Total RNA was harvested and IFN beta levels determined by RT-PCR. corrected against GAPDH and each bar represents 3 biological repeats. *?=?statistically significant difference (p?=? 0.01). Statistics were performed on raw data using the Mann-Whitney non-parametric U-test.(TIF) ppat.1003820.s002.tif (455K) GUID:?181A94C2-0502-4329-A4CE-5532544C8828 Figure S3: US25-2-3p and US25-2-5p do not target ATP6V0C. (A) Enrichment of selected top 30 targets was determined by RT-PCR following RISC-IP from cells transfected with indicted miRNA mimics (40 nM), including US25-1 mutant seed mimic. Enrichment levels converted to percentage with enrichment from AD169 infected cells set at 100%. Raw enrichment values shown above each bar. Expression levels were corrected against GAPDH and each bar represents 3 biological repeats. *?=?statistically significant difference (p?=? 0.01). Statistics were performed on raw data using the Mann-Whitney non-parametric U-test. (B) RNA levels were determined for ATP6V0C by RT-PCR, following transfection of fibroblast cells with US25-1, US25-2-3p LX7101 or US25-2-5p. RNA levels were LX7101 normalized to GAPDH and compared to cells transfected with a US25-1 seed mutant mimic.(TIF) ppat.1003820.s003.tif (331K) GUID:?1FFDDB00-7623-4CD6-AA8A-F05CAA89EFDC Figure S4: Confirmation of siRNA activity. (A) Percentage knock down of each LX7101 of the target transcripts is shown following transfection of human fibroblast cells with 40 nM of siRNA. Cells were harvested 24 hours post transfection and RT-PCR analysis performed as previously described. Percent knockdown versus cells transfected with negative control siRNA is shown. All assays were normalized against GAPDH levels and assays performed in triplicate. Stl1, 2 and 3 represent the independent siRNAs from the STL pool, targeting ATP6V0C (B) Human fibroblast cells were transfected with 40 nM siRNA or mimic and cells harvest 48 hours post transfection. Total RNA was harvested and IFN beta levels determined by RT-PCR. Positive control was RNA from cells transfected with non-infectious viral RNA. ND indicates not detected. (C) Cytotoxic effects on cells transfected with ATP6V0C siRNAs was measured using CytoTox-Glo according to manufacturer’s instructions. Fibroblast cells were transfected at 40 nM and harvested 48 hours post transfection. Results are shown as relative percentage of luciferase.(TIF) ppat.1003820.s004.tif (546K) GUID:?754821DF-3130-4754-8699-89F7C89170F3 Figure S5: Schematic representation of US25-1 targets. Untranslated regions of transcripts are shown in red, with translated region of transcript shown in green. Seed region of miRNA target interaction highlighted in red.(TIF) ppat.1003820.s005.tif (755K) GUID:?9C12F3EC-E434-4DFE-B4DC-7D0DECACE100 Figure S6: Reduction of HCMV replication from ATP6V0C knock down is not due to block in viral entry. GFP fluorescence is shown 24 hours post infection of primary fibroblast cells with GFP tagged HCMV. Cells were transfected with either negative control siRNA (A) or ATP6V0C siRNA (B) and infected 16 hours post transfection.(TIF) ppat.1003820.s006.tif (3.8M) GUID:?908F915C-B2D2-4FF9-895F-2EF958664766 Figure S7: Schematic diagram of knock virus construction. Deletion of US25-1 or US25-1 and 2 sequence regions are shown as well as the recombination event removing the KAN cassette resulting in the final Rabbit Polyclonal to BAX BAC constructs. Red boxes indicate the homologous regions of sequence where recombination occurs. Flanking transcripts US24 and US26 are shown in green.(TIF) ppat.1003820.s007.tif (569K) GUID:?D1C3E315-17DB-4F69-BCE9-D48E4C703C91 Table S1: Full data set for RISC-IP analysis of AD169 infected cells. Signal levels for total RNA and IP RNA levels are shown from LX7101 uninfected and infected pull down experiments. Final enrichment level represents analysed data after correction for false enrichment as explained in supplemental figure S1.(ZIP) ppat.1003820.s008.zip (5.9M) GUID:?57228268-0C5E-4723-897F-E7AC7C11A3A3 Table S2: Full data set for RISC-IP analysis of TR infected cells. Data analyzed as for supplemental table S1.(ZIP) ppat.1003820.s009.zip (6.5M) GUID:?94CE611D-4B2E-426E-Abdominal54-C9C377E317D4 Table S3: Full analysis of transcripts for HCMV miRNA seed focuses on. Transcript sequences were down loaded from NCBI using RefSeq ID’s. Transcript data units were searched for seed sequence matches using a Java centered script system. Seed matches for either 1 to 7 or 2 to 8 nucleotides are given. Results are demonstrated for either the ORF, 5 or 3 UTR regions of the transcripts. The position of the prospective site is given as nucleotide coordinates.(XLSX) ppat.1003820.s010.xlsx (7.0M) GUID:?71333256-723C-41AD-942D-3F80C151C254 Table S4: Summary of cloning oligonucleotides. The US25-1 target seed region for ATP6V0C is definitely highlighted in yellow and the sequence changes to produce the mutant seed region are indicated in reddish.(DOCX) ppat.1003820.s011.docx (45K) GUID:?B78AC2CA-7BAD-4023-9A17-2228C7561F58 Table S5: Small RNA sequences. Sequences of siRNA and mimics are demonstrated along with assay ID figures. For SGSH, a Dharmacon intelligent pool was used and target sequence is demonstrated under sense strand column. The seed mutation in US25-1 is definitely indicated in reddish.(DOCX) ppat.1003820.s012.docx (70K) GUID:?D7E39A11-5741-4C52-94AF-7D2347ADD0B7 Abstract Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all focuses on recognized are biologically relevant. Here, we make use of a combined.