ELF4 promotes miR-221 expression through direct promoter binding.

<p>A. Wild type (WT) or <i>Elf4</i>^{<i>─/─</i>} peritoneal macrophages were infected with VSV. 12 hours later miR-221 was analyzed by quantitative PCR. (B) iBMDM or MEF cells were infected with VSV. 8 hours later, miR-221 transcripts were measured by q-PCR. (C) HEK 293T cells were transfected with empty vector (Vec) or increasing amounts of ELF4 (50ng, 100ng, 200ng). 24 hours later miR-221 transcript levels were analyzed by quantitative PCR. (D) HEK 293T cells were transfected with miR-221 promoter-driven luciferase plasmids (wild type or mutant), and empty vector (Vec) or ELF4. (E) Wild type (WT) or <i>Elf4</i>^{<i>─/─</i>}iBMDM (immortalized Bone marrow derived macrophage) cells were infected with ELF4 or ELF4-del ETS lentivirus, and 24 hours later were infected with VSV. The data are expressed as the mean ± SEM of 4 independent experiments. (n = 4 biological replicates).</p

miR-221 negatively regulates innate anti-viral response.

<p>(A) HEK 293T cells were transfected with the indicated plasmids (the miR-221mut plasmid is derived from miR-221 plasmid by deleting 10 core nucleotides). 12 hours later, the cells were infected with VSV. 24 hours later IFNβ mRNA was measured by qPCR, supernatant from selected groups were subjected for ELISA. and the viral load (B) was analyzed by FACS. (C) HEK 293T cells were transfected with scrambled or miR-221 shRNA. 48 hours later, miR-221 transcripts were measured by q-PCR. (D) HEK 293T cells were transfected with the indicated plasmids, and infected with VSV at 24 hours. Another 24 hours later IFNβand VSV G protein transcript levels were analyzed by quantitative PCR. (E) HEK 293T cells were transfected with the indicated Oligonucleotides, and infected with VSV at 24 hours. Another 24 hours later supernatant were subjected to plaque assay and ELISA. (F) HEK 293T cells were transfected with the indicated luciferase (Luc) plasmids, and empty vector (Vec) or miR-221 and infected with VSV 24 hours later. As an internal control, 10 ng of pRL-TK was transfected simultaneously. The data are expressed as the mean ± SEM of 4 independent experiments. (n = 4 biological replicates).</p

Overexpression of miR-221 downregulated genes analysis.

<p>(A) HEK 293T cells were transfected with an IFNβ-Luc plasmid and the indicated plasmids. Cells were then given miR-221 or an empty vector (Vec) control. (B) HEK 293T cells were transfected with an IFNβ-Luc plasmid, MAVS and empty vector (Vec) or miR-221. The data are expressed as the mean ± SEM of 2 independent experiments. (n = 2 biological replicates). (C) HEK 293T cells were transfected with miR-221 or empty vector (Vec). 6 hours later the cells were harvested followed by whole genome RNA-seq analysis. (D) enrichment analysis using “<a href="http://amp.pharm.mssm.edu/Enrichr/" target="_blank">Virus Perturbations from GEO down</a>” section of “<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987924/" target="_blank">Enrichr</a>” program. (E) Network graph of downregulated genes, showing top ten clusters. (F) Heatmap of top ten clusters of downregulated genes.</p

Differentially expressed microRNAs in response to viral infection of <i>Elf4</i>^{<i>─/─</i>} macrophage.

<p>(A and B) Wild type (WT) or <i>Elf4</i>^{<i>─/─</i>} peritoneal macrophages were infected with VSV. 12 hours later miRNA transcript levels were analyzed by quantitative PCR. (C) 293T cells were transfected with the indicated luciferase (Luc) plasmids, and empty vector (Vec) or ELF4. The luciferase activity of each microRNA is indicated as fold induction. (D and E) 293T cells were transfected with the indicated plasmids. 12 hours later, the cells were infected with VSV. 24 hours later the viral load (defined by percentage of GFP-positive cells) was analyzed by FACS (D) and plaque assay (E). The data are expressed as the mean ± SEM of 4 independent experiments. (n = 4 biological replicates).</p

Additional file 5 of Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes

Additional file 5. Original western blot gel images, related to Fig. 4

Additional file 1 of Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes

Additional file 1: Table S1. An introduction to the pharmacology and toxicology of TKI drugs of different cardiotoxicity levels. Table S2. Endoplasmic reticulum stress gene markers under different conditions of sorafenib. Table S3. The ARRIVE checklist

