Autophagy is not increased in IPF.

<p>A) IPF whole lung homogenate demonstrates increased ER stress (elevated XBP1 expression) and increased phosphorylation of AMPK, factors which should drive autophagy. B) LC3-II (lower band) expression in IPF whole lung homogenate is decreased relative to control lung tissue C) Densitometry of Western blots demonstrating LC3-II level is lower in IPF than in control lung (*p = 0.05). D) Increased p62 in IPF lung suggests decreased autophagy. E) Immunofluorescence confocal microscopy of control and IPF lung tissue for p62 (green), aggresome (red), DAPI (blue) demonstrates increased p62 expression and aggresomes. F) Representative electron microscopy images from IPF (panels A, B), control (Panel C), and COPD (panel D); white arrows indicate autophagosomes. G) Quantitation of autophagic vacuoles in control, IPF, and COPD lung by EM demonstrates significantly higher numbers in COPD (*p<0.05 for IPF vs. COPD).</p

Inhibition of autophagy potentiates fibroblast activation.

<p>A) Rapamycin inhibits fibronectin and α-SMA expression in fibroblasts, and abrogates TGF-β₁ suppression of Id-1. B) Beclin and LC3 siRNA effectively inhibit protein expression. C) α-SMA and fibronectin expression increases in fibroblasts with beclin and LC3 silencing whereas collagen is unchanged. D) When LC3 is silenced, Id-1 expression is lower than control. E) Rapamycin does not modulate phosphorylation of Smad3 in response to TGF-β₁.</p

TGF-β₁ activates mTOR and TIGAR.

<p>A) In human lung fibroblasts, TGF-β₁ inhibits LC3-II formation, even in the presence of IFN-γ which induces autophagy. B) TGF-β₁ is unable to inhibit LC3-II formation in presence of mTOR inhibitor rapamycin. C) TGF-β₁ is able to activate mTORC1 which results in increased phospho-S6 but this activation does not occur in the presence of rapamycin. D) TGF-β₁ appears to activate mTORC1 by activating upstream PI3K/AKT and treatment with PI3K inhibitor LY294002 prevents TGF-β₁ induced mTOR activation. E) Western blot of phospho-mTOR (Ser2448) showing increased phospho-mTOR with TGF-β₁ in fibroblasts and inhibition by rapamycin. F) Western blot of phospho-S6 from mouse lung tissue treated with bleomycin and rapamycin. G) Densitometry of blot from 4E. (*p = 0.03 for controls vs. bleomycin, **p = 0.003 for bleomycin vs rapamycin + bleomycin). H) phospho-S6 protein levels in human lung tissue is higher in IPF patients compared with COPD patients and healthy controls. I) TIGAR is induced by TGF-β₁ in fibroblasts in a dose-responsive manner. J) Western blot demonstrating TIGAR protein levels in lung homogenate from human tissue is higher in IPF patients compared with COPD patients and healthy controls.</p

TGF-β₁ inhibits autophagy <i>in vitro</i>.

<p>A) Human lung fibroblasts were cultured and treated with varying concentrations of TGF-β₁. TGF-β₁ inhibited activation of LC3, decreasing the intensity of the lower band on the western blot, and B) increased p62. C) Inhibition of LC3 activation by TGF-β₁ with varying serum conditions. D) Fibroblasts treated with TGF-β₁ for 24 hours and measurement of autophagic flux by LC3 western blotting (using lysosomal acidification inhibitor chloroquine). E) Densitometry of western blot shown in D (p = 0.045). F) Fluorescence microscopy of fibroblasts transfected with GFP-LC3 construct and stimulated with TGF-β₁ showing that TGF-β₁ inhibits formation of LC3 puncta. G) Confocal microscopy of type II alveolar epithelial cells isolated from GFP-LC3 transgenic mice and stimulated with TGF-β₁ showing that TGF-β₁ inhibits formation of LC3 puncta (green = GFP-LC3, red = SP-C, blue = DAPI). H) Quantification of GFP puncta per cell from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041394#pone-0041394-g002" target="_blank">figure 2G</a>.</p

Effects of rapamycin on bleomycin induced fibrosis and autophagy.

<p>Hydroxyproline assay measuring lung collagen content demonstrates co-administration of rapamycin and bleomycin protects against fibrosis (*p = 0.003 for control vs. bleomycin; **p = 0.008 for bleomycin vs. rapamycin + bleomycin).</p

TGF-β1 induces mitochondrial depolarization.

