PLoS Med. 2006 October; 3(10): e420.

ImmunohistochemistryFormalin-fixed tissues were treated with anti-NRF2 antibody (H-300, Santa Cruz Biotechnology) at a dilution of 1:250 for 1 h and developed using horseradish peroxidase (Dako, Glostrup, Denmark). Non-immune rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) was used as a negative control. To demonstrate the specificity of antibody staining, we preincubated the anti-NRF2 antibody with NRF2 and luciferase in vitro transcribed and translated protein, respectively, for 30 min and then carried out immunohistochemical staining.

PLoS Med. 2006 October; 3(10): e420.

Real-Time RT-PCRTotal RNA was extracted from cells using RNeasy kit (Qiagen) and was quantified by UV absorbance spectrophotometry. The reverse transcription reaction was performed by using the Superscript First-Strand Synthesis System (Invitrogen) in a final volume of 20 μl containing 2 μg of total RNA, 100 ng of random hexamers, 1× reverse transcription buffer, 2.5 mM MgCl2, 1 mM dNTP, 10 U of RNaseOUT, 20 U of Superscript reverse transcriptase, and DEPC-treated water. Quantitative real-time RT-PCR analyses of human KEAP1, NRF2, GCLc, GCLm, GSR, PRDX1, GSTA3, GSTA2, NQO1, MRP1, and MRP2 were performed by using Assay-on-Demand primers and probe sets from Applied Biosystems. Assays were performed by using the ABI 7000 Taqman system (Applied Biosystems). β-actin was used for normalization.

PLoS Med. 2006 October; 3(10): e420.

Plasmid ConstructionPlasmid encoding human KEAP1 cDNA in pCMV6-XL5 was purchased from Origene Technologies (Rockville, Maryland, United States). Glycine-to-serine mutations, leucine-to-arginine mutations, and a single nucleotide deletion were introduced into the KEAP1 expression vector by using a site-directed mutagenesis kit (Stratagene, La Jolla, United States). Details of the site-directed mutagenesis primers used in this study are listed in Table S2.

PLoS Med. 2006 October; 3(10): e420.

Cell Culture and ReagentsHBE4, NL20, A549, H460, H1435, H292, H23, H358, H1299, H1993, H1395, and H838 cells were purchased from American Type Culture Collection (Manassas, Virginia, United States) and cultured under recommended conditions. BEAS2B cells were provided by S. Reddy (Johns Hopkins University, Baltimore, Maryland, United States). All transfections were carried out using Lipofectamine 2000 (Invitrogen).

PLoS Med. 2006 October; 3(10): e420.

Generation of Stable TransfectantsH838 cells overexpressing ARE luciferase reporter plasmid were obtained by transfecting H838 cells with 3 μg of NQO1-ARE reporter plasmid and 0.3 μg of pUB6 empty vector (Invitrogen). Stable transfectants were selected using Blasticidin at a concentration of 6 μg/ml. Stable clones were expanded and screened for the expression of ARE luciferase.

PLoS Med. 2006 October; 3(10): e420.

Luciferase AssayH838 cells stably expressing NQO1-ARE luciferase were seeded onto a 24-well dish at a density of 0.2 × 106 cells/ml for 12 h before transfection. WT-KEAP1 cDNA constructs along with the mutant cDNA constructs (G333C and L413R) were transfected into the cells along with pRL-TK plasmid expressing Renilla luciferase as a transfection control. Twenty-four hours after transfection, cells were lysed and both firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega).

PLoS Med. 2006 October; 3(10): e420.

siRNA Duplex Screening and TransfectionThe siRNA sequence targeting NRF2 corresponds to the coding region nucleotides 1903–1921 (5′-GTAAGAAGCCAGATGTTAA-3′) in the NRF2 cDNA. The NRF2 siRNA duplex with the following sense and antisense sequences was used: 5′-GUAAGAAGCCAGAUGUUAAdUdU-3′ (sense) and 3′-dUdUCAUUCUUCGGUCUACAATT-5′ (antisense). KEAP1 siRNA corresponds to the coding region nucleotides 1545–1563 (5′-GGGCGTGGCTGTCCTCAAT-3′) in KEAP1 transcript variant 2. The KEAP1 siRNA duplex with the following sense and antisense sequences was used: 5′-GGGCGUGGCUGUCCUCAAUdUdU-3′ (sense) and 3′-dUdUCCCGCACCGACAGGAGUUA-5′ (antisense). To confirm the specificity of the inhibition, the siCONTROL non-targeting siRNA 1 (NS siRNA; 5′-UAGCGACUAAACACAUCAAUU-3′) with microarray-defined signature was used as a negative control. All of the siRNA duplexes were synthesized by Dharmacon Research (Lafayette, Colorado, United States). Cells in the exponential growth phase were plated at a density of 0.2 × 106 cells/ml, grown for 12 h, and transfected twice at an interval of 48 h with 50 nM siRNA duplexes using Lipofectamine 2000 and OPTI-MEM reduced serum medium (Invitrogen) according to the manufacturer's recommendations. Concentrations of siRNAs were chosen on the basis of dose–response studies (data not shown).

