Fig 1: Genetically engineered oncogene‐driven Merlin‐deficient mammary tumors harbor elevated oxidative stress. (A) Pups of pregnant Nf2flfl MMTV Neu mice were weaned 7 days after birth, and the mice were intraductally injected (I.I) with vector‐control lentivirus (Ctr‐lenti) or Cre‐lentivirus (Cre‐lenti) in both 4th mammary glands 7 days postweaning to generate Nf2flfl MMTV Neu+ and Nf2−l− MMTV Neu + mice, respectively. I.I efficiency test with PBS with 0.2% Evans blue dye enabling the visualization of the ductal tree is shown. (B) Loss of Merlin and increased 4‐HNE staining are observed in mammary gland harvested from Nf2−l− MMTV Neu + compared to Nf2flfl MMTV Neu + by IHC; IHC score for Merlin (P = 0.0012) and 4‐HNE (P = 0.0413) is shown. Scale bar = 30 μm. Images representative of 4 different fields of each group (n = 2). (C) Mammary tumor tissue harvested from Nf2−l− MMTV Neu + showed lower levels of Merlin and increased 4‐HNE staining than Nf2flfl MMTV Neu+; IHC score for Merlin (P = 0.0452) and 4‐HNE (P = 0.0030) is shown. Scale bar = 30 μm. Images representative of 4 different fields of each group (n = 2). (D) 3D cell culture of primary mammary tumor cells harvested from Nf2−l− MMTV Neu + showed a very breached and disorganized structure compared to Nf2flfl MMTV Neu+. Circularity of the spheres is shown (P = 0.0432). Scale bar = 50 µm. Images representative of three different 3D‐culture organoids. (E) Nf2flfl MMTV Neu+‐and Nf2−l− MMTV Neu+‐derived primary tumor cells were immortalized and named 283 and 285, respectively; 285 cells had lower levels of Merlin and higher levels of NOX4, DUOX1, and DUOX2 than 283. Error bars represent ± SEM. Student's t‐test was applied for statistical analysis. β‐actin was used as loading control. Representative immunoblot band densitometry is shown.