Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: Repression of HNF1α aggravates hepatic fibrogenesis in both DMN and BDL models. (A , B) Adenovirus carrying shRNA against HNF1α (shHNF1α) or negative control (shNC) was injected into rats prior to DMN administration (A) and BDL treatment (B) , and 2 weeks later the expression of HNF1α and α-SMA in the fibrotic livers was analyzed by immunohistochemistry. Hematoxylin and eosin (HE) and Sirius red staining were used to examine pathological alterations and collagen deposition. (C) Semi-quantitative analysis of Sirius red staining in the fibrotic livers from AdshHNF1α or AdshNC-treated rats ( n = 10 rats in each group). (D) mRNA levels of HNF1α , α-SMA and COL1A1 in the livers were detected by real-time PCR Scale bar, 100 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: shRNA, Negative Control, Injection, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: HNF1α overexpression attenuates hepatic fibrosis. (A , B) A single dose of adenovirus carrying human HNF1α gene (HNF1α) or control virus (GFP) was injected into rats after DMN injection (A) or BDL operation (B). The fibrotic livers were analyzed at 4 weeks after DMN treatment or 3 weeks after BDL. The expression of HNF1α and α-SMA was assessed by immunohistochemistry. HE and Sirius red staining were used to examine pathological alterations and collagen deposition. (C) Semi-quantitative analysis of Sirius red staining in the fibrotic livers from AdHNF1α or AdGFP-treated rats ( n = 6 rats in each group). (D) mRNA levels of HNF1α , α-SMA and COL1A1 in the livers were detected by real-time PCR. Scale bars, 100 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: Over Expression, Control, Virus, Injection, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: Anti-fibrotic effect of HNF1α depends on the transcriptional activation of SHP-1. (A) Transcript level of HNF1α and SHP-1 in primary rat hepatocytes treated with AdshHNF1α or AdshNC. (B) Correlation between the mRNA levels of HNF1α and SHP-1 in human liver tissues. Each data point represents an individual sample, and the correlation coefficient (r) is shown. (C) A schematic representation of the promoter region of SHP-1 , the potential cis -acting elements for HNF1α (arrow), mutation sites and the fragment amplified in ChIP-PCR. (D) The nested deletion analysis shows the transactivation effect of HNF1α on rat SHP-1 promoter. (E) HNF1α occupancy at the SHP-1 loci detected by ChIP-PCR in freshly isolated hepatocytes. (F) Suppression of SHP-1 reverses the anti-fibrotic effect of HNF1α. AdshSHP-1 or AdshNC was simultaneously delivered with AdHNF1α into DMN-treated rats. Collagen deposition and the expression of HNF1α, SHP-1 and α-SMA were detected in the livers. (G) Hydroxyproline content was assayed in the fibrotic livers ( n = 9 rats in each group). Scale bars, 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: Activation Assay, Mutagenesis, Amplification, Isolation, Expressing

Journal: Cell Research

Article Title: An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells

doi: 10.1038/cr.2015.84

Figure Lengend Snippet: Crosstalk between HSCs and hepatocytes in vitro . (A) A schematic representation of co-culture experiments with primary HSCs and hepatocytes isolated from rats. (B) Suppression of HNF1α in hepatocytes enhances the activation of HSCs. Endogenous HNF1α level in primary rat hepatocytes pretreated with AdshHNF1α or AdshNC was detected by western blot (left). mRNA levels of α-SMA and COL1A1 in HSCs were assessed by RT-PCR (right). (C) mRNA level of α-SMA and COL1A1 in HSCs co-cultured with AdshHNF1α- or AdshNC-treated hepatocytes. Antibody against IL-6, TNFα or TGFβ1 was added into the co-culture to block the corresponding cytokine. (D) Hepatocytes overexpressing HNF1α attenuates the activation of HSCs. Expression of exogenous human HNF1α and endogenous rat HNF1α in hepatocytes treated with AdHNF1α or AdGFP analyzed by western blot is shown in the top panel; mRNA levels of α-SMA and COL1A1 in HSCs are shown in the bottom panels. (E) Western blot analysis of HNF1α and SHP-1 in hepatocytes co-cultured with quiescent or activated HSCs for 48 h. Antibodies against TNFα, IL-6 and control IgG were used to block the cytokines in co-culture. (F) Expression of HNF1α and SHP-1 in hepatocytes transfected with miRNA inhibitors and then co-cultured with quiescent or activated HSCs for 48 h.

