vertical lines) between miR-193a and the 3′ UTRs of PLAU and K-Ras, with the 8 nt seed sequence shown as a gray box. Luciferase activity of reporters containing the 3′UTR of PLAU or K-Ras 24h after transfection with miR-193a or miR negative control. D, PLAU or K-Ras mRNA levels in the indicated breast cancer cell lines transfected with miR-193a or control miRNA.

Article Snippet: 3′UTR luciferase assay Firefly luciferase reporter constructs containing the 3′ UTR of PLAU (s207136 from Switchgear Genomics) and K-Ras (HmiT010133 from Genecopoeia Inc) were transfected in MCF10A ER-Src cells along with 100 nM miR-193a or a miRNA control.

Techniques: Expressing, Transformation Assay, Sequencing, Luciferase, Activity Assay, Transfection, Negative Control, Control

Journal: bioRxiv

Article Title: Growth hormone induces mitotic catastrophe of podocytes and contributes to proteinuria

doi: 10.1101/597344

Figure Lengend Snippet: (A&B) qRT-PCR analysis showing the expression of TGF-β1 in human podocytes (HPC) treated with or without (CTL; control) GH in concentration (100 to 500 ng/ml) and time (up to 48 hr) dependent manner. mRNA levels were normalized to β-Actin and presented as fold-change on y-axis. Mean±SD. (n=5). ****p<0.0001 by 1-way ANOVA post hoc Dunnett test. (C&D) Immunoblotting analysis from HPC treated with GH in concentration (100 to 500 ng/ml) and time (up to 48hr) dependent manner. (n=3). (E) Immunofluorescence analysis for TGF-β1(purple color) and counter-stained with DAPI [4’,6-diamidino-2-phenylindole (Blue color)] in HPC treated with or without GH (500 ng/ml, 48hr). Scale bar: 50 μm, magnification x500. (F&G) Estimation of TGF-β1 in conditioned media (CM) from HPC treated with GH in concentration (100 to 500 ng/ml) and time (up to 48hr) dependent manner. Mean±SD. (n=5). ****p<0.0001 by one-way ANOVA post hoc Dunnet test. (H&I) SMAD4 luciferase activity in HPC was treated with GH in concentration (100 to 500 ng/ml) and time (up to 48 hr) dependent manner. Mean±SD. (n=6). ****p<0.0001 by one-way ANOVA post hoc Dunnet test. (J&K) qRT-PCR and immunoblotting analysis showing the expression of TGFBR1 and TGF-β1 in CTL or GH-treated (1.5mg/Kg bw) mice podocytes (MPC). Mean±SD. (n=3). ****p<0.0001 by Student t-test. Each dot represents the average value of a single mice from each group (n=6). (L) Representative images for TGFBR1 and TGF-β1 expression by DAB staining in mice glomerular sections from CTL vs GH treated mice. Scale bar:100μm, magnification x200. (n=3). (M) Quantification of TGF-β1 in urine from CTL or GH treated mice. Mean±SD. (n=3) ****p<0.0001 by Student t-test. Each dot rep-resents the average value of a single mice from each group. β-Actin served as internal control.

Article Snippet: The cells were then co-transfected with either 0.3 μg/well of Smad4 firefly luciferase reporter plasmid constructs (pLuc366 or pLuc207) or the control pGL3-Basic vector (Promega, San Luis Obispo, USA).

Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Western Blot, Immunofluorescence, Staining, Luciferase, Activity Assay

Journal: Reproductive Sciences

Article Title: High Maternal Serum Estradiol in First Trimester of Multiple Pregnancy Contributes to Small for Gestational Age via DNMT1-Mediated CDKN1C Upregulation

doi: 10.1007/s43032-021-00735-8

Figure Lengend Snippet: ( A ) Relative expression of imprinted genes in embryo tissues and CDKN1C in newborn UCBMNC and placenta tissues. ( B ) Relative DNMT1 expression in embryo tissues, UCBMNC, and placenta tissues

Article Snippet: HTR8 cells were co-transfected with DNMT1 -luc and the Renilla luciferase plasmid pRL-TK (Promega, Southampton, UK), which served as an internal transfection control.

