Sodium sulfate

All cells used in this study were obtained from the American Type Culture Collection (ATCC). HeLa (human cervical cancer, ATCC# CCL-13), HCT116 (human colorectal cancer, ATCC# CCL-247), HEK293 (adenovirus infected human embryonic kidney, ATCC# CRL-1573) and HS68 (normal HDF, ATCC# CRL-1635) cells grown as monolayers in 10 cm diameter dishes were washed in ice-cold phosphate buffer saline (PBS) pH 7.4, scraped from culture dishes on ice using a plastic cell scraper and collected in 1.5 ml micro-centrifuge tubes in 1 mL of ice-cold PBS. After centrifugation (a "pop-spin" for 10 sec in an Eppendorf table top microfuge), supernatants were removed from each sample and cell pellets were resuspended in 900 μL of ice-cold 0.1% NP40 (Calbiochem, CA, USA) in PBS and triturated 5 times using a p1000 micropipette (Gilson, WI, USA). 300 μL of the lysate was removed as "whole cell lysate" and 100 μL of 4 × Laemmli sample buffer was added to it, then kept on ice until the sonication step. The remaining (600 μL) material was centrifuged for 10 sec in 1.5 ml micro-centrifuge tubes and 300 μl of the supernatant was removed as the "cytosolic fraction". 100 μL of 4 × Laemmli sample buffer was added to this fraction and boiled for 1 min. After the remaining supernatant was removed, the pellet was resuspended in 1 ml of ice-cold 0.1% NP40 in PBS and centrifuged as above for 10 sec and the supernatant was discarded. The pellet (~20 μL) was resuspended with 180 μL of 1 × Laemmli sample buffer and designated as "nuclear fraction". Nuclear fractions and whole cell lysates that contained DNA were sonicated using microprobes (Misonix, NY, USA) at level 2, twice for 5 sec each, followed by boiling for 1 min. 10 μL, 10 μL and 5 μL of whole cell lysate, cytoplasmic and nuclear fractions, respectively, were loaded and electrophoresed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [12 (link)] and transferred to nitrocellulose membranes (Pall Life Sciences, FL, USA). Membranes were incubated with anti-pyruvate kinase (Santa Cruz, CA, USA) or anti-α-tubulin (Calbiochem, CA, USA) antibodies as cytoplasmic markers or anti-lamin A (Santa Cruz, CA, USA) or anti-nucleoporin (Santa Cruz, CA, USA) as nuclear markers after blocking with 3% bovine serum albumin in 0.1% tween 20-PBS (t-PBS). Membranes were washed with t-PBS followed by incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibody. After washing with t-PBS, target protein signals were detected by ECL (GE Healthcare, Buckinghamshire, UK) on Kodak X-ray film.

Fluorescence in situ hybridization (FISH) was performed to map repeated DNAs on the mitotic and meiotic chromosomes of H. obliquidens. Five DNA probes containing sequences of different classes of repeated DNA were used for chromosome hybridization. (i) 5S rDNA probe: complete repeat units of 5S rDNA of H. obliquidens were obtained by the polymerase chain reaction (PCR) with the primers 5SA (5'-TAC GCC CGA TCT CGT CCG ATC - 3') and 5SB (5' - CAG GCT GGT ATG GCC GTA AGC-3') designed from the rainbow trout 5S rRNA sequence [63 (link)] and successfully applied for the amplification of 5S rDNA of other cichlids [64 (link),65 (link)]. (ii) 18S rDNA probe: a segment of 1,400 base pairs (bp) of the 18S rRNA gene of H. obliquidens was obtained by PCR with the primers 18Sf 5'CCG CTT TGG TGA CTC TTG AT and18Sr 5'CCG AGG ACC TCA CTA AAC CA. The 18S primers were designed from the catfish Ictalurus punctatus (GenBank accession number AF021880) and have been successfully used to amplify 18S rRNA genes of different fish species [65 (link),66 (link)]. (iii) SATA satellite: repeated satellite DNA isolated and cloned from O. niloticus [29 (link)]; (iv) Telomeric DNA sequences: in vitro synthesized oligomers of telomeric repeats (GGGTTA)₇/(TAACCC)₇; (v) Clones BAC-C4E09 and BAC-C5E01: Bacterial artificial chromosomes containing several classes of repeated elements from the O. niloticus genome [29 (link)].
Probes were labeled by nick translation with biotin 14-dATP (Bionick labeling system-Invitrogen). After denaturation of chromosomal DNA in 70% formamide/2× SSC for 40 seconds at 70°C, hybridization mixtures containing 100 ng of denatured probe, 10 mg/ml dextran sulfate, 2× SSC and 50% formamide, in a final volume of 30 μl, were dropped on the slides and the hybridization was performed overnight at 37°C in a 2× SSC moist chamber. Post-hybridization washes were carried out at 45°C in 2× SSC/50% formamide for 15 min, followed by a second wash in 2× SSC for 15 min, and a final wash at room temperature in 4× SSC for 15 min. Detection of hybridized probes was carried out with 0.07% avidin FITC conjugate (Sigma) in C buffer (0.1 M NaHCO₃ /0.15 M NaCl) for 1 h, followed by two rounds of signal amplification using 2.5% anti-avidin biotin conjugate (Sigma) in blocking buffer (1.26% NaHCO₃, 0.018% sodium citrate, 0.0386% Triton X-100 an 1% non-fat dried milk) for 30 min. Each treatment with anti-avidin biotin conjugate was followed by a treatment with avidin-FITC. The treatments with avidin-FITC and anti-avidin-biotin were conducted in a 2× SSC moist chamber at 37°C. After each amplification step, the slides were washed three times for 5 min each in blocking buffer at 42°C. Chromosomes were counterstained with propidium iodide diluted in antifade (Vectashield Mounting Medium, Vector). Hybridized chromosomes were visualized using an Olympus BX 61 microscope, and images were captured with a digital camera Olympus DP71 with the software Image-Pro MC 6.0. Karyotypes and metaphases were arranged with Adobe Photoshop 7.0 software.

