Additional file 1: of Development and evaluation of adsorption sheet (HD safe sheet-U) using active carbon for the purpose of the preventing the contamination diffusion of urinary excreted anticancer drug

Appendix 1 and 2. Adsorption properties of the activated carbon to the urinary anticancer drug. (ZIP 103 kb

FAM5C increases ROS production and NF-κB activity.

<p><b>A and B</b>, HUVECs were transfected with 0.1 µg of FLAG (Mock) or FLAG–FAM5C (FAM5C) cDNAs and then incubated with 10 mM NAC or 10 µM TTFA for 16 h. ROS production assessed by dihydroethidium staining (<b>A</b>) and NF-κB activity (<b>B</b>) (n = 3–5) were analysed. <b>C and D</b>, HUVECs were transfected with 0.1 µg of FLAG (Mock) or FLAG–FAM5C (FAM5C) cDNAs and then incubated with 10 mM NAC, 10 µM TTFA, or 10 µM BAY 11-7085 (BAY) for 16 h. Involvement of ROS and NF-κB in the increase in FAM5C-induced ICAM-1 mRNA expression (<b>C</b>) (n = 4–5) and monocyte adhesion to HUVECs (<b>D</b>) (n = 6) were analyzed. ^†P<0.01 vs Mock, ^‡P<0.05, ^§P<0.01 vs FAM5C.</p

Involvement of FAM5C in the increases in ROS production and NF-κB activity.

<p>HUVECs transfected with control or FAM5C siRNAs were cultured for 48 h, and were then cultured in the presence of 10 ng/ml TNF-α for 6 h. The increase in TNF-α-induced ROS production (<b>A</b>) and NF-κB activity (<b>B</b>) by knockdown of FAM5C. The results shown are representative of three independent experiments (<b>A</b>). The luciferase activity was normalized by the β-galactosidase activity (<b>B</b>) (n = 4).</p

Upregulation of leukocyte adhesion molecules by FAM5C.

<p><b>A and B</b>, Total RNA was extracted from HUVECs 48 h after transfection with 0.1 µg of FLAG (Mock) or FLAG–FAM5C (FAM5C) cDNAs. An increase in FAM5C mRNA levels (<b>A</b>) (n = 3). and upregulation of ICAM-1, VCAM-1 and E-selectin mRNA levels (<b>B</b>) (n = 3) were analyzed by qPCR. <b>C</b>, 48 h after transfection, monocyte adhesion assays were performed and the numbers of monocytes that adhered to the HUVEC monolayers were analyzed (n = 5). HUVECs incubated with 10 ng/ml TNF-α for the last 16 h were used as the controls. *P<0.05, ^†P<0.01 vs Mock.</p

Upregulation of FAM5C mRNA by inflammatory stimuli in an NF-κB- and JNK-dependent manner.

<p><b>A</b>, HUVECs were cultured in the presence of 10 ng/ml TNF-α, 50 ng/ml IL-6, 10 ng/ml IL-1β, or 10 ng/ml LPS for 6 or 24 h (n = 11). <b>B and C</b>, HUVECs were incubated with 10 µM BAY 11-7085 (BAY), SP600125 (SP), SB203580 (SB), or DMSO (D) for 30 min and then cultured in the presence of 10 ng/ml TNF-α (<b>B</b>) (n = 8) or 10 ng/ml IL-1β (<b>C</b>) (n = 5) for 6 h. Values are expressed as the fold increase over the unstimulated control. ^*P<0.05, ^†P<0.01 vs control (<b>A</b>); *P<0.05, ^†P<0.01 vs DMSO (<b>B, C</b>). <b>D</b>, Upregulation of FAM5C mRNA expression in aortas of TNF-α-injected mice. RNA was extracted from mouse aortas and brains of the control (n = 5) and TNF-α-injected (n = 5) mice.</p

Expression and intracellular localization of FAM5C in cultured human endothelial cells.

<p><b>A</b>, Expression of FAM5C mRNA. The expression of FAM5A, FAM5B, FAM5C, and GAPDH mRNAs was determined with conventional RT–PCR. <b>B–E</b>, Intracellular localization of FAM5C. HUVECs were stained with the indicated antibodies. The nuclei were stained with DAPI. The results shown are representative of three to four independent experiments, with identical results obtained. ER, endoplasmic reticulum.</p

Colocalization of FAM5C with ICAM-1 and VCAM-1 in the endothelium of human coronary arteries.

<p>Serial sections of coronary arteries obtained from a 68-year-old man with chronic renal failure (<b>A</b>) and a 75-year-old man with hepatocellular carcinoma (<b>B</b>) were subjected to immunofluorescence staining for FAM5C and ICAM-1 (<b>A</b>) or VCAM-1 (<b>B</b>).</p

Involvement of FAM5C in the TNF-α-induced upregulation of leukocyte adhesion molecules.

<p><b>A</b>, Knockdown of FAM5C. HUVECs transfected with control or FAM5C siRNAs were cultured for 16 h, and were then cultured in the presence of 10 ng/ml TNF-α for 6 h (n = 6). *P<0.05, ^†P<0.01 vs control. <b>B–E</b>, Inhibition of the increase in the TNF-α-induced expression of FAM5C, ICAM-1, VCAM-1 and E-selectin mRNAs by FAM5C knockdown. HUVECs transfected with control or FAM5C siRNAs were cultured for 48 h, and were then cultured in the presence of 10 ng/ml TNF-α for 6 h. FAM5C (<b>B</b>), ICAM-1 (<b>C</b>), VCAM-1 (<b>D</b>), and E-selectin (<b>E</b>) mRNA levels were analyzed (n = 4). ^†P<0.01 vs control siRNA. <b>F–I</b>, Inhibition of the increase in the TNF-α-induced expression of ICAM-1, VCAM-1 and E-selectin proteins by knockdown of FAM5C. HUVECs transfected with control or FAM5C siRNAs were cultured for 48 h, and were then cultured in the presence of 10 ng/ml TNF-α for the indicated times. Representative blots (<b>F</b>) and densitometric analysis of band intensities of ICAM-1 (<b>G</b>), VCAM-1 (<b>H</b>) and E-selectin (<b>I</b>) were shown (n = 4). *P<0.05, ^†P<0.01 vs control siRNA. <b>J</b>, Inhibition of the increase in TNF-α-induced monocyte adhesion to HUVECs by knockdown of FAM5C. HUVECs transfected with control or FAM5C siRNAs were cultured for 48 h, and were then cultured in the presence of 10 ng/ml TNF-α for 16 h (n = 4–6). *P<0.05, ^†P<0.01 vs control.</p