Techniques: Expressing

Journal: Reproductive Sciences

Article Title: High Maternal Serum Estradiol in First Trimester of Multiple Pregnancy Contributes to Small for Gestational Age via DNMT1-Mediated CDKN1C Upregulation

doi: 10.1007/s43032-021-00735-8

Figure Lengend Snippet: E 2 increased CDKN1C and DNMT1 expression in a dose-dependent manner. These effects were attenuated by ICI, an ER antagonist. ( A ) CDKN1C expression in HTR-8 cells after treatment with different doses of E 2 for 24 h. The increased CDN1C mRNA expression levels induced by E 2 were blocked by the estrogen blocker ICI182780 (10 –7 mol/L) or co-treatment with E 2 and ER antagonist ICI (10 –7 mol/L) ( n = 9). ( B ) DNMT1 expression in HTR-8 cells after treatment with different doses of E 2 for 24 h. The increased DNMT1 mRNA expression levels induced by E 2 were blocked by ICI (10 –7 mol/L) ( n = 9). Effects of DNMT1 silencing on the expression of DNMT1 and response to E 2 induction in HTR8 cells, C and D. ( C ) HTR8 cells were transiently transfected with DNMT1 or scrambled control siRNA. Forty-eight hours after transfection, cell samples were analyzed by qRT-PCR and western blotting. ( D ) HTR8 cells were transiently transfected with siRNA against DNMT1 and then incubated with 10 −7 M E 2 . CDKN1C expression was analyzed by qRT-PCR. DNMT1 knockdown decreased the responsiveness of HTR8 cells to E 2 stimulation

Article Snippet: HTR8 cells were co-transfected with DNMT1 -luc and the Renilla luciferase plasmid pRL-TK (Promega, Southampton, UK), which served as an internal transfection control.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot, Incubation, Knockdown

Journal: Reproductive Sciences

Article Title: High Maternal Serum Estradiol in First Trimester of Multiple Pregnancy Contributes to Small for Gestational Age via DNMT1-Mediated CDKN1C Upregulation

doi: 10.1007/s43032-021-00735-8

Figure Lengend Snippet: ( A ) E 2 induced human DNMT1 transcription through an ERE proximal to the transcription start site. HTR8 cells cultured in phenol red-free medium supplemented with 5% double-charcoal stripped FCS and transiently transfected with empty pGL3-basic or pGL3-DNMT1 (1 μg) with the treatment of E 2 . Cells were harvested 24 h after transfection and assayed for luciferase activity. DNMT1 promoter-mediated luciferase values were normalized to control Renilla luciferase values. ( B ) ERα binds directly to the ERE on the DNMT1 promoter. ChIP analysis of the human DNMT1 promoter was performed after HTR8 cells were treated with E 2 for 24 h. ChIP assays were performed using IgG, anti-RNA polymerase, and anti-ERα antibodies as indicated. After cross-link reversal, the co-immunoprecipitated DNA was amplified by PCR using primers specific for the DNMT1 ERE containing region (− 659 to − 647) or a control region (− 2123 to − 2111 or − 728 to − 716). The PCR products were resolved using a 2% agarose gel. Representative data from three independent experiments are presented

Article Snippet: HTR8 cells were co-transfected with DNMT1 -luc and the Renilla luciferase plasmid pRL-TK (Promega, Southampton, UK), which served as an internal transfection control.

Techniques: Cell Culture, Transfection, Luciferase, Activity Assay, Control, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis

Journal: PLoS Pathogens

Article Title: Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

doi: 10.1371/journal.ppat.1003145

Figure Lengend Snippet: (A) Transcriptional activation of CEC1 promoter upon PGN challenge. The graph reports the z-scores calculated from the ratios of averaged RLU measurements after PGN and PBS treatments. (B and C) Transcriptional regulation of LRIM1 promoter (B) upon PGN challenge (z-scores calculated from the ratios of averaged RLU measurements after PGN and PBS treatments) and (C) in basal conditions (z-scores calculated from the RLU measured after PBS treatments). Positive hits are shown as open circles.

Article Snippet: Four days after dsRNA treatment, cells were washed and co-transfected in 50 μl of final volume with CEC1 or LRIM1 promoter firefly luciferace fusion reporter constructs (Promega pGL3 backbone) and reference pAct5C-Renilla luciferase construct using Effectene Transfection Reagent (QIAGEN) following the manufacturer's instructions.

