hek293 supertopflash  (ATCC)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

doi: 10.1016/j.omtn.2025.102453

Figure Lengend Snippet: Poly(A) tail mimetics enhance the expression of target mRNAs (A) Schematic of the basic design of the mRNA booster: a 30-nucleotide sequence complementary to a particular region of the 3′ UTR of a targeted mRNA, which bears a poly(A) tail. (B) A poly(A) mimetic enhances the expression of an in vitro transcribed Firefly luciferase reporter in HEK293 cells, with a significant (one-way ANOVA, ∗∗∗∗ p < 0.00001) 6-fold increase in FLuc over Renilla activity when the booster bears a 200-nucleotide poly(A) tail, 48 h after transfection. The results are depicted for a booster without a poly(A) tail, with a short 10-nucleotide tail, or a long 200-nucleotide tail; for two different FLuc mRNA to booster ratios (low dose, 1:36; and high dose, 1:216). (C) Targeting an endogenous mRNA confirms the booster efficiency to enhance cellular gene expression. LSM8 mRNA levels normalized to GAPDH are represented, after treatment with 20 nM final concentration of LSM8- specific booster compared to a non-specific polyadenylated control. Cells were harvested 2, 4, 6, 8, 16, and 24 h after transfection. The comparison between control vs. booster treated in each time point indicates significant increase (∗∗) after 4-, 16- and 24-h incubation. Two-way ANOVA, ∗∗ p < 0.001, ∗∗∗∗ p < 0.00001.

Article Snippet: We tested our mRNA booster technology in HEK293 (ATCC CRL 1573), HEK293 SuperTopFlash (STF), and SH-SY5Y (ATCC CRL 2266) cell lines as well as iPSC-derived neuronal cells (see below).

Techniques: Expressing, Sequencing, In Vitro, Luciferase, Activity Assay, Transfection, Gene Expression, Concentration Assay, Control, Comparison, Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

doi: 10.1016/j.omtn.2025.102453

Figure Lengend Snippet: mRNA boosters enhance haploinsufficiency-associated mRNAs (A) Schematic of the guide sequences position on the 3′ UTR of their target genes: MECP2 , CTNNB1 , PURA , and SYNGAP1 mRNAs, used throughout the study. Guide sequences spanning distinct regions were evaluated and assigned. (B) Levels of M E CP2 mRNA measured by qRT-PCR in SH-SY5Y cells transfected with 40 nM booster V.2.2 (MB2) against 3′ UTR of the M E CP2 or a control booster not targeting M E CP2 (control) and harvested 16 h after transfection. Welch’s t test, ∗ p = 0.05. (C and D) Levels of CTNNB1 (C) and PURA (D) mRNAs measured by qRT-PCR in HEK293 cells transfected with boosters V.2.2 targeting distinct regions of the 3′ UTR (B1 and B2) or a non-specific control (control), 24 h after transfection. Ordinary one-way ANOVA, ∗ p = 0.05, ∗∗ p = 0.005. (E) S YN GAP1 mRNA levels measured by qRT-PCR in SH-SY5Y cells transfected with two versions of booster V.2.0 (SB1 and SB2) targeting S YN GAP1 mRNA 3′ UTR or a non-specific control (control). SB2 showed a significant (∗) up to 4-fold increase in the mRNA level. Welch’s t test, ∗ p = 0.05.

Article Snippet: We tested our mRNA booster technology in HEK293 (ATCC CRL 1573), HEK293 SuperTopFlash (STF), and SH-SY5Y (ATCC CRL 2266) cell lines as well as iPSC-derived neuronal cells (see below).

Techniques: Quantitative RT-PCR, Transfection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

