Additional file 1: of Pirfenidone as salvage treatment for refractory bleomycin-induced lung injury: a case report of seminoma

Summary of the treatment experience of BILI with novel therapeutic regimens. PubMed search was conducted in June 2017 with query terms “bleomycin lung toxicity” and “bleomycin lung injury” and limited to clinical studies and case reports, which identified 29 literatures for the recent 15 years. Within them, only 3 reports fulfilled the following criteria; 1) containing treatment information and 2) using pharmacological intervention other than medium-dose corticosteroids for treating BILI. These cases along with our current case were summarized in supplemental table. (PDF 122 kb

Mutation of phosphorylation sites in the PTEN C-terminus modulates TGFβ-induced cell proliferation in H358 cells.

<p>(<b>A</b>) Cell proliferation was measured by WST assay for H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A in the presence of Dox. The experiment was repeated three times with similar results. (<b>B</b>) Cell proliferation was also measured for H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A treated with TGFβ in the presence of Dox. The experiment was repeated three times with similar results. N.S. indicates “not significant”. *: p<0.05 .</p

TGFβ modulates the phosphorylation levels of the PTEN C-terminus in PTEN expression in H358 cells, followed by EMT and aberrant cell motility.

<p>(<b>A</b>) H358 cells treated with vehicle or TGFβ were harvested for the analysis of fibronectin and E-cadherin. The relative expression of fibronectin to E-cadherin (F/E ratio) is shown in comparison to that in cells treated with vehicle. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 (<b>B</b>) A migration assay was performed for the H358 cell line treated with vehicle or TGFβ. Data shown represent the means ± SD. The experiment was repeated three times with similar results. *: p<0.05 The fluorescence intensities of β-catenin (red) in the treated cells were evaluated by using confocal laser scanning microscopy and imaging software. Nuclear staining was performed by Hoechst33342 (blue). The left image in (<b>C</b>) shows cells with no TGFβ stimulation. The right image in (<b>C</b>) shows cells stimulated with TGFβ. The cells incubated with isotype-matched control IgG is shown in the inset in (<b>C</b>). The upper panel in (<b>D</b>) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells with no TGFβ stimulation. The lower panel in (<b>D</b>) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of the cells stimulated with TGFβ. These figures are representative of at least three independent experiments. (<b>E</b>, <b>F</b>, and <b>G</b>) Cell extracts were harvested at the indicated periods after treatment with TGFβ for analysis of the levels of total and phosphorylated smad2 (<b>E</b>), Akt473 (<b>F</b>), Akt308 (<b>F</b>), and FAK (<b>G</b>). Results are shown for H358 naïve cells at 0 minutes (lane <b>1</b>), 5minutes (lane <b>2</b>), 20minutes (lane <b>3</b>), 1hour (lane <b>4</b>), 3hours (lane <b>5</b>), 6hours (lane <b>6</b>), 24hours (lane <b>7</b>), and 48hours (lane <b>8</b>) after treatment with TGFβ (left in E, F, and G). The ratio of phosphorylated protein to total protein is presented as the intensity level relative to that of H358 naïve cells at 0 minutes (lane <b>1</b>) after treatment with TGFβ (right in E, F, and G). Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 (<b>H</b>) Cells treated with vehicle or TGFβ for 0 minutes or 24hours were harvested for the analysis of phosphorylated PTEN (pPTEN) and total PTEN. The relative expression of pPTEN to total PTEN (pPTEN/PTEN ratio) is shown in comparison to that in the cells treated with vehicle for 0 minutes. A representative blot from three independent experiments is shown. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”. (<b>I</b>) H358 naïve cells were incubated with vehicle or SB 431542 at 10 μM for one hour before TGFβ treatment. pPTEN/PTEN ratio is shown in comparison to that in cells treated with vehicle. A representative blot from three independent experiments is shown. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”.</p

Mutation of phosphorylation sites in the PTEN C-terminus represses tumor growth in H358 cells in vivo.

<p>3x10⁶ H358ON cells expressing Dox-dependent GFP, GFPPTENWt, or GFPPTEN4A, were inoculated subcutaneously into the flank of 6-week-old female nude mice, which were then maintained on water containing Dox at a final concentration of 2mg/ml and autoclaved feed ad libitum. Tumor size was monitored for 4weeks in Dox-treated nude mice. (<b>A</b>) A representative example of nude mice with growing tumors at 4weeks after inoculation is shown (H358ON expressing GFP in <b>top</b>, and GFPPTENWt in <b>middle</b>, and GFPPTEN4A in <b>bottom</b>). Tumors were observed within the black circle. (<b>B</b>) The tumor growth rate of each cell was evaluated for 4 weeks after inoculation. *: p<0.05 Data shown represent the means ± SEM of three independent experiments. Each experiment used 5 nude mice for GFPPTENWT, 7 nude mice for GFP, and 7 nude mice for GFPPTEN4A. </p

Mutation of phosphorylation sites in the PTEN C-terminus blocks TGFβ-induced EMT and aberrance cell motility in H358 cells.