Techniques: Activation Assay

Journal: PLoS Pathogens

Article Title: Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

doi: 10.1371/journal.ppat.1003145

Figure Lengend Snippet: A total of 22 positive (green) and negative (red) regulators are shown. Dotted lines indicate modulations of CEC1 and LRIM1 gene expression upon PGN challenge, and solid lines describe the effect of genes on phagocytosis and basal expression of LRIM1 .

Article Snippet: Four days after dsRNA treatment, cells were washed and co-transfected in 50 μl of final volume with CEC1 or LRIM1 promoter firefly luciferace fusion reporter constructs (Promega pGL3 backbone) and reference pAct5C-Renilla luciferase construct using Effectene Transfection Reagent (QIAGEN) following the manufacturer's instructions.

Techniques: Gene Expression, Expressing

Journal: Oncology Letters

Article Title: Osteopontin regulates the growth and invasion of liver cancer cells via DTL

doi: 10.3892/ol.2023.14064

Figure Lengend Snippet: DTL is involved in the OPN-induced invasiveness of liver cancer cells. Cells were transfected with DTL siRNA or OPN expressing vector or treated with rhOPN for 24 h and then the cells were assessed using the Transwell assay. (A) DTL knockdown slightly inhibited invasion by both Huh7 cells and HepG2 cells, but markedly decreased the rhOPN-induced invasiveness of both Huh7 cells and HepG2 cells. These results indicated that DTL is also involved in the rhOPN-induced promotion of the invasiveness of both Huh7 cells and HepG2 cells. The data are presented as the mean ± SD of three independent experiments. (B) Similar effects were also demonstrated in the groups transfected with the vector expressing OPN. The results further indicated DTL is involved in the invasiveness of both Huh7 cells and HepG2 cells, serving a vital role in OPN-induced invasiveness of both Huh7 cells and HepG2 cells. *P<0.05. OPN, osteopontin; DTL, denticleless E3 ubiquitin protein ligase homolog; NC, negative control; rh, recombinant human.

Article Snippet: The cells were co-transfected with 0.1 μg firefly luciferase reporter construct containing the DTL promoter region (Promega Corporation), 0.01 μg pRL-TK Renilla luciferase reporter plasmid (Promega Corporation) and the pcDNA3.1-OPN vector (0.2 μg) using LipofectamineTM 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Transfection, Expressing, Plasmid Preparation, Transwell Assay, Knockdown, Ubiquitin Proteomics, Negative Control, Recombinant

Journal: Oncology Letters

Article Title: Osteopontin regulates the growth and invasion of liver cancer cells via DTL

doi: 10.3892/ol.2023.14064

Figure Lengend Snippet: OPN and the PI3K/AKT signaling pathway are transcriptionally associated with DTL expression in liver cancer cells. (A) The effects of OPN on DTL promoter activity in Huh7 and HepG2 liver cancer cells were evaluated. Luciferase reporter assays used to assess the DTL promoter activity were performed 24 h after co-transfection with constructs derived from the DTL promoter region and either OPN-overexpressing vector or OPN siRNAs. The promoter activity is presented relative to the luciferase activity. Independent experiments were repeated three times. Data are presented as the mean ± SD, normalized against the activity of the internal control vector pRL-TK. (B) Effects of rhOPN and the PI3K/AKT inhibitor LY294002 on DTL promoter activity in Huh7 and HepG2 liver cancer cells. (C) rhOPN and PI3K/AKT inhibitor LY294002 affect the expression level of DTL mRNA. Treatment with LY294002 eliminated the effects of OPN on the DTL mRNA level. The experiments were repeated three times. *P<0.05 and **P<0.01 vs. control. OPN, osteopontin; DTL, denticleless E3 ubiquitin protein ligase homolog; OE, overexpression; NC, negative control; Ctrl, control; rh, recombinant human.

Article Snippet: The cells were co-transfected with 0.1 μg firefly luciferase reporter construct containing the DTL promoter region (Promega Corporation), 0.01 μg pRL-TK Renilla luciferase reporter plasmid (Promega Corporation) and the pcDNA3.1-OPN vector (0.2 μg) using LipofectamineTM 3000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Activity Assay, Luciferase, Cotransfection, Construct, Derivative Assay, Plasmid Preparation, Control, Ubiquitin Proteomics, Over Expression, Negative Control, Recombinant