A single secreted luciferase-based gene reporter assay

Barriscale, Kathy A.; O’Sullivan, Sharon A.; McCarthy, Tommie V.

doi:10.1016/j.ab.2014.02.019

Cited by 13 publications

(7 citation statements)

References 14 publications

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“…Forty-eight hours post-transfection, luciferase signals were examined using a dual-luciferase reporter assay system. Luciferase activity was calculated as firefly luciferase activity/Renilla luciferase activity [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

RETRACTED ARTICLE: The long non-coding RNA ASMTL-AS1 promotes hepatocellular carcinoma progression by sponging miR-1343-3p that suppresses LAMC1 (laminin subunit gamma 1)

Mou

Sun

2021

Bioengineered

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Long non-coding RNAs (lncRNAs) are critical regulators of hepatocellular carcinoma (HCC) carcinogenesis and development. We aimed to identify the function of the lncRNA ASMTL-AS1 during HCC malignancy. The expression of ASMTL-AS1, miR-1343-3p, and LAMC1 (laminin subunit gamma 1) was assessed in HCC tissues and cells. Cell Counting Kit-8 (CCK8) and Transwell migration assays were performed to determine the effect of ASMTL-AS1 on HCC cell proliferation and migration. Cell apoptosis was identified by detecting Bax and Bcl-2 protein expression using Western blotting, and a xenograft assay was performed to investigate tumor growth in vivo . The interplay between miR-1343-3p and ASMTL-AS1 or LAMC1 was verified through luciferase reporter and RNA immunoprecipitation assays. ASMTL-AS1 and LAMC1 were highly expressed in HCC tissues and cells, whereas miR-1343-3p showed low expression. Clinically, miR-1343-3p expression in HCC tissues showed a negative correlation with ASMTL-AS1 or LAMC1 expression. Functional assays demonstrated that ASMTL-AS1 silencing suppressed HCC cell proliferation and migration and increased cell apoptosis. More interestingly, ASMTL-AS1 sponged miR-1343-3p and miR-1343-3p to target the 3ʹ-UTR of LAMC1, thereby interfering with the malignant behavior of HCC cells. In conclusion, ASMTL-AS1 acts as a carcinogen in HCC through competing endogenous RNA (ceRNA) activity in the miR-1343-3p/LAMC1 axis. Our findings demonstrate that regulating ASMTL-AS1/miR-1343-3p/LAMC1-mediated HCC cell malignancy might be an effective method to interfere with HCC progression.

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Section: Methodsmentioning

confidence: 99%

RETRACTED ARTICLE: The long non-coding RNA ASMTL-AS1 promotes hepatocellular carcinoma progression by sponging miR-1343-3p that suppresses LAMC1 (laminin subunit gamma 1)

Mou

Sun

2021

Bioengineered

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“…In this paper, qRT-PCR confirmed that all three TBX4 mutations affected the expression of FGF10. Dual luciferase reporter assay is widely used for the investigations on gene transcription regulation and promoter transcription activity [33]. The cDNA of Renilla luciferase (Rluc) involved in the pGL3-TK vector is used as an internal reference to eliminate the differences in cell number or transfection efficiency [34].…”

Section: Discussionmentioning

confidence: 99%

Identification and Functional Evaluation of a Novel TBX4 Mutation Underlies Small Patella Syndrome

