Fig 1: COX-2 in TAMs induces MMP-9 expression in breast cancer cells. (A) Template showing the location of the MMP/TIMP antibodies spotted onto the RayBio Human MMP Array Kit. POS: positive; NEG: negative. (B) TAMs-induced modulation of MMP/TIMP proteins in MCF-7 cells. After MCF-7 cells were co-cultured with or without TAMs for 7 days, the cell lysate was applied to the antibody array. The pixel density was measured, and the data are presented as ratios (compared to the Alone). Protein exhibiting a ratio>2 is indicated with black box. (C) MMP-9 expression in MCF-7 and MDA-MB-468 cells co-cultured with or without (Alone) TAMs for 7 days was assessed by Western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (D) MMP-9 expression in MCF-7 and MDA-MB-468 cells co-cultured with or without (Alone) TAMs transfected with adenoviral COX-2 or siRNA COX-2 for 7 days was assessed by Western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (E) MMP-9 activity in supernatant collected from different cancer cells was detected using an active human MMP-9 fluorescence assay. The experiments were performed thrice in triplicate. The data are presented as the mean ± SD. **p < 0.01 (versus Alone group).
Fig 2: Effects of VG e-cig aerosols in a large animal model (sheep). (A) Five-day exposure of sheep to 100% VG e-cig aerosols causes a significant increase in mucus concentrations (measured as % mucus solids from tracheal secretions) after five days. n = 6 from 2 sheep for baseline, n = 9 from 3 sheep for Day 5. (B) Five-day exposure of sheep to VG aerosols causes a significant increase in MMP-9 activity measured from tracheal secretions. n = 6 from 2 sheep for baseline, n = 9 from 3 sheep for Day 5. (C–E) Five-day exposure of sheep to VG e-cig aerosols does not cause a significant change in plasma levels of IL-6 (C) or IL-8 (D) proteins but causes a significant increase in the expression of TGF-β1 protein (E) in plasma. n = 6 from 2 sheep (C) or n = 9 from 3 sheep (D,E). Statistics: Data are presented as mean ± SEM. *p < 0.05, ns = not significant. Data were analyzed using a mixed-effects model.
Fig 3: MMP-9, MPO and HNE levels in early pediatric ARDS.A. Zymogram (7.5% SDS non-reduced gel) demonstrates robust gelatinase activity in tracheal aspirates from ARDS subjects. Each lane represents a sample obtained from separate ARDS subjects at 48 hours of disease onset and intubation. The higher molecular weight band at 135 kDa (black arrow) likely represents lipocalin:MMP-9 complexes, while the 82 KDa band (white arrow) corresponds with active MMP-9 isoforms. B. Endogenously active MMP-9 levels measured in tracheal aspirates of ARDS subjects at 48 hours of disease onset and intubation were elevated (16-fold) compared with control subjects (*p = 0.001). Boxes represent 25th to 75th percentile range, with the midline indicating the median; bars represent the highest and lowest values. C. MPO concentrations were elevated (7-fold) in ARDS subjects compared to controls at 48 hours of intubation (*p<0.001). Boxes represent 25th to 75th percentile range, with the midline indicating the median; bars represent the highest and lowest values. D. HNE concentrations were elevated (3-fold) in ARDS subjects compared to controls at 48 hours of intubation (*p = 0.25). Boxes represent 25th to 75th percentile range, with the midline indicating the median; bars represent the highest and lowest values.
Fig 4: Levels of MMP-9 activity and MUC5AC protein in nasal epithelial lining fluid (ELF) from volunteers who vaped VG e-liquid. (A,B) Before-after plots of levels of MMP-9 activity (A) and MUC5AC protein (B) measured from nasal ELF at baseline (BL) and day 8 from volunteers who vaped VG. n = 3.
Fig 5: Fully re-differentiated human bronchial epithelial cells (P1 HBECs) from nonsmokers and smokers. a) Quantitative mRNA expression of MUC5AC, transforming growth factor (TGF)-β1 and matrix metalloproteinase (MMP)-9 of fully re-differentiated P1 HBECs from nonsmokers and smokers. Data are shown as relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and nonsmokers (n≥4 donors for each group). b) Representative confocal images of immunofluorescent staining of P1 HBECs from a nonsmoker (upper panels) and a smoker (lower panels). MUC5AC is stained in red, nuclei were stained with Hoechst and represented in blue. Scale bar=50 μm. c) Quantification of MUC5AC-positive cells relative to nuclei (n=8 from ≥3 donors). d) Cystic fibrosis transmembrane conductance regulator (CFTR) currents measured by short circuit current changes upon CFTRinh172 (10 µM) application after 10 µM forskolin stimulation 4 h after exposure to room air or cigarette smoke (represented as ΔIsc upon CFTRinh172; thus, decreases indicate enhanced CFTR function) (n≥9 donors for each group). e) Voltage-dependent potassium (BK) currents measured upon ATP stimulation and represented as ΔIsc (n≥11 donors for each group) (decreases indicate enhanced BK function). f) Airway surface liquid (ASL) volumes measured 4–6 weeks after establishment of air–liquid interface (ALI) and represented as baselines (n=12 donors for each group). g) Mucus concentration depicted as percentage mucus solids measured 4–6 weeks after establishment of ALI and represented as baselines (n≥4 donors for each group). None of the comparisons were significant by t-test after passing Shapiro–Wilk normality test.
Supplier Page from R&D Systems, a Bio-Techne Brand for Human Active MMP-9 Fluorokine E Kit