control renilla luciferase construct prl-tk  (Promega)

Journal: iScience

Article Title: Genetic ablation of diabetes-associated gene Ccdc92 reduces obesity and insulin resistance in mice

doi: 10.1016/j.isci.2022.105769

Figure Lengend Snippet: Decreased adipocyte differentiation in Ccdc92 -deficient mesenchymal stem cells (MSCs) (A and B) Male Ccdc92 KO mice and WT littermates were fed HFD for 14 weeks, n = 5/group. (A) Representative H&E staining images of eWAT and sWAT. Scale bar = 50 μm (B) Adipocyte area was analyzed in eWAT and sWAT sections from 5 mice/group. (C-F) Ear MSCs (EMSCs) from male Ccdc92 KO mice and littermate WT mice were cultured in an adipocyte differentiation medium for 1, 3, 5, and 7 days (C) Oil Red O (ORO) staining and quantification were performed. Representative images are shown from at least 3 independent experiments. Scale bar = 100 μm. (D) Relative mRNA levels of PPAR γ and Cebpα during adipocyte differentiation were measured by RT-qPCR at the indicated time points. Representative data in D are shown from 3 independent experiments. (E) Representative immunoblots of PPARᵧ and CEBPα at the indicated time points of adipocyte differentiation. (F) Quantitative analysis of PPARᵧ and CEBPα expression from three independent experiments. (G) HEK 293 cells were co-transfected with the PPRE-luciferase reporter gene, PPARγ, and CCDC92 or pcDNA3.1 (control) vectors for 24 h. PPRE-luciferase activity was measured and normalized to Renilla luciferase activity. Representative data in G are shown from 3 independent experiments. Data are presented as mean ± SEM, ∗p < 0.05; ∗∗p < 0.01. Analysis in (B) used unpaired two-tailed t -test. Analysis in C, D, F, and G used two-way ANOVA with Bonferroni correction. See also Figures S3 and .

Article Snippet: NF-κB-luciferase vector (Agilent Technologies, 219,078-51) and pRL TK Renilla luciferase control reporter construct (Promega, Cat# E2241) were transfected into HEK-293 cells with lipofectamine 3000 (Thermo Fisher, Cat# L3000001) in Opti-MEM I medium (Thermo Fisher, Cat# 31985070).

Techniques: Staining, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Luciferase, Control, Activity Assay, Two Tailed Test

Journal: iScience

Article Title: Genetic ablation of diabetes-associated gene Ccdc92 reduces obesity and insulin resistance in mice

doi: 10.1016/j.isci.2022.105769

Figure Lengend Snippet: NF-κB signaling mediates CCDC92-dependent upregulation of NLRP3 (A) Representative immunoblots of p-p65 S536 and p65 in sWAT from Ccdc92 KO mice and littermate WT mice on HFD for 14 weeks. The p-p65/p65 ratio was quantitatively analyzed, n = 4 mice/group. (B) Representative immunoblot of IκBα and quantitative analysis in EMSCs treated with TNF-α (10 ng/mL) at the indicated time points, n = 3 independent experiments. (C) Representative immunoblot of nuclear and cytosolic p65 in EMSCs treated with TNF-α (10 ng/mL) at the indicated time points. Right panel, quantification of nuclear p65/cytosolic p65, n = 3 independent experiments. (D) Relative NF-κB-luciferase activity in HEK 293 cells transfected with CCDC92 expression vector followed by treatment with TNF-α (10 ng/mL) for 24 h. Right panel, relative NF-κB-luciferase activity in HEK 293 cells transfected with CCDC92 and p65 expression vectors for 48 h. NF-κB-luciferase was measured and normalized to Renilla luciferase activity. The data in this panel are representatives of 3 independent experiments. (E) Relative mRNA level of Nlrp3 in EMSCs infected with adenovirus encoding CCDC92 (Ad- CCDC92 ) or Ad-LacZ followed by treatment with Bay 11-7082 (10 μM), an inhibitor of κB kinase (IKK), for 24 h. The data in this panel are representative of 3 independent experiments. Data are presented as mean ± SEM, ∗p < 0.05; ∗∗p < 0.01. Analysis in A used unpaired two-tailed t-test. Analysis in B-E used two-way ANOVA with Bonferroni correction. See also Figures S7 and .

Article Snippet: NF-κB-luciferase vector (Agilent Technologies, 219,078-51) and pRL TK Renilla luciferase control reporter construct (Promega, Cat# E2241) were transfected into HEK-293 cells with lipofectamine 3000 (Thermo Fisher, Cat# L3000001) in Opti-MEM I medium (Thermo Fisher, Cat# 31985070).

Techniques: Western Blot, Luciferase, Activity Assay, Transfection, Expressing, Plasmid Preparation, Infection, Two Tailed Test

Journal: Orphanet Journal of Rare Diseases

Article Title: Bi-allelic mutation of CTNNB1 causes a severe form of syndromic microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia

doi: 10.1186/s13023-022-02239-3

Figure Lengend Snippet: Localisation, conservation and transcriptional activity of CTNNB1 c.884C>G; p.(Ala295Gly). A Sanger sequencing chromatograms confirming the presence and zygosity of the CTNNB1 c.884C>G; p.(Ala295Gly) variant in proband and parental DNA samples, in comparison to a control chromatogram which does not contain the sequence change. B CTNNB1 (NM_001904) gene (top) and β-catenin protein (bottom) schematics annotated with previously reported dominant disease-causing mutations (as reported by HGMDpro; orange text = missense mutations, green text = splice-altering mutations; blue text = nonsense mutations; purple text = small insertion, deletion or insertion/deletion mutations) and the location of the CTNNB1 c.884C>G; p.(Ala295Gly) homozygous variant identified by this study (indicated by red text). The biallelic missense mutation identified herein resides within the fourth armadillo (ARM) domain of the encoded protein. C Multispecies alignment of β-catenin (human amino acids 239–298) showing 100% conservation across 28 species of the alanine residue at position 295, found to be mutated to Glycine in the proband described in this report. Red asterisk (*) above the alignments indicates the position of human Alanine 295 in the alignment. Amino acid residues that are fully conserved are represented by an asterisk underneath the alignments; conservation between residues with strongly similar properties are represented by a colon (:); conservation between residues with weakly similar properties are represented by a period symbol (.). D Functional assessment of p.(Ala295Gly) mutant β-catenin on transcription. Bar chart detailing TOPflash luciferase assay results of STF cells transiently co-transfected with wildtype (WT) or mutant (p.(Ala295Gly)) β-catenin or empty vector (pDEST40) and renilla luciferase plasmid. Luciferase activity was measure 48 h post-transfection. Each bar indicates the recorded luciferase activity for each construct following normalisation to renilla activity and relative to the measurement recorded for cells transfected with empty vector. Results are from three independent experiments performed in triplicate. Error bars denote standard error of the mean (SEM); Y-axis represents the fold difference relative to the observed empty vector reading

Article Snippet: 90,000 cells /well were transfected with 399 ng of construct DNA plus 1 ng of Renilla luciferase control plasmid (pRL-TK) (Promega, Madison, Wi, USA), using 1.5μL of FuGENE 6 transfection reagent (Promega).

Techniques: Activity Assay, Sequencing, Variant Assay, Comparison, Control, Mutagenesis, Residue, Functional Assay, Luciferase, Transfection, Plasmid Preparation, Construct