Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Promotor Hypomethylation Mediated Upregulation of VCAN Targets Twist1 to Promote EndMT in Hypoxia‐Induced Pulmonary Hypertension

doi: 10.1161/JAHA.124.036969

Figure Lengend Snippet: A , RT‐qPCR analysis of the mRNA level of VCAN from HPAECs (n=3). ** P <0.01. ( B ) CpG sites of the promoter of VCAN gene. ( C and D ) DNA methylation levels of the VCAN promotor region in HPAECs and corresponding densitometric quantification as detected by MSP assay (n=3). *** P <0.001. ( E and F ) The protein levels and densitometric quantification of DNMT3a and DNMT3b were determined by Western blot analysis after the hypoxia treatment (n=3). * P <0.05, *** P <0.001. ( G–J ) Representative images of immunohistochemical staining confirmed that DNMT3a and DNMT3b were significantly decreased in HPH mice compared with the normal control group (n=3). Positive staining was indicated by a brown color and pointed out with black arrow. ×400, scale bar: 50 μm. *** P <0.001. ( K and L ) The protein levels and densitometric quantification of DNMTs and VCAN levels in SGI‐1027‐treated HPAECs (n=3). ** P <0.01, *** P <0.001. ( M and N ) DNA methylation levels of the VCAN promotor region in 5 μM SGI‐1027‐treated HPAECs and corresponding densitometric quantification as detected by MSP assay (n=3). *** P <0.001. An unpaired 2‐tailed Student t test was performed for comparisons between 2 groups. CpG indicates Cytosine‐phosphate‐Guanine; DNMT, DNA methyltransferase; HPAECs, human pulmonary artery endothelial cells; HPH, hypoxia‐induced pulmonary hypertension; MSP, methylation‐specific polymerase chain reaction; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; and VCAN, versican.

Article Snippet: The modified DNA was then subjected to real‐time PCR using the methylation‐specific PCR kit (Tiangen, China) to specifically amplify the bisulfite‐converted DNA of the promoter region of versican.

Techniques: Quantitative RT-PCR, DNA Methylation Assay, MSP Assay, Western Blot, Immunohistochemical staining, Staining, Control, Methylation, Polymerase Chain Reaction, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Genome Biology

Article Title: Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells

doi: 10.1186/s13059-026-03974-7

Figure Lengend Snippet: Functional correction after duplex base editing. A – D Chromatograms and quantification of HPLC analysis results after each corresponding treatment on D6 of erythropoiesis. A Chromatograms with peaks presenting HbF, HbA and HbA2 hemoglobin. B Quantification of hemoglobin levels expressed as percentage (%). The % of each hemoglobin type was calculated over the total hemoglobin tetramers, ** P= 0.0079, * P= 0.0180. C Chromatograms with peaks representing β, δ, α and γ ( G γ + Α γ) globin. D Quantification of total γ-globin but also separate G γ and A γ expression presented as ratios over α-globin, * P =0.0118 for γ-globin, * P =0.0106 for G γ and * P =0.0265 for Α γ. E – H Quantification of HPLC analysis of ( E ) γ-globin relative to all β-like globins, ( F ) γ-globin relative to β-globin, ( G ) β-globin relative to α-globin and ( H ) δ-globin relative to α-globin, ** P =0.0089, * P =0.0106. I Quantification of HBG protein after immunoblotting on D6 of differentiation normalized to HBA protein and expressed as fold change to the mock, * P =0.0405. Both proteins were first normalized to β-actin protein. J Relative γ-globin mRNA expression after RT-qPCR analysis on D3 of erythroid differentiation, normalized to α-globin and expressed as fold change relative to mock, * P =0.0471. K Quantification of BCL11A protein after immunoblotting on D0 of differentiation, normalized to β-actin protein and expressed as fold change relative to mock. L Relative BCL11A mRNA expression after RT-qPCR analysis on D0 of erythroid differentiation, normalized to endogenous β-actin and expressed as fold change relative to the mock, ** P =0.0035, * P =0.0379. All data are presented as mean±S.D. (n=3, data are based on biological replicates from three independent donors) using ordinary one-way ANOVA with Dunnett’s multiple comparisons test for P -value

Article Snippet: For the synthesis of BE (pCMV_BE4max, Addgene #112093) [ ] as mRNA, the in vitro transcription protocol was performed using the HiScribeTM T7 ARCA mRNA Kit (with tailing) (New England Biolabs) according to our published procedures [ ].

Techniques: Functional Assay, Expressing, Western Blot, Quantitative RT-PCR

Journal: Genome Biology

Article Title: Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells

doi: 10.1186/s13059-026-03974-7

Figure Lengend Snippet: RNA-seq analysis of BFU-E colonies. A Schematic diagram of the study design for RNA-seq analysis. B Flow cytometry analysis for the detection of dead and apoptotic cells after each treatment. Data are presented as % ( n =1). C BFU-E and CFU-GM colony scoring on day 14 for each treatment. Data are presented as % ( n =1). D Left: charts showing the percentage frequency of specific edits detected by Sanger Sequencing for each treatment. Y-axis labels indicate the edit in each allele, where N represents no editing in the one allele (monoallelic). Right: charts showing genotypes for the three colonies selected for further analysis. X-axis indicates the specific edits, with values representing editing status: 100=biallelic, 50=monoallelic, 0=no edit. E Relative HBG mRNA expression after RNA-seq analysis, normalized to HBA and expressed as log 2 fold change relative to mock, * P =0.0325. Data are presented as mean±S.D. ( n =3, data are based on biological replicates from three different BFU-E colonies) using Kruskal-Wallis test with Dunn’s multiple comparison test

Article Snippet: For the synthesis of BE (pCMV_BE4max, Addgene #112093) [ ] as mRNA, the in vitro transcription protocol was performed using the HiScribeTM T7 ARCA mRNA Kit (with tailing) (New England Biolabs) according to our published procedures [ ].

Techniques: RNA Sequencing, Flow Cytometry, Sequencing, Expressing, Comparison

Journal: Genome Biology

Article Title: Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells

doi: 10.1186/s13059-026-03974-7

Figure Lengend Snippet: mRNA expression of apoptotic and immune response genes after RNA-seq analysis. Normalized mRNA expression is shown as log 2 fold change of statistically significant ( A-D ) apoptotic and ( E-H ) immune response genes after each treatment. Data are presented as mean±S.D. ( n =3, data are based on biological replicates from three different BFU-E colonies). DESeq2 analysis was performed and adjusted P -values are indicated as follows: *<0.05, **<0.01, ***<0.001, ****<0.0001

Article Snippet: For the synthesis of BE (pCMV_BE4max, Addgene #112093) [ ] as mRNA, the in vitro transcription protocol was performed using the HiScribeTM T7 ARCA mRNA Kit (with tailing) (New England Biolabs) according to our published procedures [ ].

Techniques: Expressing, RNA Sequencing

Journal: Cell

Article Title: SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

doi: 10.1016/j.cell.2020.10.004

Figure Lengend Snippet:

Article Snippet: HiScribe T7 ARCA mRNA Kit , NEB , Cat. # E2060S.

Techniques: Virus, Recombinant, Transfection, RNA Sequencing Assay, Software