Journal: Advanced Science

Article Title: T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

doi: 10.1002/advs.202307648

Figure Lengend Snippet: T‐2 toxin infection increases β‐arrestin‐1 O‐GlcNAcylation and enhances β‐arrestin‐1 stability in glomerular podocytes. A) O‐GlcNAc modified proteins were analyzed by Co‐IP combined with LC‐MS, and some target proteins have higher scores, as shown in figure A. B) Endogenous interplays of β‐arrestin‐1 with OGT and O‐GlcNAc. T‐2 toxin‐exposed podocytes treated with or without MG‐132 (2.5 µM) were subjected to CoIP using β‐arrestin‐1 antibody, followed by Western blot analysis. C,D) qPCR quantification analysis of β‐arrestin‐1 along with β‐arrestin‐2 mRNA levels in mouse glomeruli C) and podocytes D) ( n = 6). E,F) Western blot analysis of β‐arrestin‐1 and β‐arrestin‐2 protein levels in mouse glomeruli E) and podocytes F) ( n = 6). G–I) Immunofluorescence staining analysis of β‐arrestin‐1 and β‐arrestin‐2 in mouse glomeruli G) and in podocytes H,I) ( n = 3). *** p < 0.001; data are expressed as mean ± SEM; unpaired t‐test was used in 2C, 2E, and one‐way ANOVA followed by Dunnett's post hoc test was used in 2D and 2F.

Article Snippet: Cells were infected with lentiviruses coding β‐arrestin‐1 ‐sgRNA (neomycin resistant) and then selected with G418 (MCE HY‐17561).

Techniques: Infection, Modification, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

doi: 10.1002/advs.202307648

Figure Lengend Snippet: β‐arrestin‐1 overexpression exacerbates podocyte injury upon T‐2 toxin exposure. A) The urine albumin‐to‐creatinine ratio in T‐2 toxin‐fed mice treated with rAAV‐CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or rAAV‐CAG‐GFP ( n = 6). B) Representative images of PAS as well as MASSON‐stained renal cortex sections were exhibited (scale bar, 50 µm) ( n = 3). C) GBM thickness. D) Number of foot processes of mice under transmission electron microscope (TEM) (Scale bar, 2 µm in upper panel; 500 nm in lower panel, n = 5). E) Foot process width. F) The infection efficiency of rAAV‐CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or rAAV‐CAG‐GFP in mice glomeruli was validated by immunofluorescence staining ( n = 3, scale bar, 20 µm). G) Quantitation of Nephrin as well as Podocin protein levels in podocytes after transfection of control siRNA or β‐arrestin‐1 siRNA and their corresponding controls by western blot ( n = 6). H) Immunofluorescence staining was used to analyze Nephrin and Podocin in mouse glomeruli infected with CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or CAG‐GFP ( n = 3, scale bar, 25 µm). *** p < 0.001, +++ p < 0.001, data are expressed as mean ± SEM; one‐way ANOVA followed by Tukey's post hoc test was used.

Article Snippet: Cells were infected with lentiviruses coding β‐arrestin‐1 ‐sgRNA (neomycin resistant) and then selected with G418 (MCE HY‐17561).

Techniques: Over Expression, Staining, Transmission Assay, Microscopy, Infection, Immunofluorescence, Quantitation Assay, Transfection, Control, Western Blot

