Journal: PloS one

Article Title: Hypoxia reduces arylsulfatase B activity and silencing arylsulfatase B replicates and mediates the effects of hypoxia.

doi: 10.1371/journal.pone.0033250

Figure Lengend Snippet: Figure 3. Total sGAG and C4S increased by hypoxia in BEC and NCM460 cells. A. In the BEC and the NCM460 cells, the total sGAG increased significantly following 10% O2624 h (p = 0.002, p = 0.013 respectively, unpaired t-test, two tailed), consistent with an effect on ARSB function. B. The majority of the increase in sGAG level following 10% O2624 h was attributable to the increases in C4S in the BEC and the NCM460 cells (p,0.001, p = 0.002, respectively). C, D. Highly significant inverse correlations were present between the sGAG content and the ARSB activity, using measurements of sGAG and ARSB at baseline, following ARSB knockdown by siRNA, following hypoxia (10% O2624 h), and following ARSB overexpression in the BEC (r = 20.96) and NCM460 cells (r = 20.999). E, F. Similar highly significant inverse correlations were present between the C4S content and the ARSB activity in the BEC (r = 20.95) and NCM460 cells (r = 20.99). [sGAG = sulfated glycosaminoglycan; BEC = bronchial epithelial cells; C4S = chondroitin-4-sulfate]. doi:10.1371/journal.pone.0033250.g003

Article Snippet: Overexpression was performed with an ARSB construct (OriGene Technologies, Inc., Rockville, MD) as previously described [3,4,6,22].

Techniques: Two Tailed Test, Activity Assay, Knockdown, Over Expression

Journal: PloS one

Article Title: Hypoxia reduces arylsulfatase B activity and silencing arylsulfatase B replicates and mediates the effects of hypoxia.

doi: 10.1371/journal.pone.0033250

Figure Lengend Snippet: Figure 4. Nuclear HIF-1a levels were increased by ARSB silencing and reduced by ARSB overexpression in BEC and NCM460 cells, and HIF-1a mRNA expression was increased by ARSB silencing in BEC. A. Activation of HIF-1a was significantly increased by silencing ARSB and significantly reduced by overexpression of ARSB in the BEC (p,0.001). B. In the NMC460 cells, ARSB silencing significantly increased the activation of HIF-1a and overexpression significantly reduced HIF-1a activation in the NCM460 cells (p,0.001). C. In the BEC, the effects of hypoxia (10% O2) on HIF-1a activation were significant at 1 h (p,0.01), 4 h (p,0.001), and 24 h (p,0.01), with the maximum increase at 4 h. D. As in the BEC, in the NCM460 cells, the effects of hypoxia (10% O2) on HIF-1a activation were significant at 1 h (p,0.01), 4 h (p,0.001) and 24 h (p,0.05), with the maximum increase at 4 h. E. In the BEC, the combined effects of ARSB silencing and hypoxia (10% O2) for 24 h had no greater effect on activation of HIF-1a than ARSB silencing alone. F. Similarly, in the NCM460 cells, the combined effects of ARSB silencing and hypoxia (10% O2) for 24 h had no greater effect on the activation of HIF-1a than ARSB silencing alone. G, H. The ARSB activity at baseline, following ARSB overexpression, following ARSB silencing by siRNA, and following hypoxia (10% O2)624 h were inversely correlated with the nuclear HIF-1a levels in the BEC and the NCM460 cells (r = 20.96; r = 20.98, respectively). I. HIF-1a mRNA expression increased to 2.5 (60.23) times the baseline following 10% oxygen64 h and to 3.73 (60.34) times the baseline following ARSB silencing by siRNA624 h (p,0.001) in the BEC. [ARSB = arylsulfatase B; BEC = bronchial epithelial cell; HIF = hypoxia inducible factor]. doi:10.1371/journal.pone.0033250.g004

Article Snippet: Overexpression was performed with an ARSB construct (OriGene Technologies, Inc., Rockville, MD) as previously described [3,4,6,22].

Techniques: Over Expression, Expressing, Activation Assay, Activity Assay