Journal: Redox Biology

Article Title: Nitrite increases mitofusin-1 levels to inhibit vascular smooth muscle cell proliferation and prevent intimal hyperplasia

doi: 10.1016/j.redox.2026.104169

Figure Lengend Snippet: Nitrite inhibits vascular smooth muscle cell proliferation in a Mfn1-dependent manner manner. (A) 3H-thymidine incorporation in RASMC pretreated with nitrite (0-25 μM) and stimulated with PDGF-BB (10 ng/mL) for 24 h (n = 4). (B) Percentage of cells in cell cycle phases in RASMC treated with (black bars) or without (white bars) nitrite (25 μM; n = 4). (C) Representative Western blot (left) and quantification of control (white bars) and nitrite treated (black bars; 25 μM) RASMC (n = 3). (D) Representative images (60X) of immunofluorescence staining in RASMC treated with or without nitrite (25 μM; 24 h) and stained for Tom20 (mitochondrial membrane). Quantification of the percentage of elongated, intermediate or fragmented mitochondria in RASMC (n = 4 biological replicates of 50-83 cells), ∗p < 0.05 with respect to elongated mitochondria. (E) Representative Western blot (top) and quantification (bottom) of RASMC treated with (black bars) or without (white bars) nitrite (25 μM). (F) Representative Western blot of RASMC treated with control or Mfn1 targeted siRNA. (G) 3H-thymidine incorporation in RASMC transfected with control (white bars) or Mfn1 siRNA (black bars) followed by PDGF (10 ng/mL) treatment in the presence or absence of nitrite (25 μM) (n = 6). Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: Female B6.129(Cg)-Mfn1 tm2Dcc /J mice carrying homozygous loxP-flanked Mfn1 alleles (Mfn1 fl/fl ) (Jackson Laboratories) were bred to male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J mice ( Myh11 -Cre ERT2 ) which carry a tamoxifen-inducible Cre recombinase under the Myh11 promoter (Jackson Laboratories.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Membrane, Transfection

Journal: Redox Biology

Article Title: Nitrite increases mitofusin-1 levels to inhibit vascular smooth muscle cell proliferation and prevent intimal hyperplasia

doi: 10.1016/j.redox.2026.104169

Figure Lengend Snippet: Nitrite increases Mfn1 levels by preventing its ubiquitination. (A) Relative Mfn1 mRNA levels in RASMC treated with or without nitrite (n = 3). (B) Representative images (left) and quantification (right) of Western blots measuring Mfn1 protein levels in RASMC treated with cycloheximide (CHX) in the presence or absence of nitrite (25 μM) for 1 h or 24 h (n = 5). (C) Proteasomal activity levels in RASMC treated with and without nitrite or the proteosomal inhibitor MG132 (n = 5). (D) Representative Western blot of ubiquitination of immunoprecipitated Mfn1 (left) and quantification (right; n = 5). (E) Representative Western blot in control and March5 knockdown RASMC 48 h after siRNA transfection. (F) Representative images (top) and quantification (bottom) of Western blot measuring Mfn1 in RASMC transfected with control or March5 siRNA followed by nitrite treatment (n = 3). (G) Representative image (top) of Western blot measuring March5 in RASMC treated with or without nitrite and quantification (bottom) of five such blots. (H) Representative Western blot of immunoprecipitated Mfn1 from RASMC, stained for March5 and pan-nitrotyrosine (top) and quantification (bottom)(n = 3). Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: Female B6.129(Cg)-Mfn1 tm2Dcc /J mice carrying homozygous loxP-flanked Mfn1 alleles (Mfn1 fl/fl ) (Jackson Laboratories) were bred to male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J mice ( Myh11 -Cre ERT2 ) which carry a tamoxifen-inducible Cre recombinase under the Myh11 promoter (Jackson Laboratories.

Techniques: Ubiquitin Proteomics, Western Blot, Activity Assay, Immunoprecipitation, Control, Knockdown, Transfection, Staining

Journal: Redox Biology

Article Title: Nitrite increases mitofusin-1 levels to inhibit vascular smooth muscle cell proliferation and prevent intimal hyperplasia

doi: 10.1016/j.redox.2026.104169

Figure Lengend Snippet: Deletion of Mfn1 leads to the increased proliferation and decreased expression of contractile genes in RASMC. (A) Representative Western blot (left) and quantification (right) of protein levels of mitochondrial dynamics proteins (Mfn1, Mfn2, OPA1, phosphorylated Drp1, total Drp1) in RASMC transfected with control (white bars) or Mfn1-targeted (black bars) siRNA. (B) Representative images and quantification of immunofluorescence in RASMC transfected with control or Mfn1 siRNA after 48 h in growth media. (C) Cell cycle analysis in control (white bars) and Mfn1 knockdown (black bars) RASMC after serum starvation. Quantification shows the percentage of cells present in either G1, S or G2 phase. (D) Representative Western blots and quantification of cell cycle regulator protein expression in RASMC transfected with control (white bars) or Mfn1 siRNA (black bars). (E – F) Relative change in (E) mRNA levels and (F) protein levels of contractile proteins in RASMC treated with control (white bars) or Mfn1-targeted (black bars) siRNA for 48 h (n = 4 in all panels); Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Female B6.129(Cg)-Mfn1 tm2Dcc /J mice carrying homozygous loxP-flanked Mfn1 alleles (Mfn1 fl/fl ) (Jackson Laboratories) were bred to male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J mice ( Myh11 -Cre ERT2 ) which carry a tamoxifen-inducible Cre recombinase under the Myh11 promoter (Jackson Laboratories.

