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Blood, Vol. 114, Issue 1, 181-186, July 2, 2009
Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells
Blood Tanno et al.
114: 181
Supplemental materials for: Tanno et al
Files in this Data Supplement:
- Figure S1. TWSG1 does not increase hepatocyte cell toxicity or alter albumin gene expression (JPG, 168 KB)
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(A) Cell toxicity was examined among HuH-7 cells (closed bars) and primary human hepatocytes (open bars) according to the manufacturer’s instructions using a cytotoxicity detection kit plus (Roche Diagnostics, Mannheim, Germany) that measures LDH leakage into the culture supernatant. Cultures containing dosed concentrations of TWSG1 (0–1000 ng/ml) were studied after 24 hours. Medium containing 2ug/ml of actinomycin D (Act.D) was utilized as a positive control. Data are presented as means ± s.e.m. (n = 3). Asterisks signifying P < .05 compared to the control (0 ng/ml). (B) To determine if TWSG1 affected albumin expression, HuH-7 cells (closed bars) and primary human hepatocytes (open bars) were cultured in the presence (+) or absence (−) of BMP2 (10 ng/ml) or TWSG1 (1,000 ng/ml).After 24 hours, total RNA was isolated and cDNA was synthesized using FastLane cell cDNA kit (Qiagen). A quantitative real-time PCR assay was performed with human albumin specific TaqMan probes (Hs99999922_s1, Applied Biosystems). Albumin mRNA relative expression (y-axis) was normalized to the control gene GAPDH mRNA expression level with the TaqMan probes (Hs99999905_m1). The relative expression level of albumin in cultures containing no supplemental BMP2 or TWSG1 was assigned a value of one for comparison.
- Figure S2. BMPs mRNA expression in human and murine liver (JPG, 194 KB)
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mRNA expressions of BMPs in (A) human and (B) murine liver. Gene expression profiles were retrieved from GEO datasets (for human, GDS596: 79 tissue sets; for mouse, GDS592: 61 tissue sets). Relative expression level of each BMP gene was calculated by dividing normalized raw intensity by median value across all the tissues used for microarray analyses. (C) Experimental confirmation of the informatics data shown in Panels A and B. Human and murine BMP2/4/6/9 mRNAs in normal liver tissues from pooled human liver (open bars) and wild type mice (closed bars) was determined by quantitative PCR. Bars represent mean values with asterisks signifying P < .05.
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