Journal: bioRxiv

Article Title: Two parallel lineage-committed progenitors contribute to the developing brain

doi: 10.1101/2025.07.02.662771

Figure Lengend Snippet: A) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with Vismodegib + PD173074 for 72 hours, in the presence or absence of WNT agonist (CHIR99021, 1-6 μM) or WNT inhibitor (XAV939, 1 μM). This revealed that 2 μM of CHIR99021 (indicated by white circle) was optimal for inducing dorsal forebrain marker EMX2 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . HH inh.: HEDGEHOG inhibitor. FGFR inh.: FGF receptor inhibitor. B) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with CHIR99021 (2 μM) + PD173074 (100 nM) for 72 hours, in the presence or absence of FGF2 (20 ng/mL) or FGFR inhibitor (PD173074, 100 nM). This revealed that FGF inhibition enhanced dorsal forebrain markers EMX2 and PAX6 , while preventing erroneous WNT-induced expression of midbrain marker EN1 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . FGFRi: FGF receptor inhibitor. C) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with CHIR99021 (2 μM) + PD173074 (100 nM) for 72 hours, in the presence or absence of BMP4 (10 ng/mL) or BMPR inhibitors (DMH1 [250 nM] or LDN193189 [100 nM]). Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . BMPRi: BMP receptor inhibitor. D) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then anterior neural ectoderm within 24 hours, followed by forebrain progenitors within 48 hours. Forebrain progenitors were then treated with XAV939 (1 μM) in the presence or absence of increasing doses of HEDGEHOG pathway agonist 21K (0.1-5 nM) for 72 hours to generate ventral forebrain. This showed that high HEDGEHOG pathway activation, together with WNT inhibition, led to maximum expression of ventral forebrain markers. Gene expression is shown relative to the sample with the highest expression in this experiment. HH: Hedgehog pathway agonist. WNTi: WNT inhibitor. E) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with HEDGEHOG inhibitor (Vismodegib, 150 nM) and WNT agonist (CHIR99021, 2 μM) for 72 hours to generate dorsal forebrain, in the presence or absence of BMP agonist (BMP4, 10 ng/mL) or BMPR inhibitor (LDN193189, 100 nM). This showed that BMP inhibition promoted expression of dorsal forebrain markers GSX2 and PAX3 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . BMPRi: BMP receptor inhibitor. F) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with HEDGEHOG pathway inhibitor Vismodegib (150 nM) in the presence or absence of increasing doses of WNT pathway agonist CHIR99021 (1-4 μM) for 72 hours to generate dorsal forebrain. This showed that moderate WNT pathway activation, together with HH inhibition, led to maximum expression of dorsal hindbrain markers GSX2 and OLIG3 . Gene expression is shown relative to reference gene YWHAZ ; 100% indicates that a gene is expressed at the same level as YWHAZ . BMPRi: BMP receptor inhibitor. G) scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. Colors denote differentiation conditions. An asterisk (*) indicates that the legend is identical to that shown in , and is reproduced here to aid the interpretation of other images shown in this subpanel. H) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with either Vismodegib (150 nM) + CHIR99021 (2 μM) + DMH1 (250 nM) for 72 hours to generate dorsal forebrain, or alternatively, 21K (5 nM) + XAV939 (1 μM) for 72 hours to generate ventral forebrain. Gene expression is shown relative to the sample with the highest expression in this experiment. I) H1 hPSCs were differentiated into definitive ectoderm within 24 hours, and then posterior neural ectoderm within 24 hours, followed by hindbrain progenitors within 48 hours. Hindbrain progenitors were then treated with WNT pathway inhibitor XAV939 (1 μM) in the presence or absence of increasing doses of HEDGEHOG pathway agonist 21K (50 pM-5 nM) for 72 hours to generate ventral hindbrain. This showed that high doses of HEDGEHOG agonist, together with WNT inhibitor, were required to maximally induce ventral hindbrain markers. Gene expression is shown relative to the sample with the highest expression in this experiment. WNT inh.: WNT inhibitor. qPCR data depicts the mean of two biological replicates, with s.e.m. shown. In vitro scRNAseq data is from a single experiment.

Article Snippet: D5, D6, and D7 dorsal forebrain media consisted of CDM2 supplemented with XAV939 (1 μM, Tocris) and SAG 21K (5 nM, Tocris).

Techniques: Marker, Gene Expression, Inhibition, Expressing, Activation Assay, Derivative Assay, In Vitro

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: bioRxiv

Article Title: Ethanol Exposure Perturbs Sea Urchin Development and Disrupts Developmental Timing

doi: 10.1101/2022.07.07.499183

Figure Lengend Snippet: A. Skeletal birefringence and morphology (DIC, inset) are shown at 48 hpf for embryos treated with cyclopamine (cyc, 0.2 µM) or with SAG (0.3 µM) with or without EtOH (E). B. The fraction of normal and perturbed embryo is plotted for the indicated conditions as the average ± s.e.m.; n ≥ 150 per condition; * p < 0.05; n.s., not significant (t test). Mild perturbations are illustrated by panels A5 and A6. See also Fig. S5.

Article Snippet: Dose-response experiments were performed to determine the optimal working doses for each perturbation reagent, which include EtOH, Acetaldehyde, Retinol, Retinoic Acid, Fomepizole (Sigma-Aldrich or Fisher Scientific), Cyclopamine (Enzo Life Sciences), and SAG (Santa Cruz Biotechnology).

Techniques:

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-SAG antibody , Proteintech , Cat #11905-1-AP; RRID: AB_10697836.

Techniques: Mutagenesis, Recombinant, CCK-8 Assay, Software