CRBL, OCTX, PUTM and WHMT samples from 12 individuals were analysed using the QG platform for validation of exon array results. We focused on three target genes for validation, leucine-rich repeat kinase 2 (LRRK2), sodium channel, voltage-gated, type VIII, alpha subunit (SCN8A), and microtubule-associated protein tau (MAPT). We selected ribosomal protein, large, P0 and ubiquitin C as housekeeping genes to normalise the target genes as they showed relatively low variability in expression levels (i.e. low coefficient of variation) in all brain regions in our dataset. The approach to the selection of reference genes is explained in previous studies (de Jonge et al. 2007 (link); Coulson et al. 2008 (link)).In addition, a recent study confirms the efficiency of using this approach in selecting housekeeping genes to normalise in different tissues (Chervoneva et al. 2010 ). A summary of the QG probes used for analysis of all five genes is provided in Table 2.
QuantiGene 2.0 Reagent System was used and the protocol in the QuantiGene 2.0 Reagent System User Manual was followed with the exception of the substrate step. Lumigen® Lumi-Phos® Plus and 10% lithium lauryl sulfate was used instead of Lumigen® APS-5 substrate. A Biotek ELx 405 select plate washer was used for all of the wash steps in the assay. The QG 2.0 plates were then read on a Molecular Devices LMax luminometer with the plate incubator set to 45°C to maintain the temperature of the Lumigen® Lumi-Phos® Plus substrate. In total, 13 QG 2.0 plates were run to cover all target genes and the house keeping genes. Each house keeping gene ribosomal protein, large, P0 and ubiquitin C was loaded in duplicates at 12.5 ng/well. In addition, target genes (LRRK2, SCN8A and MAPT) were loaded in duplicates at 75 ng/well.

Kp-reconstituted WT mice were administered Dextran Sodium Sulfate (DSS) ad-libitum in water (3% DSS in water) and sacrificed at the indicated time points. Fecal pellets were sampled at the indicated time points and were mechanically homogenized (bead beating). Seven serials 1:10 dilutions of the mixture were plated onto LB-agar supplemented with carbenicillin (100ug/ml) and neomycin (50ug/ml). After 24 h incubation at 37°C, CFUs were counted.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 12% polyacrylamide gel, according to the method described by Schägger and Jagow [29 (link)]. After electrophoresis, the gel was stained with Coomassie brilliant blue R-250.

We used 6-week-old Mptx2^−/− mice (female, n = 6; male, n = 10) and their wild-type (Wt) (female, n = 9; male, n = 6) littermates for dextran sulfate sodium (DSS)–induced colitis experiments. Mptx2^−/− (female, n = 8; male, n = 7) mice and Wt (female, n = 8; male, n = 8) mice were untreated as controls. Acute colitis was induced by administration of 2% DSS (36–50 kDa; MP Biomedicals, Solon, OH, USA) in drinking water for 7 days. We monitored changes in mouse body weight (BW) daily. To construct the DSS-induced colitis and recovery mouse model, C57BL/6 mice were induced by 3.5% DSS for 7 days and allowed to recover for 2 weeks (day 0, female, n = 8; male, n = 7), (day 1, female, n = 5; male, n = 5), (day 3, female, n = 5; male, n = 5), (day 7, female, n = 5; male, n = 5), (recovery 1 week, female, n = 5; male, n = 5), and (recovery 2 week, female, n = 6; male, n = 5).