doi: 10.1016/j.omtn.2025.102453

Figure Lengend Snippet: Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( Figure S3 ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control. To screen and select the most effective booster for CTNNB1 , HEK293-STF cells were transfected with different doses (10, 20, 30, 40, 50, and 100 nM) of five distinct CTNNB1 boosters V.1.0 (CB3, CB4, CB5, CB6, and CB7). Booster CB7 increases the protein level up to 2-fold compared to control at 40 nM booster or higher. (C) Representative western blotting and quantifications for three biological replicates showing the significant dose-dependent increase of CTNNB1 protein levels upon exposure to booster CB7 compared to control (for more blots and loading control, see Figure S3 ). Two-way ANOVA, ∗ p = 0.05, ∗∗∗ p = 0.0005. (D) qRT-PCR analysis confirms booster CB7 efficiency to increase CTNNB1 mRNA levels in a dose-dependent manner. Two-way ANOVA, ∗ p = 0.01. (E and F) mRNA levels of Wnt signaling pathway markers measured by qRT-PCR, illustrating the functional enhancement of CTNNB1 following the increase in CTNNB1 expression, in two different cell lines: HEK293-STF (E) and SH-SY5Y (F) cells. Welch’s t test, ∗ p < 0.05. (G and H) Protein levels of B-catenin and its downstream effector EN2 in β-catenin heterozygote, human iPSC-derived neurons, 48 h after transfection with LNP-packed boosters against CTNNB1 (CB7) or a non-specific control. Three different LNP formulations were tested (LNP1, LNP2, and LNP10; see Figure S2 ). (G) A representative western blot and (H) a quantitation of CTNNB1 and EN2 from western blots from two biological replicates.

Article Snippet: We tested our mRNA booster technology in HEK293 (ATCC CRL 1573), HEK293 SuperTopFlash (STF), and SH-SY5Y (ATCC CRL 2266) cell lines as well as iPSC-derived neuronal cells (see below).

Techniques: Expressing, Western Blot, Control, Transfection, Quantitative RT-PCR, Functional Assay, Derivative Assay, Quantitation Assay

Journal: Carcinogenesis

Article Title: Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic β-catenin levels to modulate Wnt signaling and intestinal homeostasis

doi: 10.1093/carcin/bgz007

Figure Lengend Snippet: BVES loss increases Wnt signaling and β-catenin protein levels. (A) TopFlash activity in 293STF cells following BVES shRNA knockdown. (B) AXIN2 fold change by qPCR in 293STF cells with BVES shRNA knockdown. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 16 h before isolation. (C) Cytoplasmic fractions of 293STF cells following shRNA knockdown of BVES. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 2 h before harvest. (D) TopFlash activity in 293STF cells following siRNA knockdown. (E) AXIN2 fold change by qPCR in 293STF cells with siRNA knockdown. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 16 h before isolation. (F) Cytoplasmic fractions of 293STF cells following siRNA knockdown of BVES. Cells were treated with 50% Wnt3a-conditioned media for 2 h before harvest. (G) β-catenin (CTNNB1) transcript levels in 293STF cells 56–72 h following BVES siRNA knockdown. (H) 293STF cells were treated with 20 μM MG132 for 4 h followed by cytoplasmic fractionation. Black arrow indicates β-catenin, and the relative intensity of only this lower band is quantified below. Bracket indicates ubiquitinated species. Western blot is representative of two independent experiments. (I) 293STF cells were treated with 100 μg/ml cycloheximide (CHX) dissolved in dimethyl sulfoxide (DMSO) or DMSO vehicle control for the indicated time points followed by cytoplasmic fractionation. Western blot is representative of two independent experiments. For (A), (B), (D) and (E), data are representative of at least four independent experiments. **P < 0.01, ****P < 0.0001 by Student’s t-test in the minus Wnt and plus Wnt conditions. For (C) and (F), data are pooled from n = 5 independent experiments, each normalized to the control minus Wnt3a condition. **P < 0.01 by Mann–Whitney test in the minus Wnt and plus Wnt conditions. For (G), data are pooled from n = 4 independent experiments. ns = non-significant by Mann–Whitney test in the minus Wnt and plus Wnt conditions.

Article Snippet: Cell lines and culture HEK293 SuperTopFlash (293STF) cells were a kind gift from Dr Ethan Lee, Vanderbilt University and J. Nathans, Johns Hopkins University ( 17 , 18 ) and 293DVL TKO were a kind gift from Dr Ethan Lee, Vanderbilt University and S. Angers, University of Toronto ( 19 ).