Journal: Advanced Science

Article Title: T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

doi: 10.1002/advs.202307648

Figure Lengend Snippet: β‐arrestin‐1 mediates histone H4 hyperacetylation via recruiting p300 in T‐2 toxin‐induced podocyte injury. (A‐B) Western blot analyzed H3ac and H4ac protein levels in glomeruli of T‐2 toxin‐ stimulated mice A) and in T‐2 toxin‐treated podocytes B) ( n = 6). C) Western blot analysis of H3ac and H4ac protein levels in glomeruli of mice transfected with CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or CAG‐GFP ( n = 6). D) Western blot analysis of H4ac protein levels in podocytes transfected with control siRNA or β‐arrestin‐1 siRNA ( n = 6). E) Western blot analyzed p300 protein levels in glomeruli ( n = 6). F) Western blot analysis of p300 protein levels in glomeruli of mice transfected with CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or CAG‐GFP ( n = 6). G) Western blot analysis of p300 protein levels in podocytes transfected with β‐arrestin‐1 siRNA ( n = 6). H) Podocytes were treated with or without T‐2 toxin in the presence or absence of MG‐132. The interaction of β‐arrestin‐1 and p300 was determined by Co‐IP combined with Western blot analysis. I) Co‐IP combined with Western blot was used to detect β‐arrestin‐1 and p300 protein interaction in glomeruli of mice transfected with CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or CAG‐GFP. J,K) Western blot analysis of J) H4ac, K) Nephrin and Podocin protein levels in podocytes after transfection with p300 siRNA ( n = 6). * p < 0.05, +++ p < 0.001, data are expressed as mean ± SEM. Unpaired t‐test was used in 4A, 4B, and 4E, and one‐way ANOVA followed by Tukey's post hoc test was used in 4C, 4D, 4F, 4G, 4J, and 4K.

Article Snippet: Cells were infected with lentiviruses coding β‐arrestin‐1 ‐sgRNA (neomycin resistant) and then selected with G418 (MCE HY‐17561).

Techniques: Western Blot, Transfection, Control, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

doi: 10.1002/advs.202307648

Figure Lengend Snippet: β‐arrestin‐1 inhibits autophagy to exacerbate podocyte damage through acetylation of H4K16 to activate mTOR pathway. A) H4 acetylation at lysine 16 was detected by LC‐MS/MS in T‐2 toxin‐exposed podocytes. (B‐C) Endogenous interactions of H4K16 with β‐arrestin‐1 and p300 in podocytes were assessed using Co‐IP assay with H4K16 B) or β‐arrestin‐1 C) antibodies. D) Chromatin immunoprecipitation sequencing (ChIP‐Seq) analysis of H4K16 targeted binding gene fragments in T‐2 toxin‐exposed podocytes. 1) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of ChIP‐Seq results showed the activation of mTOR signaling pathway in T‐2 toxin‐exposed podocytes. 2) The bubble diagram showed that H4K16ac is significantly enriched in the mTOR promoter region. 3) The Venn map showed that mTOR promoter is the only overlapping candidate gene by ChIP‐Seq and RNA‐Seq results. 4) Normalized ChIP‐seq tracks showed the enrichment of H4K16ac on mTOR promoter. Regions with differences in H4K16ac enrichment were highlighted in the lavender box. (E‐H) Western blot analysis of p‐mTOR (s2448) (E,F), p‐mTOR (s2481) (E,F), light chain 3 (LC3) (G,H), Beclin1 (G,H), and autophagy related 5 (ATG5) (G‐H) protein levels in mouse glomeruli (E and G) and podocytes (F and H) ( n = 6). I) Representative electronic micrographs showed the number of autophagosomes in glomerular podocytes ( n = 3). The arrows exhibit autophagosomes. Scale bar: 1 µm. J) Western blot analysis of p‐mTOR (s2448), LC3, Beclin1, and ATG5 in podocytes after transfection with β‐arrestin‐1 siRNA. K) LC3 staining in podocytes ( n = 3). Scale bar, 20 µm. L) Western blot analysis of p‐mTOR (s2448), LC3, Beclin1, and ATG5 in glomeruli of mice infected with rAAV‐CAG‐GFP‐Cas9‐sg β‐arrestin‐1 or rAAV‐CAG‐GFP ( n = 6). M) The number of autophagosomes in mice glomerular podocytes was shown by TEM ( n = 3). *** p < 0.001, +++ p < 0.001, data are expressed as mean ± SEM; unpaired t‐test was used in 5E, 5F, 5G, and 5H, and one‐way ANOVA followed by Tukey's post hoc test was used in 5J and 5L.

