Additional file 1: Figure S1. of RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells
Creators: name: Grabowski, Jeffrey
name: Gulia-Nuss, Monika
name: Kuhn, Richard
name: Hill, Catherine
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Inorganic Chemistry
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Summary of the process employed to select I. scapularis genes for RNAi knockdown experiments. Δ ISE6 proteins from the differential proteomic analysis at 36 hpi were analyzed. Proteins were selected based on (1) increased expression level, (2) strength of proteomic support (minimum 2 peptides identified from LC-MS-MS per protein) from proteins identified in Grabowski et al. [4], and (3) orthology to vertebrate/invertebrate proteins; * orthologous proteins identified in published proteomic studies [4–6, 8]. LGTV denotes proteins that exhibited increased expression following LGTV infection and LGTV &amp; UV-LGTV denotes proteins that exhibited increased expression following both LGTV infection and UV-LGTV treatment. + proteins that exhibited increased expression following LGTV infection as compared to UV-LGTV treatment. FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, 1-pyrroline-5-carboxylate dehydrogenase; VNN, pantetheine hydrolase; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, UMP-CMP kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195, hypothetical protein; Hypo576. The prefix “ISCW” denotes VectorBase accession IDs. Figure S2 Effect of pGEM dsRNA concentrations on ISE6 cell viability following transfection for 60 h. X-tremeGENE (Xtr) transfection reagent was used to optimize pGEM dsRNA (RNAi negative control) concentrations in ISE6 cells at 60 h post transfection. Cell viability readings were compared to the Xtr + OptiMEM (Opti) control (gray bar). Red boxes indicate increased or no significant decrease in ISE6 cell viability. RLU560,590, relative light units 560 nm excitation and 590 nm emission. Error bars represent SEM. Statistical analysis was performed using an unpaired t-test between Xtr + Opti control and each pGEM dsRNA concentration. *p value ≤ 0.05 and **p value ≤ 0.01. Results represent 3 technical replicates and 1 biological replicate (multiple biological replicates completed with 10 ng concentration). Figure S3 Effect of transfection with dsRNA on ISE6 cell viability. FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, 1-pyrroline-5-carboxylate dehydrogenase; VNN, pantetheine hydrolase; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, UMP-CMP kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195, hypothetical protein; Hypo576, hypothetical protein; pGEM, pGEM plasmid (negative control; light gray bars); LGTV 3UTR, 3’ UTR of LGTV TP21 strain (positive control; dark gray bars), RLU560,590, relative light units 560 nm excitation and 590 nm emission. ISE6 cell viability following transfection with 10ng dsRNA for 60 h normalized to the negative control pGEM dsRNA. Results represent 2–5 technical replicates and 3 biological replicates. Error bars represent SEM and unpaired t-tests for comparison of cell viability of the negative pGEM control versus each gene of interest. Table S1 T7-tagged primers used to amplify cDNA and synthesize dsRNA. Table S2 Primers used to amplify cDNA for I. scapularis genes of interest by RT-qPCR. Table S3 Enrichment/cluster analysis of ISE6 proteins that exhibited increased expression following LGTV and UV-LGTV treatment. ISE6 proteins with increased expression following LGTV infection and/or UV-LGTV treatment from [4] were searched via DAVID enrichment analysis. For each cluster, the P value represents a modified Fisher Exact P value, and EASE score implemented in DAVID gene enrichment and functional annotation analysis. Enrichment (E) score of ≥ 1.3 is equal to P value of ≤ 0.05. Table S4 Nucleotide similarity of RT-PCR products amplified from I. scapularis and ISE6 cells and IscaW1 gene models. Table S5 Summary of statistically significant values corresponding to figures. (DOCX 329 kb)