Article Snippet: Antibodies against HNF1α (ab96777, Abcam), α-SMA (BM0002, Boster, Wuhan, China), SHP-1 (ab2020, Abcam), p-Erk1/2 (Thr202/Tyr204, 4370, Cell Signaling), p-p65 (sc-101752, Santa Cruz) and p-STAT3 (Ser727, 9134, Cell Signaling) were used for immunohistochemistry.

Techniques: In Vitro, Co-Culture Assay, Isolation, Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Blocking Assay, Expressing, Control, Transfection

Journal: American Journal of Translational Research

Article Title: CircRNAs in exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells

doi:

Figure Lengend Snippet: Isolation and identification of exosomes from glomerular endothelial cells treated with high glucose and low glucose. A: Exosomal size analysis of HG-GEC-EXO and NG-GEC-EXO by NTA. B: The number of exosomes analyzed in the two different groups by NTA. C: Western blot analysis of HspA8 and Alix in the exosomes of the two different groups. ****P-value < 0.001. HG-GEC-EXO: exosomes from glomerular endothelial cells treated with high glucose. NG-GEC-EXO: exosomes from glomerular endothelial cells treated with normal (low) glucose; NTA: Nanoparticle tracer analysis.

Article Snippet: After blocking for 1 h at room temperature, the membrane was incubated with rabbit polyclonal anti-mouse HspA8 (1:1000) and Alix (1:1500) antibody (BOSTER, USA) for 12 hours.

Techniques: Isolation, Western Blot

Journal: bioRxiv

Article Title: Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

doi: 10.1101/2024.03.03.582873

Figure Lengend Snippet: Vero cells were infected with the wild-type parent B-1 (D614G) or the BK2102 vaccine candidate strains at a multiplicity of infection (MOI) = 0.01, and virus titers in the supernatants were determined for samples harvested every day, after incubating at 32 °C (A) or 37 °C (B). Infectious virus titers were determined using the TCID 50 method. Symbols indicate the average of three independent experiments, and error bars represent the SD. Days post-infection are indicated on the x-axis.

Article Snippet: Half-well protein high-binding 96-well plates (Greiner) were coated with recombinant SARS-CoV-2 (D614G) spike or nucleocapsid proteins (SinoBiologicals) dissolved in PBS (50 ng/100 μL/well) and incubated at 4 °C overnight.

Techniques: Infection, Virus

Journal: bioRxiv

Article Title: Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

doi: 10.1101/2024.03.03.582873

Figure Lengend Snippet: (A) Spike-specific IgG in the sera of BK2102-inoculated hamsters and mock-treated hamsters. Spike-specific IgG in sera obtained four weeks post-inoculation was detected by ELISA. Symbols depict data of individual hamsters, and bars correspond to the median value. The limit of dilution is indicated in the x-axis. (B) Neutralizing antibodies in the sera were induced by BK2102-inoculated hamsters and mock-treated hamsters. Neutralizing antibodies in the sera were measured at day 28 post-inoculation using the following authentic SARS-CoV-2 strains: wild-type D614G (left), Delta (middle), and BA.5 (right). Symbols represent titers of individual animals, and the bars indicate the median. The dotted line represents the assay’s limit of detection (LOD). (C) Immune response in monkeys. Neutralizing antibodies in the sera of BK2102-inoculated monkeys was measured at the indicated time points post-inoculation. The data for individual monkeys are shown. (D) Neutralizing antibodies persist in hamsters for at least 364 days. The neutralizing antibody titer against the authentic D614G wild-type strain was measured periodically in the sera of hamsters inoculated with BK2102 (once or twice at the indicated doses) for about a year. Symbols represent the mean of 9 to 10 animals, and error bars represent the SD. (E and F) Evaluation of the cellular immune response in BK2102-inoculated hamsters. Splenocytes were collected at two weeks post-inoculation and were stimulated in vitro with spike or nucleocapsid peptide pools. IFN-γ (E) and IL-4 (F) in the supernatants were measured with commercially available ELISA kits (MABTECH AB and FineTest, respectively). Symbols represent titers of individual animals, and bars indicate the median. For statistical analysis, one-way ANOVA with Tukey’s multiple comparison test was performed (ns, not significant; *, p < 0.05; **, p < 0.01).

