Figure 9.

Pink1 and parkin expression during HC11 cell differentiation and mammary gland development. (A) Expression of Prkn across differentiation in HC11 cells (n = 3). (B) Protein levels of PRKN and PINK1 during HC11 cell differentiation. Levels of PRKN and PINK1 are indicated below each lane after normalization to GAPDH. The undifferentiated sample was set to 1.00, and all other time points are presented relative to 1.00. (C) Immunohistochemical expression of PRKN and PINK1 at pregnancy day 16 (P16), lactation day 1 (L1), and lactation day 7 (L7) in the mouse mammary gland. (D) Higher magnification of cytoplasmic PRKN staining at L1. Two mice were evaluated for each time point, and two sections from each mouse were stained for comparisons. (E) Fractionation of HC11 cell lysates into mitochondrial and cytosolic components and expression of PRKN and PINK1. VDAC1 and TUBA1A expression confirm mitochondrial and cytosolic fraction purity, respectively. Levels of PRKN and PINK1 are indicated below each lane after normalization to VDAC1 for mitochondrial fractions and to TUBA1A for cytosolic fractions. The undifferentiated sample for each fraction was set to 1.00, and all other time points are presented relative to 1.00. (F) JC-1 was used to assess mitochondrial membrane polarization. A minimum of 10 images were evaluated for red and green fluorescent intensity for each time point. FCCP was used as a positive control for membrane depolarization (n = 3). U: undifferentiated; UC: undifferentiated confluent; P: 24 h primed; h: hours differentiated Scale bars: 100 μm. Data are presented as mean ± standard deviation. Statistical significance was evaluated with multiple student t-tests relative to the undifferentiated time point (U). **p < 0.01