<p>(<b>A</b>) H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A were incubated with vehicle or Dox for 24hours before TGFβ treatment. The cells were then treated with vehicle or TGFβ for a further 24hours in the absence or presence of Dox. The cells were harvested for the analysis of pPTEN (<b>top</b> panel), total PTEN (<b>middle</b> panel) and β-actin (<b>bottom</b> panel) by western blotting. A representative blot from three independent experiments is shown. (<b>B</b>) By using confocal laser scanning microscopy, the localization of GFP fluorescence in H358ON cells expressing Dox-treated GFP (<b>left panel</b>), GFP-PTENWt (<b>middle panel</b>) and GFP-PTEN4A (<b>right panel</b>) was evaluated. (<b>C</b>) The intensity levels of GFP fluorescence in both the cytoplasm and the nucleus were also quantified, by Imaging software. The fluorescence intensity was expressed as the nucleus/cytoplasm ratio for each sample. Data shown represent the means ± SEM from three independent experiments. *: p<0.05 N.S. indicates “not significant”. (<b>D</b>) H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A were treated with vehicle or TGFβ for 48hours in the absence or presence of Dox, and then harvested for the analysis of fibronectin, E-cadherin, and β-actin by western blotting. The F/E ratio is shown in comparison to that in cells treated with vehicle in the absence of Dox. A representative blot from three independent experiments is shown. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”. (<b>E</b>) A migration assay was performed for H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A in the absence or presence of Dox and/or TGFβ stimulation. Data shown represent the means ± SD. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”.</p

A FAK inhibitor targeting Tyr397 blocks TGFβ-induced aberrant cell motility, but not TGFβ-induced EMT in H358 cells.

<p>To examine the role of FAK phosphorylation at Tyr397 on TGFβ-induced EMT, Dox-treated H358ON cells expressing Dox-dependent GFP were incubated with vehicle or FAK inhibitor 14 for 24hours before TGFβ treatment. (<b>A</b>) Cell extracts were harvested 24 hours after treatment with TGFβ for analysis of the levels of total and phosphorylated FAK. Dox-treated H358ON cells expressing Dox-dependent GFP were treated with vehicle (lane <b>1</b>) or TGFβ (lane <b>2</b>, <b>3</b>, <b>4</b>, and <b>5</b>). The cells were also incubated with vehicle (lane <b>1</b> and <b>2</b>), or FAK inhibitor 14 at 0.1 nM (lane <b>3</b>), 1 nM (lane <b>4</b>), and 5 nM (lane <b>5</b>) (<b>top</b> in <b>A</b>). The ratio of phosphorylated protein to total protein is presented as the intensity level relative to that in Dox-treated H358ON cells expressing Dox-dependent GFP treated with vehicle (<b>bottom</b> in <b>A</b>). A representative blot from three independent experiments is shown. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 (<b>B</b>) Dox-treated H358ON cells expressing Dox-dependent GFP were treated with vehicle or TGFβ for 48hours in the absence or presence of FAK inhibitor 14 at 5nM, and then harvested for the analysis of fibronectin, E-cadherin, and β-actin by western blotting. The F/E ratio is shown in comparison to that in cells treated with vehicle (<b>bottom</b> in <b>B</b>). A representative blot from three independent experiments is shown. Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”. (<b>C</b>) A migration assay was performed for Dox-treated H358ON cells expressing Dox-dependent GFP treated with vehicle or TGFβ for 48hours in the absence or presence of FAK inhibitor 14 at 5nM. Data shown represent the means ± SD. The experiment was repeated three times with similar results. *: p<0.05 To evaluate the effect of FAK inhibitor 14 on localization of β-catenin in Dox-treated H358ON cells expressing Dox-dependent GFP treated with vehicle or TGFβ, the intensities of fluorescence of β-catenin in the cells were evaluated. The cells were treated with vehicle (<b>D</b> and <b>E</b>) or FAK inhibitor at 5nM (<b>F</b> and <b>G</b>). The left image in (<b>D</b> and <b>F</b>) shows cells with no TGFβ stimulation. The right image in (<b>D</b> and <b>F</b>) shows cells stimulated with TGFβ. The cells incubated with isotype-matched control IgG is shown in the inset in (<b>D</b>). Each upper panel in (<b>E</b>) and (<b>G</b>) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells with no TGFβ stimulation. Each lower panel in (<b>E</b>) and (<b>G</b>) plots the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells stimulated with TGFβ. These figures are representative of at least three independent experiments. </p

Mutation of phosphorylation sites in the PTEN C-terminus inhibits TGFβ-induced smad-independent pathways, but not the smad-dependent pathway in H358 cells.

