Proline biosynthesis is a vent for TGFβ-induced mitochondrial redox stress (2020)
Figure 6

Figure 6: Mitochondrial citrate export limits proline biosynthesis and collagen production.

(a) NIH-3T3 cells were treated with vehicle control or CTPi at increasing concentrations (0.1 mM, 0.2 mM, 0.5 mM) or TGFβ (2 ng/mL for all experiments) for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to control-treated cells. P-values represent comparison of individual samples to the control.

(b) Left: NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2.

(c) NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells.

(d) Western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 and treated with TGFβ or mock for 48h.

(e) Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.

(f) NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated empty vector expressing cells.

(g) NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.

(h) Western blot of NIH-3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h.

(i) Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated empty vector expressing cells.

(j, k) Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: (j) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827); (k) two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.

(l) Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537.

(m) IPF patients from GSE32537 were assigned into P5CSlow/SLC25A1high and P5CShigh/SLC25A1low groups based on the expression level of P5CS and SLC25A1. "High" represents patients with P5CS or SLC25A1 expression values being above the 75% percentile of the respective gene expression; "low" represents patients with expression values being below the 25% percentile of gene expression. The forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient was compared between the groups.

Data information: P-values were calculated by two-way ANOVA with Holm-Sidak's multiple comparison test (b, c, e-g, i), by one-way ANOVA with Holm-Sidak's multiple comparison test (a), by moderated t-statistics and adjustment for multiple comparisons with the Benjamini & Hochberg false discovery rate method (j, k), by Pearson correlation (l), or by two-sided, unpaired t-test with Welch's correction (m). Bars in (a-c, e-g, i) represent the mean + SD; lines in (j) represent the mean +/- SD; data in (k, m) represent the median with 50% confidence interval box and 95% confidence interval whiskers; line in (l) represents linear regression with the SD shown as dotted lines. n=3 (a-c, e-g, i); n=3 (Saline), n=5 (Bleo) (j); n=11 (Ctrl, left), n=22 (IPF, left), n=50 (Ctrl, right), n=119 (IPF, right) (k); n=169 (l); n=9 (P5CSlow/SLC25A1high), n=8 (P5CShigh/SLC25A1low) (m). A representative experiment is shown (b, d, h).