ndufs3 overexpression plasmids  (GenScript corporation)

Journal: Advanced Science

Article Title: SRSF1 Is Required for Mitochondrial Homeostasis and Thermogenic Function in Brown Adipocytes Through its Control of Ndufs3 Splicing

doi: 10.1002/advs.202306871

Figure Lengend Snippet: SRSF1 plays a key role in regulating the inclusion of constitutive exon 6 within Ndufs3 pre‐mRNA. A,B) Validation of representative exon inclusion or exclusion events influenced by SRSF1 was conducted through RT‐PCR. The regulated exon, identified with its exon number, is depicted in the green box. The exclusion/inclusion (Ex/In) ratios of RNA products are quantified (n = 3). C) A schematic diagram illustrating the Ndufs3 and Ndufs3‐T isoforms, with or without exon 6. Note that SRSF1 deletion led to the production of Ndufs3‐T. D) Analysis of the expression pattern of Ndufs3 and Ndufs3‐T isoforms in BAT samples collected from WT and KO mice using RT‐PCR analysis. E) Western blot analysis conducted on BAT samples obtained from WT and KO mice using anti‐Ndufs3 antibodies. F) Schematic diagrams illustrating the potential binding sites for SRSF1 on exon 6 of Ndufs3, indicated by the red‐marked sequence. The binding motifs of SRSF1, predicted by the RBP Suite website, are displayed at the bottom. G) q‐PCR analysis performed on Ndufs3 pre‐mRNA in samples of immunoprecipitation (IP) using anti‐SRSF1 antibodies or IgG control (left) in brown adipocytes. The western blot analysis shows SRSF1 protein levels in whole cell extracts (input) and post‐IP using the anti‐SRSF1 antibodies (right). H) Diagrams of the Nduf3 minigene construct and two of its derivatives are presented. The sequence within the exon6, highlighted in red, have been substituted with the sequences marked in yellow in the mutation minigene. I) Brown adipocytes were co‐transfected with the SRSF1‐overexpressing plasmid and either the Ndufs3 minigene or derivatives. In vitro splicing analysis of Ndufs3 E6 was performed using RT‐PCR, and the resulting Percent Spliced In (PSI) values were presented on the right graph. PSI = Inclusion/(Inclusion + Exclusion). * p < 0.05, ** p < 0.01, *** p ,0.001. Data represent the mean ± SEM.

Article Snippet: Ndufs3 overexpression plasmids were purchased from Genescript (Nanjing, China).

Techniques: Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Binding Assay, Sequencing, Immunoprecipitation, Control, Construct, Mutagenesis, Transfection, Plasmid Preparation, In Vitro

Journal: Advanced Science

Article Title: SRSF1 Is Required for Mitochondrial Homeostasis and Thermogenic Function in Brown Adipocytes Through its Control of Ndufs3 Splicing

doi: 10.1002/advs.202306871

Figure Lengend Snippet: SRSF1 regulates mitochondrial homeostasis through controlling the inclusion of Ndufs3 exon 6 in brown adipocytes. A) Primary brown adipocytes were transfected with either Ndufs3‐specific siRNAs (siNdufs3‐1, siNdufs3‐2) or control siRNA (siNC) for 48 h. Representative images of cells stained with Mitotracker green are displayed. Enlarged images reveal the presence of fragmented and degenerated mitochondria. Nuclei were stained with Hoechst. Scale bar, 5 µm. B) q‐PCR analysis was performed to assess the expression of genes involved in the regulation of mitochondrial dynamics in cells described in (A). C) mtROS detection was carried out using MitoSOX staining (red) in cells described in (A). Scale bar, 20 µm. The quantification of relative fluorescence units (RFU) was shown in the right graph. D) Mitochondrial membrane potential was assessed using JC‐1 staining in cells described in (A). Green fluorescence indicated low membrane potential, while red fluorescence indicated high membrane potential and accumulation of JC‐1 in mitochondria. Scale bar, 50 µm. The ratio of red/green fluorescence was quantified and shown in the right graph. E) Brown adipocytes were transfected with SRSF1‐specific siRNAs (siSRSF1‐1, siSRSF1‐2) or siNC for 48 h. Representative images of cells stained with Mitotracker green are displayed. Enlarged images reveal the presence of fragmented and degenerated mitochondria. F) mtROS detection was carried out using MitoSOX staining (red) in cells described in (E). Scale bar, 20 µm. The quantification of relative fluorescence units (RFU) was shown in the right graph. G) Brown adipocytes were co‐transfected with siNC and vector plasmid, siSRSF1 and vector plasmid, or siSRSF1 and Ndufs3 plasmid for 48 h. Representative images of Mitotracker staining (green, top panel) and MitoSOX staining (red, bottom panel) were shown. Scale bars: 10 µm (top panel), 20 µm (bottom panel). H) Mitochondrial membrane potential was detected using JC‐1 staining in the cells described in panel (G). I) Quantification of RFU for the experiment described in (G) was illustrated on the top graph. Quantification of the red/green fluorescence ratio for the experiment described in (H) was presented on the bottom graph. * p < 0.05, ** p < 0.01, *** p ,0.001. Data represent the mean ± SEM.

Article Snippet: Ndufs3 overexpression plasmids were purchased from Genescript (Nanjing, China).

Techniques: Transfection, Control, Staining, Expressing, Fluorescence, Membrane, Plasmid Preparation

Journal: Advanced Science

Article Title: SRSF1 Is Required for Mitochondrial Homeostasis and Thermogenic Function in Brown Adipocytes Through its Control of Ndufs3 Splicing

doi: 10.1002/advs.202306871

Figure Lengend Snippet: SRSF1‐regulated splicing of Ndufs3 plays a significant role in influencing thermogenesis in mature brown adipocytes. A,B) Primary brown adipocytes were transiently transfected with Ndufs3 siRNA or siNC, followed by induction of differentiation into mature adipocytes for the following experiments. Ndufs3 knockdown efficiency was confirmed by western blot (A). The expression of thermogenic genes was then assessed using q‐PCR in these cells (B). C) Brown adipocytes were co‐transfected with siNC and vector plasmid, siSRSF1 and vector plasmid, or siSRSF1 and Ndufs3 plasmid respectively, followed by induction of differentiation into mature adipocytes. The expression levels of genes involved in thermogenesis, mitochondrial OXPHOS, and FAO were measured by q‐PCR. The expression level of each gene was normalized to β‐Actin as an internal control (n = 3). D) Western blot analysis was conducted to assess the protein levels of Srsf1, Ndufs3, and Uc1 in the mature brown adipocytes described in panel (C). E) A functional model for SRSF1's role in the regulation of Ndufs3 splicing and its control over thermogenesis in BAT. Our model demonstrates that SRSF1 effectively binds to exon 6 of Ndufs3 pre‐mRNA, thereby promoting its inclusion in the final mRNA transcript. Conversely, the deficiency of SRSF1 results in impaired splicing of Ndufs3 and subsequently reduced levels of Ndufs3 proteins. Consequently, this deficiency triggers an increase in mtROS generation and disrupts mitochondrial function within brown adipocytes, ultimately compromising the thermogenic capacity of BAT. * p < 0.05, ** p < 0.01, *** p,0.001. Data represent the mean ± SEM.

Article Snippet: Ndufs3 overexpression plasmids were purchased from Genescript (Nanjing, China).

Techniques: Transfection, Knockdown, Western Blot, Expressing, Plasmid Preparation, Control, Functional Assay