Techniques: Expressing, Western Blot, Transfection, Control, Immunofluorescence, Cell Cycle Assay, Knockdown

Journal: Redox Biology

Article Title: Nitrite increases mitofusin-1 levels to inhibit vascular smooth muscle cell proliferation and prevent intimal hyperplasia

doi: 10.1016/j.redox.2026.104169

Figure Lengend Snippet: Deletion of Mfn1 downregulates cellular ATP production and increases cellular ROS levels. (A) Representative traces and (B) quantification of mitochondrial oxygen consumption rate (OCR) and (C) ATP levels in RASMC transfected with control (white bars) or Mfn1-targeted (black bars) siRNA for 48 h (n = 4). (D) Rate of change in MitoSOX fluorescence (mitochondrial ROS) and change in hydrogen peroxide (cellular ROS) production in RASMC transfected with control (white bars) or Mfn1-targeted (black bars) siRNA for 48 h (n = 4). (E) Representative Western blot (left) and quantification (right) of protein levels of ROS generating and antioxidant proteins in RASMC transfected with control (white bars) or Mfn1-targeted (black bars) siRNA for 48 h (n = 4-5). Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: Female B6.129(Cg)-Mfn1 tm2Dcc /J mice carrying homozygous loxP-flanked Mfn1 alleles (Mfn1 fl/fl ) (Jackson Laboratories) were bred to male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J mice ( Myh11 -Cre ERT2 ) which carry a tamoxifen-inducible Cre recombinase under the Myh11 promoter (Jackson Laboratories.

Techniques: Transfection, Control, Fluorescence, Western Blot

Journal: Redox Biology

Article Title: Nitrite increases mitofusin-1 levels to inhibit vascular smooth muscle cell proliferation and prevent intimal hyperplasia

doi: 10.1016/j.redox.2026.104169

Figure Lengend Snippet: Enhanced ROS propagates proliferation in Mfn1 deficient cells. ( A) Rate of hydrogen peroxide ( cellular ROS) generation and (B) ATP levels in RASMC transfected with control or Mfn1-targeted siRNA for 24 h followed by empty virus (null) or adenovirus-induced overexpression (2.5x106 PFU/mL) for another 24 h (n = 5). (C) Relative levels of mRNA for contractile genes in RASMC transfected with control or Mfn1 siRNA for 24 h followed by empty virus (null) or adenovirus-induced overexpression (2.5x106 PFU/mL) (n = 5). (D) Representative Western blots (top) and quantification (below) of VSMC contractile proteins in scramble control or Mfn1 siRNA cells with or without catalase overexpression (n = 3-5). (E) Representative 20X images (top) and quantification (bottom) of immunofluorescence for nuclei (DAPI), proliferation (Ki67), and smooth muscle actin (SMA) (n = 3). Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Female B6.129(Cg)-Mfn1 tm2Dcc /J mice carrying homozygous loxP-flanked Mfn1 alleles (Mfn1 fl/fl ) (Jackson Laboratories) were bred to male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J mice ( Myh11 -Cre ERT2 ) which carry a tamoxifen-inducible Cre recombinase under the Myh11 promoter (Jackson Laboratories.

Techniques: Transfection, Control, Virus, Over Expression, Western Blot, Immunofluorescence

Journal: Redox Biology

Article Title: Nitrite increases mitofusin-1 levels to inhibit vascular smooth muscle cell proliferation and prevent intimal hyperplasia

doi: 10.1016/j.redox.2026.104169

Figure Lengend Snippet: Nitrite-mediated attenuation of vascular remodeling after injury is dependent on Mfn1. (A) Images of Masson's Trichrome-stained carotid arteries from WT mice and SMC-Mfn1 −/− mice subjected to carotid ligation injury followed by administration of normal or nitrite supplemented drinking water for 3 weeks. (B) The ratio of intima area to medial area (n = 8-9 in control and n = 6 in nitrite treatment). (C) Representative images of immunofluorescence-stained carotid arteries from WT mice and SMC-Mfn1 −/− mice subjected to carotid ligation injury followed by nitrite supplemented drinking water for 3 weeks. Immunofluorescence staining shows nuclei DAPI (nuclei), YFP (SMC tracer) and Ki67 (proliferation marker). (D) Quantification of Ki67; YFP double positive cells (n = 8-10 in control and n = 5-6 in nitrite treatment).Mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: Female B6.129(Cg)-Mfn1 tm2Dcc /J mice carrying homozygous loxP-flanked Mfn1 alleles (Mfn1 fl/fl ) (Jackson Laboratories) were bred to male B6.FVB-Tg(Myh11-cre/ERT2)1Soff/J mice ( Myh11 -Cre ERT2 ) which carry a tamoxifen-inducible Cre recombinase under the Myh11 promoter (Jackson Laboratories.

Techniques: Staining, Ligation, Control, Immunofluorescence, Marker