Techniques: Activity Assay, shRNA, Knockdown, Isolation, Fractionation, Western Blot, Control, MANN-WHITNEY

Journal: Carcinogenesis

Article Title: Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic β-catenin levels to modulate Wnt signaling and intestinal homeostasis

doi: 10.1093/carcin/bgz007

Figure Lengend Snippet: LRP6 levels and phosphorylation are increased with BVES loss. (A) Cytoplasmic fractionation of 293STF cells following siRNA knockdown of BVES. Cells were treated with 50% Wnt3a conditioned media for 2 h before harvest. pLRP6 quantification is pooled from n = 5 independent experiments, and total LRP6 quantification is pooled from n = 7 independent experiments, each normalized to the control minus Wnt stimulation. (B) LRP6 fold change by qPCR in 2923STF cells with BVES siRNA knockdown. Cells were treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 8 h before isolation. (C) Cytoplasmic fractionations of 293STF cells with Wnt stimulation time course following BVES knockdown. Cells were treated with 50% Wnt3a-conditioned media for indicated time points. (D) Cytoplasmic fractionations of 293STF or 293 Dishevelled Triple-Knockout (293DVL TKO) cells following BVES knockdown. Cells were stimulated with 50% Wnt3a-conditioned media for 2 h and immunoblotted. β-catenin quantification in the 293DVL TKO is pooled from n = 3 independent experiments. Black line between lanes 6 and 7 is debris on the membrane. (E) Cytoplasmic fractionations of 293STF cells stimulated with 50% control or 50% Wnt3a-conditioned media for 2 h. At the time of Wnt3a stimulation, cells were concurrently treated with dimethyl sulfoxide (DMSO), okadaic acid (OA) at 100 nM or ceramide at 50 μM and probed for pLRP6 (S1490), pGSK3β (S9) and pCREB (S133). Data are representative of two independent experiments. For (A), (B) and (D), *P < 0.05, **P < 0.01, ***P < 0.001 by Mann–Whitney test in the minus Wnt and plus Wnt conditions. ns = non-significant by Mann–Whitney test.

Article Snippet: Cell lines and culture HEK293 SuperTopFlash (293STF) cells were a kind gift from Dr Ethan Lee, Vanderbilt University and J. Nathans, Johns Hopkins University ( 17 , 18 ) and 293DVL TKO were a kind gift from Dr Ethan Lee, Vanderbilt University and S. Angers, University of Toronto ( 19 ).

Techniques: Phospho-proteomics, Fractionation, Knockdown, Control, Isolation, Triple Knockout, Membrane, MANN-WHITNEY

Journal: Carcinogenesis

Article Title: Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic β-catenin levels to modulate Wnt signaling and intestinal homeostasis

doi: 10.1093/carcin/bgz007

Figure Lengend Snippet: BVES interacts with LRP6 and LRP5. (A) Reciprocal co-immunoprecipitation of Flag-tagged BVES (Flag-BVES) and GFP-tagged LRP6 (GFP-LRP6). HEK293T cells were transiently transfected with 4 μg of each plasmid using polyethylenimine. Filler plasmid was used to maintain equal DNA quantities. GFP-LRP6 was immunoprecipitated with GFP-binding protein magnetic beads following by immunoblotting with anti-Flag antibodies. Flag agarose resin was used to immunoprecipitate BVES followed by immunoblotting with anti-GFP antibodies. (B) Immunoprecipitation of Flag-BVES followed by immunoblotting for endogenous LRP5 and LRP6. 4 μg of vector or Flag-BVES was transfected into HEK293T cells using polyethylenimine. The LRP5 and LRP6 blots were developed using enhanced chemiluminescence and film, whereas the rest of the blots were immunoblotted using Odyssey infrared reagents as discussed in Materials and methods. (C) Schematic of the N-terminal myc-tag LRP6 (Myc-LRP6ICD) construct that spans amino acids 1364–1539 and contains the LRP6 transmembrane domain (TM) and proximal intracellular domain (ICD). Myc-LRP6ICD was transfected into HEK293T cells along with Flag-BVES as in (A) and immunoprecipitation experiments performed 48 h later. (D) Cells were transfected with 4 μg of Flag-BVES and then 48 h later treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 2 h before flag immunoprecipitation.

Article Snippet: Cell lines and culture HEK293 SuperTopFlash (293STF) cells were a kind gift from Dr Ethan Lee, Vanderbilt University and J. Nathans, Johns Hopkins University ( 17 , 18 ) and 293DVL TKO were a kind gift from Dr Ethan Lee, Vanderbilt University and S. Angers, University of Toronto ( 19 ).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Magnetic Beads, Western Blot, Construct