<p>A) Beas-2B cells stimulated with TGF-β1 and CCCP (positive controls) were stained with JC-1 for 15 min and analyzed by flow cytometric analysis. TGF-β1 treated cells showed decreased red fluorescence (mitochondrial depolarization). B) Quantification of relative MFI for experiment in A (p = 0.06). C) Beas-2B cells stimulated with TGF-β1 were stained with MitoSOX for 10 min and analyzed by flow cytometry. TGF-β1 treated cells showed increased red fluorescence (mitochondrial ROS production) and mitochondria-specific antioxidant (MitoTEMPO) reversed the effect of TGF-β1. D) Western blot of the mitochondrial fraction of Beas-2B cells stimulated with TGF-β1 (5ng/mL) +/- MitoTEMPO (200μM) showing decreased PINK1 expression in presence of MitoTEMPO.</p

Loss of PINK1 aggravates bleomycin induced lung fibrosis.

<p><i>Pink1</i>^-/- mice and their littermate controls (male, 8–10 weeks old) were treated with 3U/kg of intratracheal bleomycin sulfate or saline on day 1 and sacrificed on Day 21. <i>Pink1</i>^-/- mice in the bleomycin group showed higher levels of hydroxyproline compared to controls (85.4 μg/mL vs. 97.0 μg/mL; *p = .05, **p<0.001).</p

PINK1 Expression and Mitochondrial Dysfunction in IPF Lung Tissue.

<p>A) Western blot of mitochondrial fraction of PINK1 in human lung tissue from control and IPF patients showing increased PINK1 levels in IPF samples. B) Densitometry of blot in A. C) mRNA expression of Pink1 in human lung tissue from control (n = 5) and IPF (n = 5) samples. D) Representative transmission electron micrographs from control and IPF lung tissue showing mitochondria (*). Magnif = 18500x; scale bars = 200nm. E) Quantification of total and abnormal mitochondria. Total mitochondria per μm² in control and IPF was 0.52 ± 0.39 vs. 0.46 ± 0.34. Abnormal mitochondria per μm² in control and IPF was 0.12 ± 0.19 vs. 0.32 ± 0.32, <i>p</i> = 0.003. F) Confocal immunofluorescence against PINK1(red) and LC3 (green) in control and IPF lung (magnification 10x). G) Magnified view (60x) of confocal immunofluoresence with DAPI (blue) and PINK1 (red) in control and IPF lung.</p

TGF-β1 increases PINK1 expression and induces mitochondrial fission <i>in vitro</i>.

<p>A) TGF-β1 induced PINK1 expression of Beas-2B cells in a time- and dose-dependent manner. B) Quantification by densitometry of PINK1 expression in Beas-2B cells stimulated with TGF-β1 for 6 hours. C) Confocal microscopy of Beas-2B cells stimulated with TGF-β1 (6h) showed that TGF-β1 induces formation of PINK1 puncta (red) (magnification 120x). D) Quantification of colocalization of LC3 and PINK1 punctae (* p = 0.035, ** p = 0.001). E) Confocal microscopy of Beas-2B cells transfected with vectors staining mitochondria (green) and lysosomes (red) and with TGF-β1 stimulation for 6 hours (magnification 60x). F) Western blot of Beas-2B cells stimulated with TGF-β1 (6hrs) showed increased expression of pDRP1 (ser616). G) Confocal microscopy of Beas-2B cells stimulated with TGF-β1 (5 ng/mL, 24 hrs) showed more fragmentation (fission) of mitochondria stained with MitoTracker Green (magnification 120x).</p

Loss of PINK1 augments TGF-β1 induced cell death.

<p>A) Measurement of mitochondrial ROS in murine type II alveolar epithelial cells from <i>Pink1</i>^-/- and <i>Pink1</i>^WT mice by flow cytometry and MitoSOX staining. B) Western blot against PINK1 demonstrated knockdown of expression with PINK1 siRNA. C) Beas-2B cells were treated with siRNA (50 nM, 24hrs). Then, they were stimulated with TGF-β1 (5 ng/mL, 24hrs) and analyzed by Annexin V/PI flow cytometry. D) Quantification of cell death from experiment in C (*p = 0.0005, **p = 0.0119, ***p = 0.032). Loss of PINK1 exaggerated cell death in cells treated with TGF-β1 relative to transfection with control siRNA.</p