Journal: PLOS Biology
Article Title: Interferon regulatory factor 4 mediates nonenzymatic IRE1 dependency in multiple myeloma cells
doi: 10.1371/journal.pbio.3003096
Figure Lengend Snippet: (A) Comparative GSEA of IRE1 and IRF4. AMO1 shIRF4 Cl.1 or shNTC cells were incubated for 0, 8, 16, and 24 h with Dox (0.2 μg/mL) in biological triplicates and subjected to bulk RNA sequencing. DEGs in the shNTC samples were removed from the analysis as non-specific hits. Shown are the significant (FDR < 0.02) results stemming from Gene Set Enrichment Analyses (GSEA) using Hallmark libraries for both IRF4 and IRE1 silencing of 24 h (shIRE1 data obtained previously but re-analyzed for consistency). For GSEA analysis parameters used, see . (B) Overlap of Leading Edge Genes from GSEA in IRE1 and IRF4 knockdowns. After GSEA in , Leading Edge Genes , representing those contributing most significantly to the enrichment of the given gene sets, were extracted for both genetic backgrounds. Shown: Venn diagrams illustrating the intersection between the Leading Edge Genes for “E2F-targets” and “G2/M Checkpoint” between IRE1 knockdown (shIRE1) and IRF4 knockdown (shIRF4) genetic backgrounds. (C) Validation of cell cycle-related gene downregulation by RT-qPCR. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shNTC cells were treated with Dox (0.2 μg/mL) for the indicated times and samples were analyzed by RT-qPCR for E2F s, CDK s, Cdc25A, CDKN1A (p21 transcript), CCN s (Cyclin transcripts), and RB1 . Data represented as mean ± SEM. (D) Validation of cell cycle-related gene downregulation by IB. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were treated with Dox (0.2 μg/mL) for the indicated time and samples were analyzed by IB for cell cycle regulatory proteins. (E) Volcano plot analysis of “G2/M checkpoints” genes in a time-course of IRF4 silencing (8 h: before the onset of cell cycle defects; 16 h: at the onset of cell cycle defects; 24 h: cell cycle arrest established). E2F transcripts are indicated in red whereas Myc is in blue. Data underlying this figure can be found in , , and GEO archive ( GSE288674 ).
Article Snippet: GAPDH-HRP (#2118), IRF4 (#4964), MITF (#12590), Aiolos (#12720), Cofilin-HRP (#8503), Histone H3 #9715), Lamin B2 (#12255), Ubiquitin (#3936), CDK2 for immunoprecipitation (#18048), Rb (#9309), p21 (#2947), actin-HRP (#5125), Lamin A/C (#2032), PARP1 (#9532), Caspase 3 (#9662), Caspase 8 (#9746), p53 (#18032), pT160 CDK2 (#2561), CDK1 (#9116), CDK6 (#13331), Cyclin E2 (#4132), Cyclin B1 (#4138), p27 (#2552), pS608 Rb (#2181), pS795 Rb (#9301), PERK (#5683), eIF2α #9722), and CHOP (#2895) were from Cell Signaling Technology (CST).
Techniques: Incubation, RNA Sequencing, Knockdown, Biomarker Discovery, Quantitative RT-PCR
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