Additional file 3 of Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes

Additional file 3: Figure S12. Cell viability of NRCMs in response to afatinib, sorafenib, and ponatinib, related to Fig. 4. Figure S13. Low dose of TKIs induce ER stress over time in NRCMs, measured by mRNA fold changes, related to Fig. 4. Figure S14. Effects of ponatinib and sorafenib on heart weight, body weight, heart-to-body weight ratio, and Ddit3 expression in rat hearts, related to Fig. 4. Figure S15. Lipid peroxidation levels in NRCMs treated with cumene hydroperoxide and ethanol, related to Fig. 5. Figure S16. The effect of trolox on lipid peroxidation and ER stress induced by TKIs, related to Fig. 5. Figure S17. ISRIB and 4μ8c affected gene targets of Atf4 and Xbp1s induced by TKIs in NRCMs, related to Fig. 6. Figure S18. ISRIB and 4μ8c did not rescue NRCMs from cell death induced by the 3 TKIs, related to Fig. 6. Figure S19. The effects of ISRIB and 4μ8c on TKI-induced cell death examined using fluorescence imaging and quantification, related to Fig. 6. Figure S20. Persistent eIF2α phosphorylation up-regulated Nfkb1 and Il6 expression induced by 3 TKIs in H9C2 cells, but not Il1b or Tnf, related to Fig. 6

Schematic models of UBXN1 interaction with Cul1.

<p><b>(A)</b> Cartoon rendering of how UBXN1 may be interfering with Cul1 in the canonical NFκB signaling pathway; <b>(B)</b> and <b>(C)</b> additional schematics of how UBXN1 may somehow be interfering with Cul1 activity either by steric hindrance <b>(B)</b> or allosteric effect <b>(C)</b>. UBXN1 is known to multimerize and interacts with both N and C termini of Cul1; stoichiometry between UBXN1 and Cul1 is not known (dashed ovals). In both models Skp1 and Rbx1 interact with the N and C termini of Cul1, respectively, and UBXN1 interacts with Rbx1 but not Skp1. These models do not exclude the possibility that another factor or protein (e.g., multimerized ubiquitin) mediates the interaction between UBXN1 and Cul1/Rbx1.</p

Additional file 2 of Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes

Additional file 2: Figure S1. EC50s of response calculated based on a four-parameter log-logistic model in the ATP fold change, related to Fig. 1. Figure S2. Seahorse experiment on acute effects of TKIs on mitochondrial oxygen consumption and extracellular acidification, related to Fig. 1. Figure S3. Mitochondrial membrane potential changes in response to TKIs observed by TMRE staining and its fold change, related to Fig. 1. Figure S4. Clustering of TKI-induced transcriptome data based on tSNE analysis, related to Fig. 2. Figure S5. Cluster 0, 2, 3, 4, 6 contained over 10 significant DEGs found by log2-based fold changes, related to Fig. 2. Figure S6. Expression of genes related to tRNA aminoacylation for protein translation in different clusters or in response to different drugs, related to Fig. 2. Figure S7. Good quality and consistency of 3’DGE-UMI RNA-seq, related to Fig. 2. Figure S8. The Jackstraw plot of the top 15 principal components in the tSNE analysis, related to Fig. 2. Figure S9. The number of unique genes, total counts, and proportion of mitochondrial DNA present in the 3'DGE-UMI RNA-seq data, related to Fig. 2. Figure S10. Correlation analysis between mitochondrial DNA and total counts or between unique genes and total counts in 3’DGE-UMI RNA-seq data, related to Fig. 2. Figure S11. Comparison of differentially expressed genes detected by 3'DGE-UMI and bulk RNA-seq for sorafenib and sunitinib treatments, related to Fig. 2

Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

<div><p>UBXN proteins likely participate in the global regulation of protein turnover, and we have shown that UBXN1 interferes with RIG-I-like receptor (RLR) signaling by interacting with MAVS and impeding its downstream effector functions. Here we demonstrate that over-expression of multiple UBXN family members decreased lentivirus and retrovirus production by several orders-of-magnitude in single cycle assays, at the level of long terminal repeat-driven transcription, and three family members, UBXN1, N9, and N11 blocked the canonical NFκB pathway by binding to Cullin1 (Cul1), inhibiting IκBα degradation. Multiple regions of UBXN1, including its UBA domain, were critical for its activity. Elimination of UBXN1 resulted in early murine embryonic lethality. shRNA-mediated knockdown of UBXN1 enhanced human immunodeficiency virus type 1 (HIV) production up to 10-fold in single cycle assays. In primary human fibroblasts, knockdown of UBXN1 caused prolonged degradation of IκBα and enhanced NFκB signaling, which was also observed after CRISPR-mediated knockout of UBXN1 in mouse embryo fibroblasts. Knockout of UBXN1 significantly up- and down-regulated hundreds of genes, notably those of several cell adhesion and immune signaling pathways. Reduction in UBXN1 gene expression in Jurkat T cells latently infected with HIV resulted in enhanced HIV gene expression, consistent with the role of UBXN1 in modulating the NFκB pathway. Based upon co-immunoprecipitation studies with host factors known to bind Cul1, models are presented as to how UBXN1 could be inhibiting Cul1 activity. The ability of UBXN1 and other family members to negatively regulate the NFκB pathway may be important for dampening the host immune response in disease processes and also re-activating quiescent HIV from latent viral reservoirs in chronically infected individuals.</p></div