Journal: International Journal of Molecular Sciences

Article Title: Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

doi: 10.3390/ijms26167774

Figure Lengend Snippet: EMD-LNPs efficiently deliver cargo in vitro. ( A ) Emerin-null myogenic progenitors dosed with 2.5 pg/cell EMD-LNPs (EMD −/y + LNP) or PBS (EMD −/y + PBS) for 22 h. Immunofluorescence microscopy was performed using emerin antibodies (green). DAPI (blue), DNA. ( B ) Greater than 97% of EMD-LNP-treated emerin-null progenitors retain emerin protein expression after 4 days. ( C ) Western blotting was performed on wildtype (WT) or emerin-null proliferative myogenic progenitors treated with EMD-LNPs (EMD −/y + LNP) or PBS (EMD −/y + PBS) over four days. ( D ) Quantitation of emerin protein expression normalized to endogenous emerin in wildtype progenitors. Scale bar represents 100 µm. Error bars represent S.D. ( n = 4).

Article Snippet: Cells were washed and blocked with 3% BSA in PBS for 1 h. The primary emerin antibody (1:500; ProteinTech #10351-1-AP; Rosemont, IL, USA) was diluted in blocking buffer and incubated for 2 h at room temperature or overnight at 4 °C.

Techniques: In Vitro, Immunofluorescence, Microscopy, Expressing, Western Blot, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

doi: 10.3390/ijms26167774

Figure Lengend Snippet: EMD-LNPs rescue differentiation of emerin-null myogenic progenitors. ( A ) Line graph showing the timing of LNP treatment (green dot), differentiation induction, and sample collection (pink dot). Wildtype myogenic progenitors treated with PBS (WT + PBS), emerin-null progenitors treated with PBS (EMD −/y + PBS), or emerin-null progenitors treated with 2.5 pg/cell of EMD-LNPs (EMD −/y + LNP) were incubated for 22 h and induced to differentiate. ( B ) After 24 h, the cells were incubated with EdU for 2 h. The cells were fixed and incubated with emerin antibodies. EdU, red; emerin, green; DAPI, DNA (blue). ( C ) Quantification of EdU-positive nuclei. ( D , F ) Immunofluorescence microscopy was performed after 48 h ( D ) or 72 h ( F ) to detect MyHC (red) or emerin (green). DAPI, DNA (blue). ( E ) The differentiation index was quantified by determining the percentage of MyHC-positive cells. ( G ) The fusion index represents the percentage of nuclei present in a shared (≥3 nuclei/cell) MyHC-positive cytoplasm. ( H ) Western blot images of myogenesis markers in differentiating myogenic progenitors. Representative images of normal exposure (i) or increased exposure (ii) are shown. Scale bars represent 100 µm. Error bars represent S.D. ( n = 4; ≥100 nuclei per biological replicate); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Cells were washed and blocked with 3% BSA in PBS for 1 h. The primary emerin antibody (1:500; ProteinTech #10351-1-AP; Rosemont, IL, USA) was diluted in blocking buffer and incubated for 2 h at room temperature or overnight at 4 °C.

Techniques: Incubation, Immunofluorescence, Microscopy, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

doi: 10.3390/ijms26167774

Figure Lengend Snippet: EMD-LNPs partially rescue H4K5ac and H3K9me2 levels in emerin-null myogenic progenitors. Whole-cell lysates were collected from wildtype cells treated with PBS (WT + PBS), emerin-null cells were treated with 2.5 pg/cell of EMD-LNPs (EMD −/y + LNP), and emerin-null cells were treated with PBS (EMD −/y + PBS) 24 h after treatment. ( A ) Western blotting for emerin, H3K9me2, H4K5ac, and γ -tubulin. ( B , C ) Quantification of H3K9me2 and H4K5ac Western blots. Levels of each protein were normalized to γ -tubulin and then normalized to wildtype cells. Error bars represent S.D. ( n = 3); # p ≤ 0.08; * p ≤ 0.05; ** p ≤ 0.01.

Article Snippet: Cells were washed and blocked with 3% BSA in PBS for 1 h. The primary emerin antibody (1:500; ProteinTech #10351-1-AP; Rosemont, IL, USA) was diluted in blocking buffer and incubated for 2 h at room temperature or overnight at 4 °C.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

doi: 10.3390/ijms26167774

Figure Lengend Snippet: EMD-LNPs efficiently deliver cargo in vitro. ( A ) Emerin-null myogenic progenitors dosed with 2.5 pg/cell EMD-LNPs (EMD −/y + LNP) or PBS (EMD −/y + PBS) for 22 h. Immunofluorescence microscopy was performed using emerin antibodies (green). DAPI (blue), DNA. ( B ) Greater than 97% of EMD-LNP-treated emerin-null progenitors retain emerin protein expression after 4 days. ( C ) Western blotting was performed on wildtype (WT) or emerin-null proliferative myogenic progenitors treated with EMD-LNPs (EMD −/y + LNP) or PBS (EMD −/y + PBS) over four days. ( D ) Quantitation of emerin protein expression normalized to endogenous emerin in wildtype progenitors. Scale bar represents 100 µm. Error bars represent S.D. ( n = 4).

