Journal: Science Advances

Article Title: Enterococcus faecalis redox metabolism activates the unfolded protein response to impair wound healing

doi: 10.1126/sciadv.aeb5297

Figure Lengend Snippet: ( A and B ) Gene expression of UPR markers ( Xbp1s , Chop , and Herpud1 ) in infected with E. faecalis at MOI of 800 (A) or 600 (B) or Tm treated NIH-3T3 mouse fibroblasts (A) or HaCaT human keratinocytes (B) ( n = 3). See Materials and Methods for multiplicities of infection (MOI) optimization description. ( C ) Gene expression of IRE1 downstream gene ( EDEM1 ) in cells treated as in (A) ( n = 3). ( D ) Quantitative analysis and representative immunoblots showing levels of XBP1s and BiP in HaCaT cells treated as in (A). ( E ) Scratch wound assay quantification for uninfected, infected, and Tm-treated cells (positive control). Shaded areas represent 1 SD for each condition ( n = 4 biological replicates). ( F ) Gene expression of XBP1s from HaCaT cells treated with the dimethyl sulfoxide (DMSO) control (open circles) or the IRE1 inhibitor (IRE1i) 4μ8c (closed circles) ( n = 3, one-way ANOVA, Dunnett’s test). ( G ) Scratch wound assay quantification for uninfected and infected cells treated with 0.5% DMSO control (open circles) or IRE1i (close circles) ( n = 4 biological replicates). Shaded areas represent 1 SD for each condition. Significance was determined using one-way ANOVA Dunnett’s test [(A) to (C)], two-way ANOVA Tukey’s test [(E) and (G)], or one-way ANOVA Tukey’s test (F) (* P < 0.05, ** P < 0.01, *** P < 0 0.001, and **** P < 0.0001).

Article Snippet: Unless otherwise specified, the following final concentrations of reagents were used: Tm at 0.2 μg/ml (for qPCR), 2.5 μg/ml (for scratch wound assays), or 5 μg/ml (for immunoblot/microscopy); the IRE1i 4μ8c at 50 μM (MedChemExpress, #HY-19707), added 1 hour before infection; H 2 O 2 at 250 μM; catalase at 100 U/ml; and SOD (Sigma-Aldrich, #S5395) at 100 U/ml.

Techniques: Gene Expression, Infection, Western Blot, Scratch Wound Assay Assay, Positive Control, Control

Journal: Science Advances

Article Title: Enterococcus faecalis redox metabolism activates the unfolded protein response to impair wound healing

doi: 10.1126/sciadv.aeb5297

Figure Lengend Snippet: ( A ) E. faecalis OG1RF Tn screen in which a library of 14,976 mutants was screened against an NIH-3T3 cell line expressing the Xbp1-mApple reporter system (3T3R). ( B ) Representative epifluorescence microscopy images of 3T3R under different conditions (uninfected, WT, and Tm-treated) at 21 hpi. Scale bar, 100 μm. ( C ) Diagram showing the pathways in which a subset of UPR-defective mutants (genes/proteins with red font) were identified. DHNA, 1,4-dihydroxy-2-naphthoic acid; GPP, geranyl diphosphate; HPP, heptaprenyl diphosphate; ETC, electron transport chain. ( D ) Validation of UPR-defective mutants with 3T3R ( n = 3). ( E ) UPR induction by WT and ΔT7SS in a 3T3(R) background ( n = 3). ( F and G ) Scratch wound assay quantification for HaCaT infected with (F) WT and ΔEET, which were also (G) treated with either 0.5% DMSO control (open circles) or IRE1i (close circles). WT data are identical to and replicated here for ease of comparison ( n = 4 biological replicates). The data underlying and (F) and (G) were generated in the same experimental sets and can therefore be directly compared against each other. Shaded areas represent 1 SD for each condition. ( H ) UPR induction at 24 hpi in HaCaT after treatment with 0.5% DMSO control (open circles) or IRE1i (closed circles) under uninfected, WT-infected, and ΔEET-infected conditions. Uninfected and WT-infected findings are identical to and replicated here for ease of comparison ( n = 3). Significance was determined using one-way ANOVA Dunnett’s test [(D) and (E)], two-way ANOVA Tukey’s test [(F) and (G)], or one-way ANOVA Tukey’s test (H) (*** P < 0 0.001 and **** P < 0.0001).

