JSS participated in the microarray assays. recovery graph. An asterisk indicates a cell made up of a plasmid. Scale bar: 10 m. 1471-2199-15-12-S1.tiff (17M) GUID:?E4123582-E459-4A41-86AC-9A626EB10678 Additional file 2: Figure S2 Real-time imaging of DZIP1-GFP aggregation with granules. (A) HeLa cells were transfected with DZIP1-GFP and cultured for 18 hours at 37C, and were then shifted to 20C. Fluorescence images were taken at one-second intervals (the time after the temperature shift is usually indicated in each panel). (B-C) Indirect immunofluorescence staining was carried out to detect the Rabacfosadine colocalization of DZIP1 (green) and TIA1 (red) or DCP1 (red) in HeLa cells. Nuclei were counterstained with DAPI (blue). (B) Oxidative stress with 0.5 mM sodium arsenite. (C) Oxidative stress with 2 mM sodium arsenite. Scale bar: 10 m. 1471-2199-15-12-S2.tiff (10M) GUID:?9903763D-3C31-49B5-A03C-768D2E81F405 Additional file 3: Table S1 mRNA Targets of DZIP1. Ratios of Signal Intensity Were Calculated and Genes that Have Fold Change (IP-DZIP1/IP-Control) Greater or Equal to 2.0 Were Listed. 1471-2199-15-12-S3.xls (261K) GUID:?F278BCAF-210B-48AA-B045-8925BF7D70F7 Additional file 4: Physique S3 DZIP1 did not interact robustly with the RNA probe. (A) Western-blot analysis of DZIP1-GFP and GFP levels in protein extracts from cells transfected with the corresponding plasmids. The band detected proximally at 120 kDa corresponds to DZIP1-GFP and the band detected at 27 kDa corresponds to free GFP. (B) Western-blot analysis against MYC tag: eluates (1C4) for affinity purification from cells transfected with a construct encoding DZIP1 fused to a histidine tail and a MYC tag (pSECTAG2). These eluates were used in electrophoretic mobility shift assays (EMSA). (C) We investigated whether DZIP1 interacted Rabacfosadine directly with these RNAs, by performing EMSA with purified DZIP1 protein and polyr A and C (U and G C not shown) probes. TcRBP40 is an RNA-binding protein used as a positive control. IP, immunoprecipitation. 1471-2199-15-12-S4.tiff (641K) GUID:?A81C718A-2166-4BDD-A47A-7333B6D63606 Additional file 5: Figure S4 Expression of and its mRNAs targets is affected by Hh pathway blockaded. (A) HeLa cells were incubated for several time periods, with various concentrations of cyclopamine. Proliferation was evaluated by BrdU incorporation. (B) We analyzed and mRNA levels in cells treated with various concentrations of cyclopamine by quantitative RT-PCR. (C-D) No change in the percentage apoptotic cells was observed after treatment of the cells with 300 nM cyclopamine for 24?hours. FACS-based apoptosis analysis showed that cyclopamine caused no significant change in the percentages of live, apoptotic and dead cells with respect to control cells. Dot plots for (C) control and (D) cyclopamine-treated cells. Cells were treated with Alexa Fluor 488 annexin V and propidium iodide (Molecular Probe), and subjected to flow cytometry. (E-F) FACS-based cell cycle analysis exhibited that cyclopamine treatment did not affect the percentages of cells in the G1, S and G2 phases of the cell cycle. A representative histogram of control cells (E) and cyclopamine-treated cells (F) based on the Dean-Jett-Fox model. 1471-2199-15-12-S5.tiff (872K) GUID:?76B628C5-0553-4148-BCA2-12413CB65C17 Additional file 6: Physique S5 knockdown and overexpression do not affect the accumulation or stability of mRNAs associated with DZIP1-containing complexes but modified quantity of stress granules per cell. (A) Quantitative RT-PCR analysis of and expression 24, 48 and 72?h after the transfection of cells with 1 nM DZIP1 duplex mix (siDZIP1) or 1 nM Scrambled-negative control duplex (siNC1). (B-C) The percentage of apoptotic cells was comparable in knockdown caused no significant change in the percentages of live, apoptotic and dead cells with respect to control cells (siNC1). Dot plot of (B) control and (C) knockdown had no effect on the percentages of cells in the G1, S Rabbit polyclonal to ITPK1 and G2 phases of the cell cycle. A representative histogram of control cells (D) and knockdown. Cells were counted at the indicated time points and the mean??SD values of three independent experiments are shown. (G) Representatives images of fields used to count stress granules. Rabacfosadine Stress granules were labeled with anti-TIA1 antibody in knockdown cells and control (siNC1). 1471-2199-15-12-S6.tiff (2.7M) GUID:?F85583B9-3F74-48BF-B340-1E93E3D5D56F Additional file 7: Physique S6 Half-life of and mRNAs in gene, also referred to as (DAZ-interacting protein 1), has three protein isoforms, each with a single C2H2 zinc finger domain but no other defined domain.