Article Snippet: Cells were infected with lentiviruses coding β‐arrestin‐1 ‐sgRNA (neomycin resistant) and then selected with G418 (MCE HY‐17561).

Techniques: Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, ChIP-sequencing, Binding Assay, Activation Assay, RNA Sequencing, Western Blot, Transfection, Staining, Infection

Journal: Advanced Science

Article Title: T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

doi: 10.1002/advs.202307648

Figure Lengend Snippet: Modification of β‐arrestin‐1 with O‐linked N‐acetylglucosamine stabilizes β‐arrestin‐1 by preventing its ubiquitin‐dependent proteolysis. A) Expression of β‐arrestin‐1 binding proteins in normal or T‐2 toxin‐exposed podocytes was shown in a heat map. B) Endogenous interplays of β‐arrestin‐1 with MIB1 and ubiquitin. T‐2 toxin‐exposed podocytes with or without MG‐132 were subject to Co‐IP using a β‐arrestin‐1 antibody, followed by Western blot analysis with MIB1 and ubiquitin antibodies. (C‐D) Co‐IP and Western blot analyzed β‐arrestin‐1 ubiquitination levels in podocytes cultured with or without T‐2 toxin transfected with control siRNA, MIB1 siRNA C) or OGT siRNA D). E) GlcNAc‐modified β‐arrestin‐1 at site threonine 98 was detected by LC‐MS/MS in T‐2 toxin‐exposed podocytes as compared with normal controls. F) Five candidate O‐GlcNAcylated residues of β‐arrestin‐1, identified by MS, were exhibited in the context of protein domains, secondary structure elements, along with interacting partners of β‐arrestin‐1. The figure below shows the predictive β‐arrestin‐1 domain for MIB1 binding. G) Co‐IP combined with Western blot analysis of the MIB1 binding domain in β‐arrestin‐1 in podocytes transfected with Flag‐tagged MIB1 overexpressed plasmid or different HA‐tagged β‐arrestin‐1 truncation plasmids. H) Molecular docking using PyMol showed that the O‐GlcNAcylation of β‐arrestin‐1 at sites of threonine 98 leads to the dissociation of MIB1 from β‐arrestin‐1. I) Myc‐tagged β‐arrestin‐1 constructs and Flag‐tagged MIB1 were transfected into Crispr/Cas9 β‐arrestin‐1 ‐KO podocytes, and then incubated with T‐2 toxin and MG‐132 in the presence or absence of OSMI‐1, and anti‐β‐arrestin‐1 antibody was used for IP. Precipitates were analyzed by Western blot analysis with indicated antibodies.

Article Snippet: Cells were infected with lentiviruses coding β‐arrestin‐1 ‐sgRNA (neomycin resistant) and then selected with G418 (MCE HY‐17561).

Techniques: Modification, Ubiquitin Proteomics, Expressing, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Transfection, Control, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Construct, CRISPR, Incubation

Journal: Advanced Science

Article Title: T‐2 Toxin‐Mediated β‐Arrestin‐1 O‐GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation

doi: 10.1002/advs.202307648

Figure Lengend Snippet: OGT deletion reduces β‐arrestin‐1 expression and alleviates mTOR pathway‐mediated glomerular podocyte injury in T‐2 toxin‐fed mice. A) Representative images of PAS and MASSON‐stained renal cortex sections (scale bar, 40 µm, n = 3). B) GBM thickness. C) TEM showed podocyte morphology (Scale bar, 500 nm, n = 5) and number of autophagosomes (Scale bar, 200 nm, n = 5). D) Foot process width. (E‐G) Western blot analyzed Nephrin E), Podocin E), β‐arrestin‐1 F), p300 F), H4K16ac F), p‐mTOR (s2448) G), LC3 G), Beclin1 G) and ATG5 G) protein levels in glomeruli ( n = 6). H) The interplays of β‐arrestin‐1 with MIB1, O‐GlcNAc, and β‐arrestin‐1 ubiquitination and O‐GlcNAcylation in isolated glomeruli were analyzed by Co‐IP. I) The interaction of β‐arrestin‐1 with p300 and H4K16ac in isolated glomeruli was analyzed by Co‐IP. *** p < 0.001, +++ p < 0.001, data are expressed as mean ± SEM; one‐way ANOVA followed by Tukey's post hoc test was used.

