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) Effect of IRF4, IRE1, or XBP1 silencing on in vitro spheroid growth of AMO1. Cells were stably transfected with plasmids encoding Dox-inducible shRNAs against either IRF4 (purple) or non-targeting control (blue). Growth of these cells in the absence (closed symbols) or presence (open symbols) of Dox (0.2 μg/mL) was compared to that of cells expressing shRNAs against IRE1 or XBP1. Spheroid growth, depicted as FC confluence, was monitored by time-lapse microscopy in an IncuCyte instrument and values represent mean ± SEM. (B) Effect of IRF4, IRE1, or XBP1 silencing on number of cell divisions. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were stained with CFSE-type dye and incubated in the absence (filled curves) or presence (open curves) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dashed line) was used as a non-proliferative control. Representative experiment out of 3 independent replicates. (C) Effect of IRF4, IRE1, or XBP1 silencing on DNA replication. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were pulsed with BrdU (10 μM) and incubated in the absence (filled bars) or presence (open bars) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dotted line) was used as a non-proliferative control. Data represented as mean ±SEM. (D) Effect of IRE1 or IRF4 silencing on cell cycle progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence (filled symbols) or presence (open symbols) of Dox (0.2 μg/mL) for the indicated timepoints, EtOH-fixated and PI stained before analyzed by flow cytometry. The indicated cell cycle phases were determined according to univariate (DNA content) modeling. Representative experiment out of at least 3 independent replicates. (E) Effect of IRE1 or IRF4 silencing on the rate of G2/M progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were pre-incubated with 9 μM RO-3306 CDK1 inhibitor (synchronization to G2/M phase) in the absence or presence of Dox (0.2 μg/mL). Cells in G2/M phase were collected and their cell cycle progression during indicated time points post-sorting was analyzed by flow cytometry as before. The indicated cell cycle phases were determined according to DNA content and EdU incorporation to accurately decipher S phase. (F) Effect of IRE1 or IRF4 silencing on CDK2 activation. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence or presence of Dox (0.2 μg/mL) for 24 h. CDK2 was purified by immunoprecipitation. The top band is inactive CDK2 and the bottom band is the active form . Additionally, binding of the CDK2 substrate, Rb, is reduced by IRE1 or IRF4 silencing while binding of p21, the CDK inhibitor, is increased. Ig represents an isotype control for Ig detection. (G) Effect of IRE1 or IRF4 silencing on subcellular abundance of CDK2. Samples from and samples from AMO1 shIRF4 Cl.1 cells were analyzed by IB for CDK2 protein. Subcellular fractions: C—cytoplasmic, M—Membrane, SN—Soluble Nuclear, CN—Chromatin-bound Nuclear. Nuclear fractions were analyzed by IB for IRE1 and IRF4 while Cofilin, Histone H3, and Lamin B2 served as fractionation internal controls. The blots for IRE1, IRF4, Cofilin, and Lamin B2 from are shown here again for direct comparison. Data underlying this figure can be found in and .
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: In Vitro, Stable Transfection, Transfection, Control, Expressing, Time-lapse Microscopy, Staining, Incubation, Flow Cytometry, Activation Assay, Purification, Immunoprecipitation, Binding Assay, Membrane, Fractionation, Comparison
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