C3f in human serum samples.

<p>(A) Measurements of C3f in μg/ml in serum samples from OA patients (n = 13), RA patients (n = 13) and NC (n = 13). All samples were analysed at 1 in 50 dilution. Concentration of C3f was significantly increased in the RA group in comparison to the OA and NC group (***p>0.0001) and no increase was observed for the OA group. Data were analysed using Kruskal-Wallis with post hoc Dunn’s analysis and plotted as means ± SEM. (B) Schematic representation of human C3 complement cleavage to generated C3a, C3b, C3f and iC3b fragments. (C) C3f sandwich ELISA performed on filtered serum samples from the 3 study groups. RA patient’s sample (n = 5) and 1 NC sample were filtered using 3kDa and 10kDa cut-off filter and assessed by C3f sandwich ELISA. Unfiltered serum were tested as control. Data plotted as means ± SEM.</p

C3f and V65 ELISA development and validation.

<p>(A) A typical standard curve for C3f sandwich ELISA. Graph represent the log of C3f peptide concentration (from 0–1 μg/ml) against reading of optical density (OD) values fitted with nonlinear regression curve (Four Parameters Logistic Regression(4PL)). (B) Recovery of C3f peptide from spiked normal human serum diluted 1:50 to 1:400. C3f peptide was added at 1 μg/ml into assay buffer as positive control (C3f control) and to human serum diluted at 1:50, 1:100, 1:200 and 1:400 to test recovery. Diluted serum without spiked C3f was tested as negative control. Recovery of spiked C3f in serum was calculated in comparison to C3f control and showed 65%, 86%, 88% and 96% recovery at 1:50, 1:100, 1:200 and 1:400 dilutions respectively. Data plotted as means ± SEM. (C) A typical standard curve for V65 competitive ELISA. Graph represent the log of V65 peptide concentration (from 0–4 μg/ml) fitted with nonlinear regression curve (4PL). (D) Graph represent the concentration of V65 peptide against OD values. Spiking experiment was carried out with 2-fold serial dilution starting at 1μg/ml of V65 peptide using normal human serum (at 1:100 or 1:400). Data plotted as means ± SEM.</p

Western blot of C3f and V65.

<p>(A) Reactivity of monoclonal and polyclonal antibodies anti-C3f (mAb-C3f and pAb-C3f respectively) to C3b (Complement C3b) and human vitronectin protein (V) on western blots. (B) Reactivity of polyclonal antibodies anti-V65 (pAb-V65) to C3b and vitronectin protein on western blots. (C) Reactivity of monoclonal antibody anti-C3f on Western blots of depleted and concentrated serum samples from OA and RA patients and synthetic C3f peptide. OA sample 1 and 2 were loaded at 21ng and 25ng per lane respectively. RA and C3f samples were loaded at 42ng and 7.5μg per lane respectively.</p

Additional file 4: Figure S3. of Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF)

Expression of VEGF-A receptor and co-receptor mRNA and proteins in response to hypoxia and hyperoxia in normal (NF) and fibrotic (FF) fibroblasts. a) Quantitative RT-PCR of VEGFR1, neuropilin (NP) 1, and NP2 mRNA expression in total RNA cell lysates in NF and FF. VEGFR1 (***p < 0.001) and NP2 (*p < 0.05) mRNA levels were significantly up-regulated in NF in response to exposure to hypoxia, whilst NF NP1 mRNA levels were significantly downregulated (*p < 0.05). Similarly, FF VEGFR1 (*p < 0.05) and NP2 (****p < 0.0001) mRNA levels were significantly upregulated in response to hypoxia, but NP1 mRNA levels were unaffected. Hyperoxia had no significant effect on VEGFR1 or NP2 mRNA levels in neither NF or FF, whilst hyperoxia significantly upregulated (*p < 0.05) FF NP1 mRNA levels. Data are presented as mean fold change in expression (2-△△CT) with SEM, data analysis performed on △△CT values (NF and FF n = 6). b) The effect of hypoxia and hyperoxia on VEGFR1 protein expression in NF and FF as measured by western blotting (above) and densitometric analysis (below). VEGFR1 protein expression was significantly upregulated (***p < 0.001) in response to hypoxia in NF but not in FF. Hyperoxia had no statistically significant effect on VEGFR1 expression in NF or FF. c) Hypoxia resulted in the significant down-regulation of NP1 protein expression in NF cell lysates (*p < 0.05), but had no significant effect on FF. Hyperoxia up-regulated NP1 protein expression in FF (p* < 0.05), but had no significant effect on NF. d) Hypoxia and hyperoxia had no significant effect in the expression of NP2 protein. Data presented as means with SEM (n = 4, n = 2 shown in each western blot image). Normal: Normal fibroblasts, Fibrotic: Fibrotic fibroblasts, N: Normoxia, HO: Hypoxia, HE: Hyperoxia. Tubulin: loading control. Analysis of variance with post hoc Dunnett’s multiple comparisons analysis used throughout. (JPEG 114 kb

Additional file 3: Figure S2. of Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF)

a) Expression of HIF-1α in normal (NF) and fibrotic (FF) fibroblast cultures following exposure to hypoxic-like growth conditions with Cobalt Chloride. Representative western blot of NF and FF cultures treated with (HO) or without (N) Cobalt Chloride (CoCl2) for 24 h (above) with densitometric analysis (below). A specific band was detected for HIF-1α in cells exposed to CoCl2, that was absent in normoxic fibroblast cultures (*p < 0.05). Hypoxic-like growth conditions increased HIF-1α expression to a greater extent in NF compared to FF (*p < 0.05), unpaired t-test, n = 4 performed, n = 1 shown. Tubulin was used as the loading control. L: Protein Ladder, N: Normoxia, HO: Hypoxia. b) Quantitative RT-PCR of Fibronectin and Procollagen-1α mRNA in NF and FF total RNA lysates following exposure to hypoxia and hyperoxia. Fibronectin mRNA levels were significantly increased in total RNA lysates of NF and FF fibroblasts exposed to 24 h of hypoxia (NF *p < 0.05, FF **p < 0.01) and in FF exposed to 24 h of hyperoxia (*p < 0.05) when compared to normoxia using qRT-PCR. In contrast, hypoxia and hyperoxia had no significant effect on procollagen-1α mRNA levels. Data are presented as mean fold change in expression (2-△△CT) with SEM, data analysis performed on △△CT values (NF and FF n = 6). Statistical analysis: analysis of variance with post hoc Holm-Sidak multiple comparisons analysis used throughout. (JPEG 53 kb

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Primer sequences used for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). VEGFR1: Vascular endothelial growth factor receptor 1, VEGFR2: Vascular endothelial growth factor receptor 2, Neuropilin 1 and 2: NP1 and NP2, For: Forward, REV: Reverse. (JPEG 90 kb