PLoS Med. 2006 October; 3(10): e420.

MTT Cell Viability AssayThe in vitro drug sensitivity to etoposide and carboplatin was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Cells were plated at a density of 10,000 cells for BEAS2B, 5,000 cells for A549, H460, and H838, and 20,000 cells for H1435 in 96-well plates. They were allowed to recover for 12 h and then exposed to various concentrations of etoposide and carboplatin for 72 h. After 72 h, drug cytotoxicity was evaluated by using a MTT reduction conversion assay (Sigma, Saint Louis, Missouri, United States). Forty microliters of MTT at 5 mg/ml concentration was added to each well, and incubation was continued for 4 h. The formazan crystals resulting from mitochondrial enzymatic activity on MTT substrate were solubilized with 200 μl of dimethyl sulfoxide, and absorbance was measured at 570 nm by using a SpectraMAX microplate reader (Molecular Devices, Sunnyvale, California, United States). Each combination of cell line and drug concentration was set up in eight replicate wells, and the experiment was repeated three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as means ± standard deviation (SD).

PLoS Med. 2006 December; 3(12): e486.

Quantitative RT-PCRExpression of LOX, NRCAM, BNC1, CCNA1, MAF, ALDH1A3, CTSZ, IRX4, MSX1, KLF11, SERPINB5, TKTL1, GAPDH, r18s, and CDKN2A was analyzed by quantitative real-time RT-PCR. Primers and probes were purchased from Applied Biosystems assay-on-demand, with the exception of p16, which was an assay-by-design (Hs00923893_m1) (http://www.appliedbiosystems.com). All samples were run on the Chromo 4 Real Time Detector (MJ Research [http://www.bio-rad.com]) twice, each time in duplicate. We averaged expression of GAPDH and r18s as internal reference genes to normalize input cDNA. Quantitative real-time reverse-transcriptase-PCR (QPCR) was performed in a reaction volume of 20 μl including 1 μl of cDNA. We used the comparative Ct method to compute relative expression values.

PLoS Med. 2006 December; 3(12): e486.

RNA Quality and Microarray Analysis RNA from primary lung cancers were obtained as part of collaborations with William Gerald at Memorial Sloan-Kettering Cancer Center (New York dataset) and Chi-Leung Lam and Maria Wong at the University of Hong Kong. All samples were collected with appropriate consent and internal review board approval. Cell line RNA was extracted from cell lines maintained in the Minna laboratory at UT Southwestern Medical Center at Dallas as described above. The quality of total RNA for all samples was analyzed by formaldehyde gel and/or by capillary electrophoresis on the Experion System (Bio-Rad). Total RNA was labeled and amplified by our genomics core facility, according to manufacturer's instructions (Affymetrix [http://www.affymetrix.com]). cRNA was reanalyzed after labeling to ensure optimal amplification for most of the samples. cRNA was hybridized to U133 Plus 2.0 (~47,000 transcripts) or U133A (~18,400 transcripts) (Affymetrix), and scanned by our microarray core facility (http://microarray.swmed.edu). Expression analysis of microarray data was performed using several algorithms: Robust Multichip Averaging (RMA) [31,32], Microarray Analysis Suite 5.0 (Affymetrix), MATRIX 1.29 (an array analysis program written by GL [unpublished data]; see below), NIH-DAVID [33], Cluster, and TreeView [34]. After scanning, arrays were checked for quality using GCOS (Gene Chip Operating Software) from Affymetrix and then normalized using either RMA or MATRIX 1.29. For log ratio calculations using MAS5 normalization (MATRIX 1.29), the only requirement was that the numerator be present (Affymetrix p-value < 0.065). Data were then logged and renormalized. For RMA normalization, all data were compiled using RMA Express, or RMA through R or BRBArrayTools. MATRIX (MicroArray TRansformation In eXcel) is a Microsoft Visual Basic program that allows import of multiple CHP files (saved as text file format) from Affymetrix MicroArray Suite 5.0 into an Excel spreadsheet where median normalization, comparison of arrays using log ratios and t-tests, color display, and hierarchical clustering can be performed. Specifically, expression signals are first log2-transformed and color coded such that higher signals are displayed as darker (blue) colors. Absent (high detection p-value) signals are optionally coded separately on a gray scale. For comparison of samples or classes of samples, log2 ratios (i.e., difference of log2-transformed signals) are calculated. If samples are compared, the stronger signals must have a present call (detection p-value < 0.05). If classes of samples are compared (as log ratios of the means), the median of the detection p-values for the class with the highest mean expression value must be less than 0.05. Two-sample t-tests are further calculated to filter out univariate non-significant differential expression. Hierarchical clustering was performed using average linkage with a Pearson correlation metric. All analyses are performed using extensive gene annotation and all probes are BLAST-verified. MATRIX has not been released, as it is still under development. While this program was used extensively in these studies, all analyses were reproduced using publicly available software. Please contact Luc Girard (Luc.Girard@utsouthwestern.edu ) for further details.

Adv Urol. 2009; 2009: 341268.

