Journal: Gut Pathogens

Article Title: Hypoxia exacerbates intestinal injury and inflammatory response mediated by myeloperoxidase during Salmonella Typhimurium infection in mice

doi: 10.1186/s13099-023-00586-5

Figure Lengend Snippet: Hypoxia exacerbates bacterial translocation in S. Typhimurium-infected mice. Bacterial translocation to the spleen ( A ) and liver ( B ) was determined 72 h after infection via one-way ANOVA with Tukey’s multiple comparison test. The CFU number per gram of tissue was displayed (n = 8–10/group; * p < 0.05; ** p < 0.01; *** p < 0.001). ( C ) Immunofluorescence microscopy revealed the presence of Salmonella (in green) in spleen sections (×200). The spleen ( D ) and liver ( E ) were stained with hematoxylin and eosin [( D ) ×200; ( E ) ×400]. The blue arrow represents lymphocyte hyperplasia. The black arrow means red marrow congestion. The yellow arrow indicates that the structure of the liver plate is unclear. The green arrow represents focal abscess. ( F – I ) Relative mRNA expression of TNF-α ( F , H ) and IL-1β ( G , I ) in the spleen and liver tissues was detected by qPCR. ( J - M ) Levels of TNF-α ( J , L ) and IL-1β ( K , M ) were detected by ELISA. Data are presented as mean ± SEM (n = 8 /group) and analyzed via one-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: ELISA capture antibodies (BD Cat. #557,516) and biotinylated secondary antibodies for TNF-α (BD Cat. #558,415), and standard curve was constructed using recombinant murine TNF-α (BD Cat. #554,589) antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Translocation Assay, Infection, Comparison, Immunofluorescence, Microscopy, Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Gut Pathogens

Article Title: Hypoxia exacerbates intestinal injury and inflammatory response mediated by myeloperoxidase during Salmonella Typhimurium infection in mice

doi: 10.1186/s13099-023-00586-5

Figure Lengend Snippet: Hypoxia augments Salmonella -induced mucosal injury and colitis in mice. ( A ) Hematoxylin and eosin staining of the colon (×100, ×400). ( B ) Immunofluorescence microscopy data show the distribution of Salmonella (in green) in colon sections (×200). Levels of TNF-α ( C ) and IL-1β ( D ) were determined by quantitative PCR. Cytokine levels of TNF-α ( E ) and IL-1β ( F ) were detected by ELISA. Colon levels of KC ( G ), MCP1 ( H ), and MIP2 ( I ) were analyzed by qPCR. Data represent mean ± SEM (n = 8 /group) and one-way ANOVA followed by Tukey’s multiple comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: ELISA capture antibodies (BD Cat. #557,516) and biotinylated secondary antibodies for TNF-α (BD Cat. #558,415), and standard curve was constructed using recombinant murine TNF-α (BD Cat. #554,589) antibodies purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Staining, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

doi: 10.1016/j.apsb.2022.08.019

Figure Lengend Snippet: Loss of hepatic FXR underlies compromised efficacies of FXR agonists in injured livers. (A, B) Reduced levels of FXR protein under various liver injuries, including BDL-induced cholestasis, CCl 4 -induced acute liver injury, HFHC-and MCD diet-induced NASH by IHC (A) and Western blot (B). (C) FXR protein degradation in apoptotic HepG2 cells triggered by ActD/TNF α , CHX/FasL, and TRAIL. (D–F) Attenuated hepatoprotective effects of OCA in FXR knock-down mice caused by specific AAV Fxr shRNA injection. (G–I) Attenuated hepatoprotective effects of OCA in FXR down-regulated mice caused by CCl 4 injury. (J–L) Restored hepatoprotective effect of OCA in CCl 4 -injured mice with reinforced injection of Ad- Fxr . (D, G and J) Mouse experiment procedure schemes. (E, H and K) Serum ALT and AST levels. (F, I and L) Representative H&E, Sirius Red and Masson staining of liver sections. Scale bar, 100 μm. n = 6 biologically independent samples within these experiments. Results are mean ± SEM, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ns, statistically not significant, as assessed with ANOVA.

Article Snippet: Recombinant murine TNF α (14-8321-63) was purchased from Bioworld Technology.

Techniques: Western Blot, shRNA, Injection, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

doi: 10.1016/j.apsb.2022.08.019

Figure Lengend Snippet: Cytoplasmic CHIP mediates FXR degradation upon apoptotic stimulation. (A) Degradation of FXR protein in DRs-triggered apoptotic HepG2 cells were blocked by MG132, but not bafilomycin A1. (B) Ubiquitinated FXR accumulated in ActD/TNF α -treated HepG2 cells. (C, D) Interaction between CHIP and FXR by GST pull-down assay (C) and BLI assay (D). (E) FXR bound to the U-Box region of the CHIP protein. (F) CHIP bound to the DBD region of the FXR protein. (G, H) Knock-down of CHIP precluded the ubiquitination (G) and degradation (H) of FXR protein in ActD/TNF α -treated HepG2 cells. (I, J) CHIP WT but not CHIP H260Q, an E3 ligase activity mutant, overexpression enhanced poly-ubiquitination (I) and degradation (J).

Article Snippet: Recombinant murine TNF α (14-8321-63) was purchased from Bioworld Technology.