Article Snippet: Cells were infected with lentiviruses coding β‐arrestin‐1 ‐sgRNA (neomycin resistant) and then selected with G418 (MCE HY‐17561).

Techniques: Expressing, Staining, Western Blot, Ubiquitin Proteomics, Isolation, Co-Immunoprecipitation Assay

Journal: Science immunology

Article Title: The tumor-intrinsic role of the m 6 A reader YTHDF2 in regulating immune evasion

doi: 10.1126/sciimmunol.adl2171

Figure Lengend Snippet: ( A and B ) The tumor growth of WT and Ythdf2 -KO MC38 tumors ( n = 5) and B16-OVA tumors ( n = 6) in C57BL/6 mice. C57BL/6 mice were subcutaneously injected with either 1 × 10 6 WT and Ythdf2 -KO MC38 cells (A) or 1 × 10 6 WT and Ythdf2 -KO B16-OVA cells (B). Tumor sizes were measured every other day starting from the 12th or 14th day after tumor inoculation. ( C and D ) Overall survival of immunocompetent mice subcutaneously implanted with WT or Ythdf2 -KO B16-OVA tumor cells ( n = 14 for WT and n = 15 for KO) (C) or MC38 tumor cells ( n = 8 for WT and n = 10 for KO) (D). ( E and F ) WT or Ythdf2 -KO B16-OVA tumors were transplanted from Rag1 −/− to C57BL/6 mice ( n = 4). Tumor volume (E) and tumor weight (F) were measured on the 14th day after the tumor was transplanted. ( G ) A total of 1 × 10 6 WT or Ythdf2 -KO B16-OVA tumor cells were subcutaneously implanted in C57BL/6 mice pretreated with anti–immunoglobulin G (IgG), anti-NK1.1, anti-CD4, and anti-CD8 antibodies ( n = 5 or 6). Tumor growth was monitored by measuring tumor sizes every other day starting from the eighth day after tumor inoculation. ( H and I ) WT or Ythdf2 -KO MC38-OVA (H) and B16-OVA (I) tumor growth in Rag1 −/− mice that were adoptively transferred with OT1 CD8 + T cells. The mice were subcutaneously injected with either 1 × 10 6 WT or Ythdf2 -KO tumor cells. Tumor sizes were measured every other day starting from the sixth day after tumor inoculation ( n = 5). ( J ) WT or Ythdf2 -KO B16F10 tumor growth in Rag1 −/− mice that were adoptively transferred with gp100-stimulated pmel-1 splenocytes. The mice were subcutaneously injected with either 1 × 10 6 WT or Ythdf2 -KO tumor cells. Tumor sizes were measured every other day starting from the sixth day after tumor inoculation ( n = 5). Data are represented as means ± SD. Statistical analysis was performed using two-way ANOVA with a mixed-effects model with P values adjusted by a Holm-Šídák method for multiple comparisons (A, B, G, H, I, and J), unpaired two-tailed t test (E and F), or Kaplan-Meier survival analysis and log-rank test (C and D). The data presented represent one of two or three independent experiments. * P < 0.05, ** P < 0.01, and **** P < 0.001.

Article Snippet: Splenocytes isolated from pmel-1 T cell receptor transgenic mice were stimulated with gp100 25–33 (5 μg/ml; MedChemExpress) and mouse recombinant IL-2 (100 IU/ml) in RPMI 1640 complete medium at 37°C for 24 hours.