Between December 2006 and July 2007, twenty patients affected with anterior prolapse were included in this prospective survey. All the patients were referred to our Urology Unit because of their voiding problems. Mean age was 52 years (36–76). Mean parity, 3 vaginal childbirths. Sixteen patients with anterior vaginal wall prolapse reported socially annoying type II or III urinary stress incontinence, 4 patients reported voiding difficulty and related a history of urinary incontinence that has resolved with worsening of their prolapse. These patients were considered at risk for development of de novo stress incontinence after the prolapse is repaired. Twelve patients reported symptoms related to prolapse including the sensation of a vaginal mass or bulge, pelvic pressure, low back pain, and sexual difficulty. Twelve patients were sexually active, 8 had sexual difficulty (Table 1). The examination was first performed with the patient supine in lithotomy position. A retractor or Sims speculum was used to depress the posterior vagina to aid in visualizing the anterior vagina. After the resting examination, the patient was instructed to strain down forcefully or to cough vigorously. During this maneuver, the order of descent of the pelvic organs and their relationship at the peak of straining were noted. If physical findings did not correspond to symptoms or if the maximum extent of the prolapse could not be confirmed, the woman was reexamined in the standing position.

Adv Urol. 2009; 2009: 341268.

Surgical Technique All patients received spinal anesthesia and cephalosporin and metronidazol as antibiotic prophylaxis. The patient is placed in the lithotomy position and her thighs flexed approximately 90 degrees. A 16 Fr Foley catheter is placed to empty the bladder.

Adv Urol. 2008; 2008: 173694.

A 63-year old male was referred to our practice with recurrent, low-grade, bulbar urethra transition cell carcinoma which had recurred after two previous excisions. The patient was taken to the operating room for a third excision, where several urethral tumors were removed. Pathology showed T1, Grade 1 urothelial carcinoma. A large caliber urethral stricture in the area was also treated with urethrotomy at the same time. After surgical excision, he underwent 6 weekly installations of 40 mg Mitomycin C reconstituted in 80 cc of saline. While the usual method of intravesical chemotherapy involves installation through a bladder catheter, in this case we used a catheter-tip “Toomey” syringe (Bard; Covington, Ga, USA) to gently instill the medication into the urethral meatus, then used a penile clamp (Storz; Culver City, Calif, USA) to keep the column of medication in the urethra for 15 minutes. The patient suffered usual side effects of Mitomycin, including self-limited fatigue, dysuria, daytime urinary frequency up to every hour, and nocturia 2-3 X nightly [5]. All symptoms but nocturia resolved by the 6th treatment. He had cystoscopy and voided cytology examinations at 2, 6, and 12 and 18 months without recurrence of stricture or development of urethral cancer recurrences. (Previous recurrences X3 had been evident within 3 months of resection). The urethra appeared largely pink and healthy, with only small areas of white scar tissue marking the areas of previous urethral tumor resection.

Adv Urol. 2008; 2008: 213516.

三段论形式：1 病例入选原则，情况2 研究的目的3 MRI诊断方法标准 Following IRB approval, female patients diagnosed with urethral diverticulum from 1999 to 2004 were identified by a retrospective chart review using electronic medical records and paper charts. Information about presenting symptoms, urological history, diagnosis method, imaging studies, and outcomes at last followup was documented. Surgical operative reports and pathology reports were also reviewed for final diagnosis. Cases were reviewed to assess the method of initial diagnosis (physical exam, MRI, VCUG, cystoscopy, or urethrography). Our goal was to determine how the diverticula were diagnosed and to determine what studies were most sensitive at making the diagnosis. Therefore, any additional studies, as well as their contribution to diagnosis and surgical planning, were recorded. Outcomes at last followup were correlated with the type of imaging modality used for diagnosis and/or surgical planning. MRI studies (if obtained) were performed at this institution using a 1.5 Tessla magnet with a phase-array pelvic coil. Axial, coronal, and sagittal T2 weighted sequences were obtained using fast spin echo technique. Axial and sagittal T1 weighted sequences were obtained before and after intravenous gadolinium contrast. Computer tomographic (CT) scans were used in four cases due to clinical contraindications for MRI.

Am J Pathol. 2008 July; 173(1): 30–41.

Antibodies and Reagents The primary antibodies were obtained from different sources: anti-RANTES (sc-1410), anti-tumor necrosis factor (TNF)-α (sc-8301), anti-IκBβ (sc-946), anti-interleukin (IL)-6 (sc-7920), and anti-actin (sc-1616) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-IκBα (Ser32/36), anti-IκBα, anti-phospho-p65 NF-κB (Ser536), anti-p65 NF-κB, anti-phospho-GSK-3β (Ser9), anti-GSK-3β (Cell Signaling Technology, Beverly, MA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX). Recombinant human HGF, TNF-α, and IL-1β were purchased from R&D Systems (Minneapolis, MN). Cell culture media, newborn calf serum, and supplements were obtained from Invitrogen (Carlsbad, CA). Chemical inhibitor wortmannin and cell-permeable inhibitor peptide NF-κB SN50 were purchased from Calbiochem (La Jolla, CA). All other chemicals were of analytic grade and were obtained from Sigma (St. Louis, MO) or Fisher (Pittsburgh, PA) unless otherwise indicated.