Techniques: Pull Down Assay, Activity Assay, Mutagenesis, Over Expression

Journal: Acta Pharmaceutica Sinica. B

Article Title: SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

doi: 10.1016/j.apsb.2022.08.019

Figure Lengend Snippet: Reduced KPNA3 interaction with FXR limits FXR nuclear import in apoptotic HepG2 cells. (A, B) Subcellular location of FXR in apoptotic HepG2 cells by Western blot (A) and immunofluorescence analysis (B). (C) Alignment of the FXR region containing predicted NLS sequences from various species. Predicted NLSs were indicated in boldface type. (D) Alignment of human FXR amino acids 101–120 and 201–220 around the predicted NLSs. Predicted NLSs were indicated in boldface type and the mutants were in red. (E) Mutation of predicted NLS2 but not predicted NLS1 of FXR caused its cytoplasmic accumulation. (F) Mutation of FXR NLS impaired its function in nuclear import as shown by cargo reporter. (G) Mutation of FXR NLS promoted its degradation upon apoptotic trigger. (H, I) Association between FXR and KPNA3 were detected by GST pull-down analysis (H) and BLI analysis (I). (J) Mutation of FXR NLS impaired the interaction between FXR and KPNA3 by Co-IP assay. (K) Reduced FXR-KPNA3 interaction in apoptotic HepG2 cells by PLA. (L) Knock-down of KPNA3 induced cytoplasmic accumulation of FXR. (M) KPNA3 knock-down accelerated FXR degradation triggered by ActD/TNF α . Scale bar, 20 μm.

Article Snippet: Recombinant murine TNF α (14-8321-63) was purchased from Bioworld Technology.

Techniques: Western Blot, Immunofluorescence, Mutagenesis, Co-Immunoprecipitation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

doi: 10.1016/j.apsb.2022.08.019

Figure Lengend Snippet: Enhanced CRM1/FXR interaction promotes nuclear export of FXR in apoptotic HepG2 cells. (A) Alignment of the FXR region containing predicted NES sequences from various species. Predicted NESs were indicated in boldface type. (B) Alignment of human FXR amino acids 404–450 around the predicted NESs. Predicted NESs were indicated in boldface type and the mutants were in red. (C) Mutations of FXR NESs caused its nuclear retention. (D) Mutations of FXR NESs impaired their functions in nuclear export of NLS cargo reporter. (E) Mutations of FXR NESs prevented its degradation upon apoptotic trigger. (F–H) Association between FXR and CRM1 was detected by Co-IP analysis (F), GST pull-down analysis (G) and BLI analysis (H). (I) Mutations of FXR NESs impaired the interaction between FXR and CRM1. (J) Leptomycin B, a CRM1 inhibitor, prevented nuclear export of FXR protein. (K and L) Enhanced FXR-CRM1 interaction in apoptotic HepG2 cells by Co-IP assay (K) and PLA (L). (M) CRM1 knock-down by specific siRNA transfection prevented FXR degradation stimulated by ActD/TNF α . Scale bar, 20 μm.

Article Snippet: Recombinant murine TNF α (14-8321-63) was purchased from Bioworld Technology.

Techniques: Co-Immunoprecipitation Assay, Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

doi: 10.1016/j.apsb.2022.08.019

Figure Lengend Snippet: FXR posttranslational modification controls its nucleocytoplasmic shuttling. (A) The lysine residue (K217 in human) closed to FXR NLS is evolutionarily conserved in vertebrates. Alignment of the FXR sequence surrounding K217 (human) from various species. The NLSs were indicated in boldface type and the lysine residues were in red. (B) Acetylation at FXR K217 increased in ActD/TNF α -treated HepG2 cells. (C) Subcellular distribution of FXR K217R-NLS cargo and FXR K217Q-NLS cargo compared with FXR WT-NLS cargo by fluorescence images. (D) Subcellular location of acetylation defective mutation K217R compared with FXR WT in ActD/TNF α -treated HepG2 cells by cell fractionation. (E) FXR K217 acetylation impaired FXR-KPNA3 interaction while promoted FXR-CHIP interaction by Co-IP analysis. (F) FXR K217 acetylation impaired the interaction between FXR NLS peptide and KPNA3 by BLI assay. (G) FXR K217 acetylation promoted its ubiquitination. (H) FXR K217 acetylation accelerated its degradation caused by ActD/TNF α . (I) FXR acetylation increased promptly and gradually upon apoptotic stimulation. Scale bar, 20 μm.

Article Snippet: Recombinant murine TNF α (14-8321-63) was purchased from Bioworld Technology.

Techniques: Modification, Sequencing, Fluorescence, Mutagenesis, Cell Fractionation, Co-Immunoprecipitation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR

doi: 10.1016/j.apsb.2022.08.019

Figure Lengend Snippet: SIRT1 deacetylates FXR and governs its phosphorylation, nuclear export, ubiquitination and degradation. (A) Association between P300/SIRT1 with FXR as detected by Co-IP analysis. (B) Reduced association between FXR and SIRT1 in ActD/TNF α -treated HepG2 cells by Co-IP analysis. (C) SIRT1 activation by SRT1720 promoted its association with FXR and inhibited FXR acetylation, and vice versa . (D) SRT1720 treatment enhanced FXR phosphorylation, and vice versa . (E) SRT1720 treatment promoted FXR–KPNA3 interaction while attenuated FXR–CHIP interaction, and vice versa . (F) SIRT1 activation by SRT1720 increased nuclear accumulation of FXR, and vice versa . (G) SIRT1 activation by SRT1720 decreased ubiquitination of FXR, and vice versa . (H) SIRT1 activation by SRT1720 stabilized the protein expression of FXR, and vice versa .

Article Snippet: Recombinant murine TNF α (14-8321-63) was purchased from Bioworld Technology.

Techniques: Co-Immunoprecipitation Assay, Activation Assay, Expressing