Lan

Zhang

et al. 2022

IJMS

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Small patella syndrome (SPS) is a rare autosomal dominant disorder caused by mutations in TBX4 gene which encodes a transcription factor of FGF10. However, how TBX4 mutations result in SPS is poorly understood. Here, a novel TBX4 mutation c.1241C>T (p.P414L) was identified in a SPS family and series of studies were performed to evaluate the influences of TBX4 mutations (including c.1241C>T and two known mutations c.256G>C and c.743G>T). Results showed that mesenchymal stem cells (MSCs) with stable overexpression of either TBX4 wild-type (TBX4wt) or mutants (TBX4mt) were successfully generated. Immunofluorescence study revealed that both the overexpressed TBX4 wild-type and mutants were evenly expressed in the nucleus suggesting that these mutations do not alter the translocation of TBX4 into the nucleus. Interestingly, MSCs overexpression of TBX4mt exhibited reduced differentiation activities and decreased FGF10 expression. Chromatin immunoprecipitation (ChIP) study demonstrated that TBX4 mutants still could bind to the promoter of FGF10. However, dual luciferase reporter assay clarified that the binding efficiencies of TBX4 mutants to FGF10 promoter were reduced. Taken together, MSCs were firstly used to study the function of TBX4 mutations in this study and the results indicate that the reduced binding efficiencies of TBX4 mutants (TBX4mt) to the promoter of FGF10 result in the abnormal biological processes which provide important information for the pathogenesis of SPS.

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“…Samples (10 ml) were assayed for Gaussia secreted luciferase activity with 1.43 mM Coelenterazine (NanoLight Technology) substrate in PBS on the Veritas Microplate Luminometer (Turner Biosystems) as described previously. 67 The luciferase activity directed by each ITM2A promoter fragment was normalized to that of the pGluc(PB)basic empty vector construct to account for the weak promoter activity of the piggyBac transposable arms in the vector backbone. 66 Adipogenesis was assessed at day 8 of differentiation when cells were fixed and stained with Oil Red O as described above.…”

Section: Real-time Qpcrmentioning

confidence: 99%

Itm2a silencing rescues lamin A mediated inhibition of 3T3-L1 adipocyte differentiation

Davies

Ryan²,

O‘Connor

et al. 2017

Adipocyte

Self Cite

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Dysregulation of adipose tissue metabolism is associated with multiple metabolic disorders. One such disease, known as Dunnigan-type familial partial lipodystrophy (FPLD2) is characterized by defective fat metabolism and storage. FPLD2 is caused by a specific subset of mutations in the LMNA gene. The mechanisms by which LMNA mutations lead to the adipose specific FPLD2 phenotype have yet to be determined in detail. We used RNA-Seq analysis to assess the effects of wild-type (WT) and mutant (R482W) lamin A on the expression profile of differentiating 3T3-L1 mouse preadipocytes and identified Itm2a as a gene that was upregulated at 36 h post differentiation induction in these cells. In this study we identify Itm2a as a novel modulator of adipogenesis and show that endogenous Itm2a expression is transiently downregulated during induction of 3T3-L1 differentiation. Itm2a overexpression was seen to moderately inhibit differentiation of 3T3-L1 preadipocytes while shRNA mediated knockdown of Itm2a significantly enhanced 3T3-L1 differentiation. Investigation of PPARγ levels indicate that this enhanced adipogenesis is mediated through the stabilization of the PPARγ protein at specific time points during differentiation. Finally, we demonstrate that Itm2a knockdown is sufficient to rescue the inhibitory effects of lamin A WT and R482W mutant overexpression on 3T3-L1 differentiation. This suggests that targeting of Itm2a or its related pathways, including autophagy, may have potential as a therapy for FPLD2.

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A single secreted luciferase-based gene reporter assay

Cited by 13 publications

References 14 publications

RETRACTED ARTICLE: The long non-coding RNA ASMTL-AS1 promotes hepatocellular carcinoma progression by sponging miR-1343-3p that suppresses LAMC1 (laminin subunit gamma 1)

RETRACTED ARTICLE: The long non-coding RNA ASMTL-AS1 promotes hepatocellular carcinoma progression by sponging miR-1343-3p that suppresses LAMC1 (laminin subunit gamma 1)

Identification and Functional Evaluation of a Novel TBX4 Mutation Underlies Small Patella Syndrome

Itm2a silencing rescues lamin A mediated inhibition of 3T3-L1 adipocyte differentiation

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