AFAP1L1 expression in sarcoma cell lines.

<p>(<b>A</b>) <b>mRNA expression of the </b><b><i>AFAP1L1</i></b><b> gene in sarcoma cell lines.</b> Reverse transcribed cDNA from each cell line was used as a template for PCR with primers specific for the <i>AFAP1L1</i> gene. The <i>β-actin</i> gene was used as a control. (B) Quantitative analysis of the gene expression of <i>AFAP1L1</i>. qPCR was performed with a Taqman probe and the primers listed in Table S1. Expression levels were calculated as fold changes relative to U2OS. (C) Protein expression of AFAP1L1. Total cell lysate from each cell line was used for Western blotting. β-tubulin was used as a control. Error bars indicate standard deviations.</p

Binding of Sp transcription factors to the core-promoter region of the <i>AFAP1L1</i> gene <i>in vitro</i>.

<p>EMSA was performed to analyze the binding ability of putative transcription binding sites. Nuclear extracts were prepared from U2OS cells. Cold competitor experiments were conducted by the addition of 25- and 50-fold excess amounts of unlabeled SBS1WT or SBS1MUT to nuclear extracts before incubating with labeled SBS1WT (<i>lanes c</i>–<i>f</i>). Supershift experiments were conducted by the addition of anti-Sp1 or anti-Sp3 antibody to protein-OND complexes (lanes <i>g</i> and <i>h</i>). Non-immune IgG was used as a control (lane <i>i</i>). Open and closed arrowheads indicate the Sp3-OND and Sp1-OND complex, respectively. Single and double asterisks indicate bands supershifted by the addition of Sp1 or Sp3 antibody, respectively.</p

Restoration of down-regulated AFAP1L1 expression by an siRNA-resistant Sp3 expression vector.

<p>U2OS cells stably expressing the Sp3 mRNA resistant to Sp3#1 and Sp3#2 siRNA was established and treated with these siRNAs. U2OS cells stably expressing the <i>EGFP</i> or <i>LacZ</i> gene were employed as a control. After 48-h-treatment with siRNAs, RNA was extracted from each cell and the expression of <i>Sp3</i> and <i>AFAP1L1</i> was analyzed by RT-PCR (A). Knocking down of the endogenous <i>Sp3</i> gene was confirmed by PCR using a set of primers located in the 3′ UTR of the <i>Sp3</i> gene (Table S1). The <i>β-actin</i> gene was used as a control. Protein was extracted after 72 h of treatment and used for Western blotting (B). β-tubulin was used as a control. Error bars indicate standard deviations. Single and double asterisks indicate the long and short forms of the Sp3 protein, respectively.</p

Identification of Sp1-binding sites as essential sequences for <i>AFAP1L1</i> transcription.

<p>(A) Identification of core domains for transcriptional activity. Open and closed circles represent wild-type and mutated EBS and open and closed rectangles represent wild-type and mutated SBS. PGV-vectors containing various segments of the <i>AFAP1L1</i> promoter were transfected into U2OS cells, and their luciferase activities were measured. (B) The effect of exogenous Sp1 and Sp3 on the transcriptional activity of the core promoter region of the <i>AFAP1L1</i> gene. The luciferase activity of the core promoter region (−224 to +75) was evaluated after Sp1 or Sp3-expressing vectors were co-transfected into U2OS cells. The total amount of transfected plasmid DNA was equalized by the addition of pcDNA3.1(+), an empty vector. Error bars indicate standard deviations.</p

Inhibition of Sp3 expression reduces cell migration and invasiveness in U2OS cells.

<p>Numbers of cells migrating through the uncoated 8-micron membrane pores (A) and through the Matrigel-coated membranes (B) were counted in five randomly chosen fields at a magnification of ×100. (C) A cell invasion index was calculated as the ratio of the number of cells migrating through the matrigel to the number migrating through the uncoated membrane.</p

Identification of Sp3 as a major transcription factor for <i>AFAP1L1</i>.

<p>(A) and (B) Binding of Sp transcription factors to the core-promoter region of the <i>AFAP1L1</i> gene <i>in vitro</i>. ChIP assays were performed using anti-Sp1 and anti-Sp3 antibodies or control IgG and the precipitated DNA was PCR-amplified using a pair of primers located in the core-promoter region (Table S1) (A), and the precipitated genome was quantified by qPCR (B). (C) The effect of mithramycin A treatment on Sp3 binding. U2OS cells were treated with mithramycin A or DMSO for 48 h, and immunoprecipitated DNA by Sp3 antibody was quantified by qPCR. (D) The effect of mithramycin A on the expression of the <i>AFAP1L1</i> gene. RNA was extracted from U2OS cells treated with mithramycin A or DMSO for 48 h, and RT-PCR was performed to semi-quantify the expression of each gene. The <i>β-actin</i> and <i>GAPDH</i> genes were used as a control. Error bars indicate standard deviations.</p

Linking of Sp3 with AFAP1L1 by siRNA experiments.

<p>(A) The specificity of siRNA. U2OS cells were treated with siRNA targeting <i>Sp1</i>, <i>Sp3</i>, or <i>Sp4</i> for 48 h, and the expression of these genes as well as the <i>AFAP1L1</i> gene was analyzed by PCR. Two different siRNAs targeting the <i>Sp1</i> and <i>Sp3</i> genes were designed and used. <i>β-actin</i> was used as a control. (B) Down-regulation of <i>AFAP1L1</i> expression by siRNA targeting the <i>Sp3</i> gene at the mRNA level. U2OS cells were treated with siRNAs targeting each gene for 48 h and the expression of <i>AFAP1L1</i> was analyzed by qPCR and indicated as fold changes relative to that in untreated cells. (C) Down-regulation of AFAP1L1 expression by siRNA targeting the <i>Sp3</i> gene at the protein level. U2OS cells were treated with siRNA targeting each gene for 72 h and proteins were extracted and used for Western blotting. β-tubulin was used as a control.</p

Additional file 2: of A comparison of nephrotoxicity between patients with a solitary-functioning kidney and those with bilateral-functioning kidneys in cisplatin-based chemotherapy for advanced urothelial carcinoma: a Japanese retrospective multi-institutional study

Figure S2. Comparison of the number of patients with nephrotoxicity between the four groups during four courses of cisplatin-based chemotherapy. A: Post first course, B: Post second course, C: Post third course, D: Post fourth course. (PDF 102 kb

Representative RNA-FISH images of fusion-positive case TKB014.

<p>The small boxed areas are enlarged in the adjacent large boxes. Scale bars in the figure are 10 μm.</p

<i>FGFR3</i> mutation and <i>FGFR3-TACC3</i> fusion status.

<p>(A) The heatmap shows the distribution of <i>FGFR3</i> mutations and <i>FGFR3-TACC3</i> fusions with respect to T stage and pathological grade. (B) Subgroup analysis of NMIBC by T stage.</p