Article Snippet: Membranes were stained with primary antibodies against emerin (1:3000; ProteinTech #10351-1-AP; Rosemont, IL, USA), γ -tubulin (1:10,000; Invitrogen #MA1-850; Carlsbad, CA, USA), MyoD (1:200; Santa Cruz #sc-304; Dallas, TX, USA), MyoG (1:250: Abcam #ab1835; Cambridge, UK), H3K9me2 (1:1500; Active Motif #39239; Carlsbad, CA, USA), or H4K5ac (1:1500; Sigma-Aldrich #07-327; St. Louis, MO, USA) diluted in 0.5% milk in PBST.

Techniques: In Vitro, Immunofluorescence, Microscopy, Expressing, Western Blot, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

doi: 10.3390/ijms26167774

Figure Lengend Snippet: EMD-LNPs rescue differentiation of emerin-null myogenic progenitors. ( A ) Line graph showing the timing of LNP treatment (green dot), differentiation induction, and sample collection (pink dot). Wildtype myogenic progenitors treated with PBS (WT + PBS), emerin-null progenitors treated with PBS (EMD −/y + PBS), or emerin-null progenitors treated with 2.5 pg/cell of EMD-LNPs (EMD −/y + LNP) were incubated for 22 h and induced to differentiate. ( B ) After 24 h, the cells were incubated with EdU for 2 h. The cells were fixed and incubated with emerin antibodies. EdU, red; emerin, green; DAPI, DNA (blue). ( C ) Quantification of EdU-positive nuclei. ( D , F ) Immunofluorescence microscopy was performed after 48 h ( D ) or 72 h ( F ) to detect MyHC (red) or emerin (green). DAPI, DNA (blue). ( E ) The differentiation index was quantified by determining the percentage of MyHC-positive cells. ( G ) The fusion index represents the percentage of nuclei present in a shared (≥3 nuclei/cell) MyHC-positive cytoplasm. ( H ) Western blot images of myogenesis markers in differentiating myogenic progenitors. Representative images of normal exposure (i) or increased exposure (ii) are shown. Scale bars represent 100 µm. Error bars represent S.D. ( n = 4; ≥100 nuclei per biological replicate); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Membranes were stained with primary antibodies against emerin (1:3000; ProteinTech #10351-1-AP; Rosemont, IL, USA), γ -tubulin (1:10,000; Invitrogen #MA1-850; Carlsbad, CA, USA), MyoD (1:200; Santa Cruz #sc-304; Dallas, TX, USA), MyoG (1:250: Abcam #ab1835; Cambridge, UK), H3K9me2 (1:1500; Active Motif #39239; Carlsbad, CA, USA), or H4K5ac (1:1500; Sigma-Aldrich #07-327; St. Louis, MO, USA) diluted in 0.5% milk in PBST.

Techniques: Incubation, Immunofluorescence, Microscopy, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

doi: 10.3390/ijms26167774

Figure Lengend Snippet: EMD-LNPs partially rescue H4K5ac and H3K9me2 levels in emerin-null myogenic progenitors. Whole-cell lysates were collected from wildtype cells treated with PBS (WT + PBS), emerin-null cells were treated with 2.5 pg/cell of EMD-LNPs (EMD −/y + LNP), and emerin-null cells were treated with PBS (EMD −/y + PBS) 24 h after treatment. ( A ) Western blotting for emerin, H3K9me2, H4K5ac, and γ -tubulin. ( B , C ) Quantification of H3K9me2 and H4K5ac Western blots. Levels of each protein were normalized to γ -tubulin and then normalized to wildtype cells. Error bars represent S.D. ( n = 3); # p ≤ 0.08; * p ≤ 0.05; ** p ≤ 0.01.

Article Snippet: Membranes were stained with primary antibodies against emerin (1:3000; ProteinTech #10351-1-AP; Rosemont, IL, USA), γ -tubulin (1:10,000; Invitrogen #MA1-850; Carlsbad, CA, USA), MyoD (1:200; Santa Cruz #sc-304; Dallas, TX, USA), MyoG (1:250: Abcam #ab1835; Cambridge, UK), H3K9me2 (1:1500; Active Motif #39239; Carlsbad, CA, USA), or H4K5ac (1:1500; Sigma-Aldrich #07-327; St. Louis, MO, USA) diluted in 0.5% milk in PBST.

Techniques: Western Blot