Fig. 7: Mitochondrial dysfunction augments TGFβ-ALK5-SMAD2 signaling.

HUVECs were transfected with control, Tfam, Cox10, or Trx2 siRNAs for 48 h. a Cell lysates were subjected to Western blotting with respective antibodies. Protein levels were quantified and presented as fold changes by taking control siRNA as 1.0. n = 3 (three independent experiments). b mRNA expression of Alk5, Tgfbr2, Smad1, and Smad2 was determined by qRT-PCR with specific primers. Relative mRNA levels were quantified and presented as fold changes by taking control siRNA as 1.0. n = 3 (three independent experiments). c Cells were treated with cycloheximide (10 μg/ml) for indicated times. Cell lysates were subjected to Western blotting with respective antibodies. Protein levels were quantified and presented as fold changes by taking control siRNA as 1.0. n = 3 (three independent experiments). d, e Cells were subjected to indirect immunofluorescence staining for SMAD2 or phosphor-SMAD2 and mitochondrial marker TOM20. Arrowheads and arrows (d) indicate SMAD2^- and SMAD2⁺ nuclei, respectively. % nuclear SMAD2 was quantified (e). f Arrowheads and arrows indicate phosphor-SMAD2^- and phosphor-SMAD2⁺ mitochondria, respectively. % phosphor-SMAD2⁺ mitochondria were quantified (g). n = 6 (duplicates from three independent experiments; 10 cells were counted for each biological sample). h, i Immuno-EM. HUVECs were transfected with control or Tfam siRNAs for 48 h and subjected to immunogold electron microscopy for TFAM and phosphor-SMAD2. Arrows and arrowheads indicate immunogold particles (5 nm) outside and inside mitochondria, respectively. %phosphor-SMAD2⁺ mitochondria were quantified (h). 10 EM section per sample. n = 3 per group. j–l ECs were pre-treated with TGFβR1 inhibitors (5 μM each) for 60 min followed by treatment with TGF-β (10 ng/ml) for 30 min. l ECs were pre-treated with LY3214996 (ERK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor) or GS444217 (ASK1 inhibitor) (5 μM each) for 2 h. Cell lysates were subjected to Western blotting with respective antibodies. Protein levels were quantified and presented as fold changes by taking control siRNA as 1.0. Ratios of p-SMAD2/SMAD2 and p-Akt/Akt are also presented. n = 3 (three independent experiments). Data are means ± SEM. P values are indicated, using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 50 μm (d); 10 μm (f); 150 nm (h). Source data are provided as a Source Data file.