Article Snippet: Unless otherwise specified, the following final concentrations of reagents were used: Tm at 0.2 μg/ml (for qPCR), 2.5 μg/ml (for scratch wound assays), or 5 μg/ml (for immunoblot/microscopy); the IRE1i 4μ8c at 50 μM (MedChemExpress, #HY-19707), added 1 hour before infection; H 2 O 2 at 250 μM; catalase at 100 U/ml; and SOD (Sigma-Aldrich, #S5395) at 100 U/ml.

Techniques: Expressing, Epifluorescence Microscopy, Biomarker Discovery, Scratch Wound Assay Assay, Infection, Control, Comparison, Generated

Journal: Nature Communications

Article Title: Endothelial IRE1α promotes thrombospondin-1 mRNA decay and supports metabolic stress adaptation of pancreatic islets

doi: 10.1038/s41467-025-68276-1

Figure Lengend Snippet: a MS1 cells were pre-cultured at 5 mM glucose for 4 hours before stimulation with 16 mM glucose for 12 hours in the absence or presence of 10 μM 4μ8C. Quantitative RT-PCR analysis of Xbp1 mRNA splicing and Thbs1 mRNA abundance (n = 3 independent experiments). b HEK293T cells with IRE1α depletion (HEK293T-KO) were transiently transfected for 36 hours with vector control (-) or THBS1-Myc plasmid, or co-transfected with THBS1-Myc plus IRE1α or XBP1s-Flag plasmids. Cells transfected as indicated were also treated with 10 μM 4μ8C for 12 hours. Immunoblot analysis of IRE1α, XBP1s and TSP1-Myc protein. Shown also is quantification of TSP1-Myc protein level after normalization to β-Actin control ( n = 3 independent experiments). c , d HEK293T-KO cells were transiently transfected for 36 hours with vector control, IRE1α-WT or its RNase-deficient K907A mutant plasmids. c Quantitative RT-PCR analysis of human XBP1 s, BLOC1S1 and THBS1 mRNA levels ( n = 3 independent experiments). d Immunoblot analysis of IRE1α, TSP1-Myc and XBP1 protein with α-Tubulin as a loading control. Shown also is quantification of TSP1-Myc protein levels (n = 3 independent experiments). e Consensus sequence (underlined) of the putative RIDD region in human and mouse TSP1 mRNAs within the stem-loop structure predicted by RNAstructure Version 6.2. f Agarose gel analysis of IRE1α-mediated cleavage of human THBS1 mRNA. In vitro transcription-derived mRNA fragments of THBS1 (nt 1-1770 and nt 1771-3510) were incubated for 1 hour with recombinant human IRE1α protein (1 μg) in the presence of DMSO (-) or 10 μM 4μ8C, followed by 3% agarose gel analysis. Human XBP1 mRNA was used as a positive control. Red arrows indicate the probable major RNA cleavage products. g Synthetic RNA substrates for wild-type THBS1 WT (nt 2771-2808) or THBS1 Mut with the indicated G-to-C mutation were incubated for 1 hour with human IRE1α protein (1 μg) pre-mixed for 1 hour with DMSO (-) or 10 μM 4μ8C, followed by 20% TBE-Urea PAGE gel analysis. The red arrow indicates the RNA cleavage product. Data are representative of 2 independent experiments in ( f , g ). Results are shown as mean ± SEM by unpaired two-tailed Student’s t -test ( a – d ).

Article Snippet: The stability of mRNAs of interest in MS1 cells was determined by addition of 5 μg/mL actinomycin D (Act D) (MedChemExpress, HY-17559) together with 10 μg/mL tunicamycin (Tm) after cells were pre-treated with DMSO or 10 μM 4μ8C.

Techniques: Cell Culture, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control, Western Blot, Mutagenesis, Sequencing, Agarose Gel Electrophoresis, In Vitro, Derivative Assay, Incubation, Recombinant, Positive Control, Two Tailed Test