Additional file 1: of The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

Figure S1. Immunohistochemistry staining of CSE, CBS and MPST in bronchial biopsies from non-smokers, smokers and COPD patients. Photomicrographs showing representative photomicrographs of cystathionine-γ-lysase (CSE), cystathionine-β-synthase (CBS) and 3-mercaptopyruvate sulphur transferase (MPST) staining in the bronchial mucosa from control non-smokers, control smokers with normal lung function and mild/moderate COPD patients. Immune-stained airway smooth muscle cells are indicated by brown staining. Results are representative of those from 13 non-smokers, 14 smokers with normal lung function, 15 mild/moderate COPD patients. Calibration bar represents 20 μm. Graphical representation of the results are shown in the right hand panels. (JPG 332 kb

Additional file 2: of The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

Figure S2. Further examples of the effect of the H2S donor, NaSH on CBS and MPST protein expression, and activation of extracellular signal–regulated kinase (ERK)–1/2 and p38 mitogen-activated protein kinase (MAPK) in human ASM cells from non-smokers, smokers and COPD patients. ASM cells were incubated with FCS (2.5%) for 1 h and NaSH (100 μM) was added for another 24 h. CBS, MPST (A), Total and phospho–ERK-1/2, total and phospho-p38 and β-actin (B) were detected by Western blotting. (JPG 212 kb

Photomicrographs showing the parietal pleura (A, B) and positive control tonsil (C) immunostained for identification of FOXP3+ cells (A–C).

<p>FOXP3 positive cells are stained brown. Photomicrograph D shows the negative control slide of tonsil immunostained using normal nonspecific immunoglobulins. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.</p

Photomicrographs showing the parietal pleura (A and B) immunostained for GATA-3+ cells or the parietal pleural (C and D) double immunostained for GATA-3+ cells and tryptase+ cells.

<p>GATA-3 is stained in red in A and B and brown in C and D. Tryptase is stained in red. Results are representative of those from 14 patients with PLTB (A and C) and 12 patients with NSP and (B and D). Original magnification: 400×. The scale bar represents 50 µm.</p

Summary of the double staining immunohistochemical procedures.

<p>DAB: 3,3′-diaminobenzidine tetrahydrochloride.</p

Photomicrographs showing the parietal pleura (A, B) and positive control nasal polyp (C) immunostained for CRTH2 (A–C).

<p>CRTH2+ cells are stained in red. Photomicrograph D shows negative control slide of nasal polyp immunostained using normal nonspecific immunoglobulins. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.</p

Photomicrographs showing the parietal pleura (A, B) and control tonsil (C and D) immunostained for identification of CD68+ cells (A–C).

<p>Photomicrograph D shows negative control slide of tonsils stained with normal nonspecific immunoglobulins of the same animal species of the primary antibody used at the same protein concentration as the primary antibody. CD68+ cells are stained in brown. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.</p

Summary of the immunohistochemical procedures used to detect the Th1, Th2 and Tregs cells.

<p>Th1: T-helper type 1; Th2: T-helper type 2; Tregs: regulatory T cells; CCR: chemokine CC motif, receptor; CXC: chemokine CXC motif, receptor; T-bet: T box expressed in T cells; CRTH2: chemoattractant receptor-homologous molecule expressed on Th2; STAT: signal transducer and activator of transcription; GATA3: GATA motif binding protein 3; FOXP3: Forkhead box protein 3. AEC: 3-amino-9-ethylcarbazole; DAB: 3,3′-diaminobenzidine tetrahydrochloride.</p

Photomicrographs showing the parietal pleura (A, B) and control tonsil (C and D) immunostained for identification of neutrophil elastase+ cells (A–C).

<p>Photomicrograph D shows negative control slide of tonsils stained with normal nonspecific immunoglobulins of the same animal species of the primary antibody used at the same protein concentration as the primary antibody. Neutrophil elastase+ cells are stained in red. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.</p

Photomicrographs showing the parietal pleura (A, B) and positive control tonsil (C) immunostained for identification of STAT4+ cells (A–C).

<p>STAT4, positive cells are stained brown. Photomicrograph D shows the negative control slide of tonsil immunostained using normal nonspecific immunoglobulins. Results are representative of those from 14 patients with PLTB (A) and 12 patients with NSP (B). Original magnification: 400×. The scale bar represents 50 µm.</p