Article Snippet: Half-well protein high-binding 96-well plates (Greiner) were coated with recombinant SARS-CoV-2 (D614G) spike or nucleocapsid proteins (SinoBiologicals) dissolved in PBS (50 ng/100 μL/well) and incubated at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Comparison

Journal: bioRxiv

Article Title: Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

doi: 10.1101/2024.03.03.582873

Figure Lengend Snippet: (A and B) BK2102 protects hamsters against homologous and heterologous virus challenges. Hamsters that received a full vaccination protocol with the indicated doses of BK2102 were challenged with wild-type D614G (A) or BA.5 (B) strains, and their body weight was monitored for four days. Body weight is expressed as a percentage of the initial weight. Two-way ANOVA with Tukey’s multiple comparison test was performed for statistical analysis (**, p < 0.01; ****, p < 0.0001). (C, D, F and G) The infectious virus titer in the lungs and nasal wash specimens taken on day four post-challenge was measured via a plaque assay for the wild-type D614G strain (C and D) and for the BA.5 strain (F and G). One-way ANOVA with Dunnett’s multiple comparison test was performed for statistical analysis (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (E and H) Lung inflammation scores were determined via H&E staining of D614G- (E) and BA.5-challenged (H) hamsters. The percentage of the disrupted area in the entire visual field was classified as 0: not remarkable (< 10%); 1: minimal (10–50%); and 2: mild (50–70%). One-way ANOVA with Tukey’s multiple comparison test was performed for statistical analysis (ns, not significant; *, p < 0.05). (I) Weight changes after the challenge assay one year post-inoculation with BK2102. Hamsters inoculated with BK2102 were challenged with the wild-type D614G strain at 3×10 5 PFU on 420 days. Nine-month-old elder hamsters were used as the naïve group. The symbols represent the average weight of the hamsters, and error bars indicate the mean SD. Two-way ANOVA with Tukey’s multiple comparison test was performed for statistical analysis (****, p < 0.0001).

Article Snippet: Half-well protein high-binding 96-well plates (Greiner) were coated with recombinant SARS-CoV-2 (D614G) spike or nucleocapsid proteins (SinoBiologicals) dissolved in PBS (50 ng/100 μL/well) and incubated at 4 °C overnight.

Techniques: Virus, Comparison, Plaque Assay, Staining

Journal: bioRxiv

Article Title: Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

doi: 10.1101/2024.03.03.582873

Figure Lengend Snippet: (A) Scheme for the evaluation of tissue damage in acute infection with BK2102 in a hamster model. The wild-type D614G strain was used as a positive control. (B) Inflammation score of nasal cavity sections and lungs determined via H&E. The percentage of the disrupted area in the entire visual field was classified as 0: not remarkable (< 10%); 1: minimal (10–50%); 2: mild (50–70%), respectively. (C) SARS-CoV-2 spike protein staining in the nasal cavity sections and lungs determined via immunohistochemistry using a SARS-CoV-2 spike RBD-specific antibody. The proportion of positive cells in the entire visual field was classified as 0: not remarkable (< 10%); 1: minimal (10–50%); and 2: mild (50–70%), respectively. (D) Scheme for the evaluation of BK2102 transmission via in vivo passage in hamsters. The TS-strain A50-18 was used as a positive control. (E) Ct values obtained for the RT-PCR performed using RNA extracted from the nasal wash specimens.

Article Snippet: Half-well protein high-binding 96-well plates (Greiner) were coated with recombinant SARS-CoV-2 (D614G) spike or nucleocapsid proteins (SinoBiologicals) dissolved in PBS (50 ng/100 μL/well) and incubated at 4 °C overnight.

Techniques: Infection, Positive Control, Staining, Immunohistochemistry, Transmission Assay, In Vivo, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

doi: 10.1101/2024.03.03.582873

Figure Lengend Snippet: (A and B) Survival rate of Tg mice infected with the wild-type D614G, B-1 ΔFCS, L50-33, and A50-18 TS strains (A) and BK2102 (B).

Article Snippet: Half-well protein high-binding 96-well plates (Greiner) were coated with recombinant SARS-CoV-2 (D614G) spike or nucleocapsid proteins (SinoBiologicals) dissolved in PBS (50 ng/100 μL/well) and incubated at 4 °C overnight.

Techniques: Infection