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

Effects of PDGF on Intracellular Ca²⁺ Concentrations.

<p>Effects of PDGF-BB on [Ca²⁺]_i. Representative changes in the F₃₄₀/F₃₈₀ ratio with 10 ng/mL PDGF-BB in the normal solution (<b>A</b>) or in the nominally Ca²⁺-free solution (<b>B</b>). <b>C</b>: The peak F₃₄₀/F₃₈₀ ratios in response to PDGF-BB in the normal solution (control) or the nominally Ca²⁺-free solution. Bars represent the means ± SD (n = 6). *Significantly different from the baseline values without PDGF-BB treatment (P<0.05). <b>D</b>: The sustained F₃₄₀/F₃₈₀ ratios (plateau phases) in response to PDGF-BB in the normal solution (control) or the nominally Ca²⁺-free solution. Effects of EGTA (2 mM) on sustained increase in the F₃₄₀/F₃₈₀ ratio with 10 ng/mL PDGF-BB are also shown. Bars represent the means ± SD (n = 6). *Significantly different from the values with PDGF-BB alone (P<0.05).</p

Roles of STIM1 and Orai1 in Cell Migration Induced by PDGF.

<p>Roles of STIM1 and Orai1 in cell migration induced by PDGF-BB (10 ng/mL, 6 h) are shown. Cell migration was assessed by a chemotaxis assay. <b>A</b>: Effects of PDGF-BB on migrated cell numbers with or without (control) 1 mM EGTA treatment (n = 6). Baseline (black column) denotes the time-matched number of cells that migrated without PDGF-BB treatment. Significantly different from the values of the baseline (*) and by PDGF-BB alone (#) (P<0.05). <b>B</b>: Effects of siRNA treatment targeting STIM1, STIM2, Orai1, and both STIM1 and Orai1 on migrating cell numbers induced by PDGF-BB (n = 6). *Significantly different from the values of the time-matched control cells treated with PDGF-BB plus scrambled siRNA (negative control, NC) (P<0.05). Bars represent means ± SD.</p

Expression of STIM1, STIM2, and Orai1.

<p><b>A</b>: Expression of STIM1, STIM2, Orai1, and GAPDH mRNAs detected by RT-PCR in human airway smooth muscle (ASM) cells is shown. Negative indicates a negative control. The product sizes for STIM1, STIM2, Orai1, and GAPDH were 481bp, 498bp, 483bp, and 498bp, respectively. <b>B</b>: Effects of siRNA-targeted knockdown of STIM1, STIM2, and Orai1 mRNAs on the change in mRNA expression over control normalized to the reference gene GAPDH are shown (n = 4). Changes in mRNA expression were assessed by quantitative real-time PCR. Effects of siRNA transfection targeting STIM1 (siSTIM1) (<b>C</b>), STIM2 (siSTIM2) (<b>D</b>), and Orai1 (siOrai1) (<b>E</b>) on changes in protein levels were assessed by Western blot. STIM1, STIM2, and Orai1 protein levels expressed as the target protein/actin ratio in the cells transfected with siSTIM1 (<b>C</b>), siSTIM2 (<b>D</b>), or siOrai1 (<b>E</b>) and scrambled siRNA (negative control) are compared (n = 3). The control value without siRNA transfection is defined as 100%. *Significantly different from the values of the scrambled siRNA condition (P<0.05). Bars represent means ± SD.</p

Role of STIM1 in PDGF-Induced Intracellular Ca²⁺ Elevation.

<p>Roles of STIM1 and Orai1 in [Ca²⁺]_i elevation induced by PDGF-BB. Representative changes in the F₃₄₀/F₃₈₀ ratios with 10 ng/mL PDGF-BB in the cells transfected with siSTIM1 (<b>A</b>) and siOrai1 (<b>B</b>), and siSTIM2 (<b>C</b>) are shown. Transient (peak) (<b>D</b>) and sustained increases (plateau phase) (<b>E</b>) in the F₃₄₀/F₃₈₀ ratio in response to PDGF-BB with or without (control) siRNA treatment are compared. Bars represent the means ± SD (n = 6). *Significantly different from the values of the control cells treated with 10 ng/mL PDGF-BB plus scrambled siRNA (P<0.05).</p

Role of STIM1 and Orai1 in Store-Operated Ca²⁺ Entry.

<p>Roles of STIM1 and Orai1 in SOCE. Representative changes in the F₃₄₀/F₃₈₀ ratio due to 5 μM thapsigargin (TPG) in cells transfected with siRNA targeting STIM1 (<b>A</b>) and Orai1 (<b>B</b>). After the cells were treated with thapsigargin in the nominally Ca²⁺-free solution, 2 mM Ca²⁺ was added to the solution. <b>C</b>: The F₃₄₀/F₃₈₀ ratios in response to 5 μM thapsigargin in the normal solution due to SOCE with or without (control) siRNA treatment. The cells transfected with scrambled siRNA, siSTIM1, siOrail, both siSTIM1 and Orai1, or siSTIM1. Bars represent the means ± SD (n = 6). *Significantly different from the values of the cells transfected with scrambled siRNA (P<0.05).</p

Store-Operated Ca²⁺ Entry Induced by Thapsigargin.

<p>Store-operated Ca²⁺ entry (SOCE) activated by thapsigargin. <b>A</b>: Representative traces of the F₃₄₀/F₃₈₀ ratio, a measure of intracellular Ca²⁺ concentrations ([Ca²⁺]_i), by 5 μM thapsigargin (TPG). After the cells were treated with 5 μM thapsigargin in the nominally Ca²⁺-free solution, 2 mM Ca²⁺ was added to the solution. At the end, 2 mM EGTA was added. <b>B</b>: The F₃₄₀/F₃₈₀ ratios in nominally Ca²⁺-free solution (Ca²⁺-free), in response to 5 μM thapsigargin in the Ca²⁺-free solution due to Ca²⁺ release, in the normal solution containing 2 mM Ca²⁺ with thapsigargin due to SOCE (the plateau phase), and in the normal solution with thapsigargin and 2 mM EGTA (SOCE+EGTA). Bars represent the means ± SD (n = 6). Significantly different from values in the Ca²⁺-free solution (*) and of SOCE (#) (P<0.05).</p

Fluorescent images of organization of F-actin and microtubules in the static or stretched cells.

<p>Cells were in the static condition (<i>upper panels</i>) or subjected to 2 h uniaxial cyclic stretch (<i>lower panels</i>). F-actin was visualized with rhodamine-phalloidin (red). Microtubules were visualized with FITC conjugated secondary antibody following immunostaining with anti-α-tubulin antibody (green). Cell nuclei were stained with DAPI (cyan).</p

Effects of nocodazole and paclitaxel on the organization of F-actin and microtubules.

<p>Fluorescent images of the organization of F-actin stained with rhodamine-phalloidin (red) and microtubules immunostained with anti-α-tubulin antibody (green) in the static or stretched cells. Cell nuclei were stained with DAPI (cyan). The cells were pretreated with either 1 µM nocodazole (NDZ; <b>A</b>) or 1 µM paclitaxel (PTX; <b>B</b>). Arrows indicate stretch direction.</p

Schematic of how the angle of orientation (θ) of the long axis was measured.

<p>The angle is always between 0° and 90° with respect to the stretch axis (arrows).</p