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Proteasomal inhibition induces both viral and autophagy gene transcriptions.

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posted on 2020-02-24, 18:52 authored by Chandrima Gain, Samaresh Malik, Shaoni Bhattacharjee, Arijit Ghosh, Erle S. Robertson, Benu Brata Das, Abhik Saha

(A-D) ~10 x 106 two LCL clones–LCL#1 and LCL#89 were either left untreated (DMSO control) or treated with 1 μM MG132. 12 h post-treatment cells were harvested for (A and C) total RNA or (B and D) genomic DNA isolation as described in the “Materials and Methods” section. (B and D) LCLs were treated with 3 mM sodium butyrate (NaBu) in combination with 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h to induce viral lytic cycle as positive control. (A and C) Total RNA was subjected to cDNA preparation followed by quantitative real-time PCR (qPCR) analyses for the selected viral genes. The relative changes in transcripts (log10) using the 2−ΔΔCt method are represented as bar diagrams in comparison to DMSO control using GAPDH, B2M and RPLPO as housekeeping genes. Two independent experiments were carried out in similar settings and results represent as an average value for each transcript. (B and D) qPCR was performed for the detection of EBV DNA (BamHW fragment) using the genomic DNA isolated from each sample. The average fold increase of two independent experiments represented as bar diagrams was calculated in comparison to DMSO control using the 2−ΔΔCt method taking GAPDH as genomic control. (A-D and G-H) Average values +/- SEM are plotted. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. (E) The cDNA samples similarly prepared in (B and D) were subjected to whole transcriptome analyses (RNA-Seq) using Ion S5 XL System as described in the “Materials and Methods” section. RNASeqAnalysis plugin (v5.2.0.5) was utilized to perform the analysis after align with human genome (hg19) and produce gene counts for all the samples. Differential gene expressions were performed based on p-value as < = 0.05 and log2 Fold Change as 2 and above (upregulated, red) and -2 and below (downregulated, blue). (F) Differentially expressed gene sets were uploaded on DAVID v6.8 webserver for functional analysis. Gene Ontology (GO) was selected from the hits table for DAVID clustering. The bar diagrams (upregulated: red; downregulated: blue) represent top 15 most significantly affected pathways. (G-H) qPCR analyses of the selected cellular genes as described in (A and C).

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