<p>Cell extracts from H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A in the absence or presence of Dox were harvested for analysis of the levels of total and phosphorylated for smad2 (<b>A</b>), Akt473 (<b>B</b>), Akt308 (<b>B</b>), and FAK (<b>C</b>) at the indicated periods after treatment with vehicle or TGFβ (1hour for smad2, 1hour for Akt473, 1hour for Akt308, and 24hours for FAK, respectively). A representative blot from three independent experiments is shown (<b>top</b> in <b>A</b>, B and C). The ratio of phosphorylated protein to total protein is presented as the intensity level relative to that in H358ON cells expressing Dox-dependent GFP treated with vehicle in the absence of Dox (<b>bottom</b> in <b>A</b>, B and C). Data shown represent the means ± SE. The experiment was repeated three times with similar results. *: p<0.05 N.S. indicates “not significant”.</p

Mutation of phosphorylation sites in the PTEN C-terminus inhibits TGFβ-induced EMT via blockade of β-catenin translocation, but not by modulation of EMT-related genes in H358 cells.

<p>The expression levels of EMT-related genes were evaluated in H358 naïve cells treated with vehicle or TGFβ for 24hours. (<b>A</b>) snail mRNA and (<b>B</b>) twist mRNA were analyzed and normalized to GAPDH mRNA, by using real-time PCR. The relative expression of each targeted gene is shown in comparison to that in cells treated with vehicle. Data shown represent the means ± SEM from three independent experiments. *: p<0.05 N.S. indicates “not significant”. (<b>C</b>) H358ON cells expressing Dox-dependent GFP, GFP-PTENWt, or GFP-PTEN4A were incubated with vehicle or Dox for 24hours before TGFβ treatment. The cells were then treated with vehicle or TGFβ for a further 24hours in the absence or presence of Dox. Snail mRNA was analyzed and normalized to GAPDH mRNA, by using real-time PCR. The relative expression of the snail gene is shown in comparison to that in cells treated with vehicle in absence of Dox. Data shown represent the means ± SEM from three independent experiments. The fluorescence intensity of β-catenin in H358ON cells expressing Dox-dependent GFP (<b>D</b>), GFP-PTENWt (<b>F</b>), or GFP-PTEN4A (<b>H</b>) was evaluated. Each left image in (<b>D</b>), (<b>F</b>), and (<b>H</b>) shows cells with no TGFβ stimulation. Each right image in (<b>D</b>), (<b>F</b>), and (<b>H</b>) shows cells stimulated with TGFβ. Each upper panel in (<b>E</b>), (<b>G</b>), and (<b>I</b>) plotted the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells with no TGFβ stimulation. Each lower panel in (<b>E</b>), (<b>G</b>), and (<b>I</b>) plotted the fluorescence intensity of β-catenin (red) and nucleus (blue) over a cross section of cells stimulated with TGFβ. These figures are representative of at least three independent experiments. </p

Ophthalmological findings from a patient with paraneoplastic retinopathy (PR) associated with lung cancer.

<p>(A) Threshold of static visual field (Humphrey, 30-2 program) plotted on a gray scale showing severely decreased sensitivities within the central 30 degrees of the visual field. (B) Fundus photographs of the patient showing a nearly normal fundus. (C) Fluorescein angiograms showing periphlebitis of the retinal vessels (arrows). (D) Spectral-domain optical coherence tomographic (SD-OCT) image of a 9 mm horizontal scan of the retina of our patient. The retinal structure in each retinal layer is normal.</p

Western blot analysis of human TRPM1 using sera from the MAR patients.

<p>(A, B) Immunoblots of the transfected cell lysates using sera from MAR patient #8 (A) and MAR patient #23 (B). HEK293T cells were transfected with pCAGGS or pCAGGS-human TRPM1-3xFlag plasmid, and cells were harvested after 48 hrs. Arrowheads indicate the TRPM1-3xFlag protein bands. β-actin (β-act) was used for a loading control.</p