Techniques: Injection, Two Tailed Test

Journal: The Tohoku journal of experimental medicine

Article Title: Osteopontin as a key mediator for vasculogenic mimicry in hepatocellular carcinoma.

doi: 10.1620/tjem.224.29

Figure Lengend Snippet: Fig. 7. Immunohistochemical staining for VM in HCC and normal liver tissues. (A) VM expression was identified in a HCC lesion by laminin immunostain (red arrow). (B) The same HCC lesion in (A) showing VM expression by laminin im- munostain did not express the endothelial cell marker CD31. Hepatic sinusoids were CD34 positive (yellow arrow). (C) The same HCC lesion in (A) was positive for hepatocyte. (D) A HCC lesion without VM expression by laminin im- munostain. Bars = 50 μm.

Article Snippet: After washing in 0.1 M phosphate-buffered saline (PBS), pH 7.4, and exposure to 3% normal goat serum for 20 min to reduce non-specific binding, the slides were incubated for 2 h at 37°C with a 1:100 dilution of anti-laminin monoclonal antibody (Millipore), or a 1:100 dilution of anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or a working solution of anti-hepatocyte antibody (Zhongshan Goldenbridge Biotechnology CO Ltd, ready to use), or a 1:300 dilution of anti-OPN antibody (Santa Cruz), or a 1:300 dilution of anti-MMP-2 antibody (Santa Cruz), or a 1:300 dilution of anti-MMP-9 antibody (Santa Cruz), or a 1:300 dilution of anti-uPA antibody (Santa Cruz).

Techniques: Immunohistochemical staining, Staining, Expressing, Marker

Journal: Hepatology (Baltimore, Md.)

Article Title: Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination

doi: 10.1002/hep.30844

Figure Lengend Snippet: The virions produced under NA treatment were mainly disabled to infect PHHs. (A,B) HBeAg and HBsAg levels in the media of PHH cultures infected with virions produced by HepAD38 cells treated with DMSO (NT) or 2.5 μM of ETV were detected 2, 4, and 6 days postinfection, respectively. (C,D) HBeAg and HBsAg levels in the media of PHH cultures infected with virions from sera of CHB patients with or without NA therapy were detected 2, 4, and 6 days postinfection, respectively. Negative control (NC) is media harvested from uninfected cells. The dashed lines indicated the LLoD of HBeAg and HBsAg, respectively. PE IU/mL, Paul Ehrlich Institute Units.

Article Snippet: Media of HepG2‐NTCP and PHH cultures were harvested at 2, 4, and 6 days post‐HBV infection, and levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in culture media were detected by time‐resolved fluoroimmunoassay, according to the manufacturer’s instructions (PerkinElmer, Waltham, MA).

Techniques: Produced, Infection, Negative Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination

doi: 10.1002/hep.30844

Figure Lengend Snippet: HBV virions produced under NA treatment were mainly disabled to infect HepG2‐NTCP cells. (A) Schematic for the treatment of HepAD38 cells and HBV‐DNA quantification of the supernatant with or without ETV treatment. (B) HBV capsid level with adjusted HBV DNA was detected by western blotting of HBV core (HBc) protein. (C) Immunofluorescence demonstration of NTCP expression in HepG2‐NTCP cells cultured in the presence of Dox (right). (D‐F) HBeAg, HBsAg, and HBV DNA in the media of HepG2‐NTCP cultures infected with virions produced by HepAD38 cells treated with DMSO (NT) or ETV at the indicated MOI were tested. Negative control (NC) is media harvested from uninfected cells. (G) HBeAg levels in the media of HepG2‐NTCP cultures infected with virions produced by HepAD38 cells treated with different concentrations (0, 100, 300, 600, 1,200, and 2,500 nM) of ETV were detected 2, 4, and 6 days postinfection, respectively. (H) The IC 50 of ETV in inhibiting progeny HBV infectivity was calculated by nonlinear regression of log (inhibitor) versus response. PE IU/mL, Paul Ehrlich Institute Units.

Article Snippet: Media of HepG2‐NTCP and PHH cultures were harvested at 2, 4, and 6 days post‐HBV infection, and levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in culture media were detected by time‐resolved fluoroimmunoassay, according to the manufacturer’s instructions (PerkinElmer, Waltham, MA).

Techniques: Produced, Western Blot, Immunofluorescence, Expressing, Cell Culture, Infection, Negative Control