Fig. 2: SMAD3 is a positive nuclear transcription factor of PINK1.

a HEK293T WT and SMAD3 knockdown cells were treated with O/A (1/1 μM) for 4 h and harvested for western blotting analysis with the indicated antibodies. b PINK1 mRNA level was quantified using RT-PCR in HEK293T SMAD3 knockdown cells followed with or without O/A (1/1 μM) induction for 4 h. c Schematic presentation of SMAD3 and PINK1 binding sites. The scheme represents the PINK1 promoter construct lacking different SMAD3 response elements (ARE1, ARE2, ARE3). PINK1-ARE-0 is shown as a negative control. d Different PINK1 promoter constructs (PINK1-ARE-1-2-3, PINK1-ARE-2-3, PINK1-ARE-3) were co-transfected in HEK293T cells with the pRL-TK reporter gene (normalize transfection efficiencies) for 24 h. Data are expressed as a percentage of firefly luciferase activity (promoter constructs-transfected cells)/Renilla luciferase activity (pRL-TK-transfected cells) by Dual-Glo luciferase assay system and presented as the means ± SD of 3 independent experiments performed in triplicates. e ChIP analysis of SMAD3 binding to the PINK1 locus in HEK293T cells. GAPDH was used as a negative control. f Different PINK1 promoter construct (PINK1-ARE-1-2-3, PINK1-ARE-2-3, PINK1-ARE-3) were co-transfected in HEK293T cells or HEK293T SMAD3 knockdown cell with the pRL-TK reporter gene (normalize transfection efficiencies) for 24 h. Data are expressed as a percentage of firefly luciferase activity (promoter constructs-transfected cells)/Renilla luciferase activity (pRL-TK-transfected cells) by Dual-Glo luciferase assay system and presented as the means ± SD of 3 independent experiments performed in triplicates. g HeLa WT cells were treated with OA (1/1 μM) for 4 h and analyzed by confocal microscopy followed by immune-staining with SMAD3 (red) and DAPI. Scale bars, 10 μm. h The colocalization of SMAD3 with DAPI in g was quantified by Pearson’s correlation overlapping coefficient. i Subcellular fractionation was performed to isolate the cytosolic and nuclear fractions in HeLa WT cells treated with O/A (1 μM) for 4 h. The samples were then analyzed by western blotting with the indicated antibodies. Lamin A/C and GAPDH were used as nuclear and cytosolic markers, respectively. j SMAD3 in nucleus was quantified by normalizing to that in cytosol of cells treated as in i. Data shown represent the means ± SD of three biological replicates, ***P < 0.001, ****P < 0.0001; significance was determined by one-way ANOVA test followed by Tukey’s correction (b, d, e, f, h, j).