backbone plasmid  (Addgene inc)

Journal: NPJ Regenerative Medicine

Article Title: Long noncoding RNA LncBAR enhances BRG1 protein to promote cardiomyocyte cell cycle progression and cardiac repair

doi: 10.1038/s41536-025-00439-6

Figure Lengend Snippet: a Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of LncBAR expression in murine hearts at indicated time points (embryonic day (E) 13.5; postnatal days (P) 1 to P14, and 4 and 12 weeks (W)) ( n = 4 mice). b Relative LncBAR expression in hearts at 7 (AR7) and 21 (AR21) days post-apical resection (AR), determined by RT-qPCR. RZ: remote zone; BZ: border zone; AZ: apex zone ( n = 3 mice). c Relative LncBAR expression in murine hearts at 4 weeks post-myocardial infarction (MI) by RT-qPCR. RZ: remote zone; BZ: border zone; IZ: infarct zone. ( n = 3 mice). d ICC analysis of EdU-incorporated NMCMs for cTnI (cardiomyocyte marker). Cells were transduced with sh LncBAR , control shNT, or PBS and cultured with EdU for 1 day prior to staining. White arrows indicate EdU+ NMCMs. Scale bars, 50 µm. e Proportions of total (cTnI+) and proliferating (EdU+ cTnI+) NMCMs 4 days after treatment with sh LncBAR , shNT, or PBS. ( n = 15 fields). f Representative ICC and quantification of cTnI+ cells in EdU-incorporated NMCMs following transduction with lentivirus harboring LacZ or LncBAR , or treatment with PBS (vehicle control), after a 24 h EdU pulse. White arrows indicate EdU+ NMCMs ( n = 15 fields). Scale bars, 50 µm. g Representative ICC and quantification of cTnI and Ki67 in NMCMs treated with or without lentiviral LncBAR . PBS was used as non-viral treatment control. White arrows indicate Ki67+ NMCMs ( n = 15 fields). Scale bars, 50 µm. h Representative ICC and quantification of cTnI and Aurora B in NMCMs treated with or without lentiviral LncBAR . PBS was used as non-viral treatment control. White arrows indicate Aurora B+ NMCMs ( n = 5 biological replicates). Scale bars, 50 µm. i, j RT-qPCR analysis showing the expression of genes positively regulating the cell cycle and proliferation upon LncBAR knockdown ( i ) or overexpression treatment ( j ) ( n = 3 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For gene overexpression, pLenti- LncBAR was cloned into lentiviral backbone that was originally purchased from Addgene (#17448).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Marker, Transduction, Control, Cell Culture, Staining, Knockdown, Over Expression

Journal: NPJ Regenerative Medicine

Article Title: Long noncoding RNA LncBAR enhances BRG1 protein to promote cardiomyocyte cell cycle progression and cardiac repair

doi: 10.1038/s41536-025-00439-6

Figure Lengend Snippet: a Schematic depiction of experimental design using NMCMs treated with sh LncBAR or shNT for RNA-seq ( n = 2 biological replicates). b Volcano plot showing differentially expressed genes (DEGs) induced by LncBAR knockdown (sh LncBAR ) in NMCMs compared to those treated with shNT. DEGs were defined by P < 0.05 and |fold change| ≥ 2 comparing sh LncBAR -NMCMs to shNT-NMCMs. c Gene Ontology (GO) analysis of significantly enriched pathways in sh LncBAR -treated NMCMs compared with shNT-treated NMCMs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showing significantly upregulated ( d ) and downregulated ( e ) pathways enriched in sh LncBAR -NMCMs compared to shNT control cells. f Heatmap showing the expression levels of representative Brg1 downstream factors involved in proliferation in sh LncBAR - or shNT-NMCMs ( n = 2 biological replicates). g–i Western blot showing the expression of total and phosphorylated form of PI3K and AKT in NMCMs treated with or without LncBAR overexpression. Quantification of indicated protein expression was shown in ( h ) and ( i ) ( n = 3 biological replicates). Representative ICC images and quantification for cTnT+ pH3+ ( j ) and cTnT+ Ki67+ ( k ) NMCMs treated with LncBAR along with small molecule LY294002 (PI3K/AKT pathway inhibitor). Cells infected with lentiviral LacZ and treated with DMSO were used as control. White arrows indicate proliferating NMCMs ( n = 15 fields). Scale bars, 100 µm. l RT-qPCR analysis showing expression of genes involved in positive regulation of cell cycle in LncBAR overexpressed NMCMs treated with or without LY294002 inhibitor ( n = 3 biological replicates). Data are presented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t test or two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For gene overexpression, pLenti- LncBAR was cloned into lentiviral backbone that was originally purchased from Addgene (#17448).

Techniques: RNA Sequencing, Knockdown, Control, Expressing, Western Blot, Over Expression, Infection, Quantitative RT-PCR, Two Tailed Test