Fig 1.
PIK-III attenuates ECM production and myofibroblast differentiation in activated HDFs.
A) HDFs were stimulated with TGF-β1 (10 ng/ml) in the presence or absence of PIK-III (2.5, 5 and 10 μM). 24 hours later, cells were lysed to conduct Western blot to measure FN (FN1), Col1 (COL1A1/COL1A2) and αSMA (ACTA2) expression (n = 3). B) Cells were mixed with collagen 1 solution as described in Methods and treated with TGF-β1 (10 ng/ml) in the presence or absence of PIK-III (5 μM). Gel size was measured at 0 and 24 h after collagen gelation (n = 3 for each condition). C) Cells were treated as described in (A). After treatment, cells were lysed for total RNA isolation. mRNA levels of collagen1a1 (COL1A1), fibronectin1 (FN1) and αSMA (ACTA2) were measured by qRT-PCR (n = 3 for each condition). Data are mean ± SEM. P < 0.05; significant comparisons by one-way ANOVA:* vs. unstimulated, † vs. TGF-β1 alone.
Fig 2.
PIK-III attenuates the phosphorylation of p38 in activated HDFs.
A) HDFs were stimulated with TGF-β1 (10 ng/ml) in the presence or absence of PIK-III (5 μM). 24 hours later, cells were lysed for Western blot to measure phospho-p38 (Thr180/Tyr182) (n = 3). B) HDFs were stimulated with TGF-β1 (10 ng/ml) in the presence or absence of SB203580 (10 μM). 24 hours later, cells were lysed for Western blot to measure Fibronectin, Type I Collagenand αSMA expression (n = 3). E) HDFs were lentivirally transfected with pLenti-C-Myc-DDK (empty vector, EV) or pLenti-p38-Myc-DDK (p38-DDK). Then, cells were stimulated with TGF-β1 (10 ng/ml) in the presence or absence of PIK-III (5 μM). 24 hours later, cells were lysed for Western blot. Data are mean ± SEM. P < 0.05; significant comparisons by one-way ANOVA:* vs. unstimulated, † vs. TGF-β1 alone.
Fig 3.
PIK-III inhibits dermal fibrosis in mice.
Mice were subjected to Bleo-induced dermal fibrosis with or without treatment with PIK-III (5 mg/kg) as described in Methods. A and B) 28 days after first Bleo administration, 6 mm skin biopsies were harvested, fixed and subjected to Trichrome and H&E staining. Dermal thickness was measured in H&E stained skin samples using ImageJ software (n = 4 or 6). C) Hydroxyproline content was measured in the 6 mm punch skin biopsies of Vehicle (n = 8) and PIK-III treated mice (n = 8) exposed to Bleo (gray bars) and Vehicle treated mice exposed to PBS (n = 5) (white bar). D) Gene expression of Fn1 and Col1a1, and Acta2 in lesional skin samples was measured using qRT-PCR (n = 4 or 6). E) Skin samples were homogenized and lysed for western blot to measure phospho-p38 and total p38 (n = 3 or 5). Data are mean ± SEM. P < 0.05; significant comparisons by one-way ANOVA:* vs. Vehicle/PBS, † vs. Bleo alone.
Fig 4.
PIK-III inhibits lung fibroblast activation and pulmonary fibrosis.
A) HLFs were stimulated with TGF-β1 (10 ng/ml) in the presence or absence of PIK-III (2.5, 5 and 10 μM). 24 hours later, cells were lysed for Western blot to measure fibronectin (FN1), Collagen 1 (COL1A1/COL1A2) and αSMA (ACTA2) expression (n = 3). B) HLFs were mixed with collagen 1 solution as described in Methods and treated with TGF-β1 (10 ng/ml) in the presence or absence of PIK-III (5 μM). Gel size was measured at 0 and 24 h after collagen gelation (n = 3). C and D) Mice were subjected to Bleo-induced lung fibrosis with or without treatment with PIK-III (5 mg/kg) as described in Methods. 21 days after Bleo administration, lungs were harvested and subjected to Trichrome stain (C) and hydroxyproline assay (D) (n = 5–9). E) Lung samples were homogenized and lysed for western blot to measure phospho-p38 and total p38 (n = 3 or 5). Data are mean ± SEM. P < 0.05; significant comparisons by one-way ANOVA:* vs. unstimulated or Vehicle/PBS, † vs. TGF-β1 or Bleo alone.