Journal: bioRxiv

Article Title: ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

doi: 10.1101/2021.12.15.472753

Figure Lengend Snippet: (A) Expression of HIF1α, ZEB2, TG2 , and TRPC6 was analyzed between healthy volunteers (n=3) and chronic kidney disease patients (n=5) in the validated set group of Nakagawa CKD kidney database in Nephroseq ( www.nephroseq.org ). Immunoblotting analysis of HIF1α, TG2, and β-actin in human glomerular podocytes treated with (B) 1,5,10, and 15 µM of FG4592 for 12h and ( C ) 10µM FG4592 for 3,6,12, and 24h. (D) Immunofluorescence detection of TG2 in control vs. FG4592 treated podocytes. Scale bar 20μm. Nephroseq data was validated in human podocytes exposed to 10µM of FG4592 for 24h by (E) immunoblotting and ( F ) qRT-PCR. Error bars indicate mean ± SE; n=3. *, p < 0.02; **, p <0.001; ***, p < 0.0002; ****, p < 0.0001 by student t-test.

Article Snippet: TRPC6 siRNA (#SC-42672), ZEB2 siRNA (#SC-38641) and HIF1α inhibitor (#sc-205346) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Western Blot, Immunofluorescence, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

doi: 10.1101/2021.12.15.472753

Figure Lengend Snippet: Immunoblotting analysis of HIF1α, ZEB2,TRPC6,TG2, vimentin, fibronectin, and α-SMA in podocytes (A) treated with FG4592 in the presence or absence of metformin or (B) exposed to hypoxia (1% oxygen) in the presence or absence of metformin, and (C) exposed to hypoxia (1% oxygen) in the presence or absence of HIF1α inhibitor. Immunofluorescence detection of (D) HIF1α and (E) TG2 in podocytes treated with FG4592 with or without metformin treatment. Scale bar 10μm.

Article Snippet: TRPC6 siRNA (#SC-42672), ZEB2 siRNA (#SC-38641) and HIF1α inhibitor (#sc-205346) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

doi: 10.1101/2021.12.15.472753

Figure Lengend Snippet: (A) Immunoblotting analysis of HIF1α, ZEB2, TRPC6, TG2 and mesenchymal markers in glomerular lysate from mice administered with FG4592 and treated with or without metformin. (B) Masson’s Trichrome Staining (MTS), PAS, and H&E staining of glomerular regions of mice administered with FG4592 and treated with or without metformin. Scale bar 20μm. (C) Glomerular injury analysis was performed as described in the methods section. Data presented as mean ± SE; n=9. ***, p < 0.0005; ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison test. (D) TEM images of podocytes from mice administered with FG4592 and treated with or without metformin. Arrow marks indicate the podocyte FPE. Scale bar 1μm.

Article Snippet: TRPC6 siRNA (#SC-42672), ZEB2 siRNA (#SC-38641) and HIF1α inhibitor (#sc-205346) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Western Blot, Staining, Comparison

Journal: bioRxiv

Article Title: ZEB2 transduces HIF1α dependent regulation of Transglutaminase 2 in glomerular podocytes

doi: 10.1101/2021.12.15.472753

Figure Lengend Snippet: (A) Silver staining of urinary proteins from mice administered with FG4592 and metformin. (B) UACR from mice administered with FG4592 and treated with or without metformin. Data presented as mean ± SE; n=6. ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison test. (C) Representative images of immunohistochemical staining for TG2 and ZEB2 in glomerular sections from people with or without DN. Scale bars indicate 20μm. (D) Albumin levels were estimated from the human urine samples in healthy vs DN patients. Data presented as mean ± SE; n=12. ****, p < 0.0001 by student t-test. (E) A scheme depicting the mechanism of regulation of TG2 by HIF1α/ZEB2 axis.

Article Snippet: TRPC6 siRNA (#SC-42672), ZEB2 siRNA (#SC-38641) and HIF1α inhibitor (#sc-205346) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Silver Staining, Comparison, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Metal-organic polyhedra maintain the self-renewal of embryonic stem cells

doi: 10.1038/s41467-025-63811-6

Figure Lengend Snippet: A The photo of MOP-1 aqueous solution (left). Monodisperse structure of MOP-1 in H 2 O (right). Color codes: V, green; O, red; C, gray; N, blue; Cl, bright blue. The large pink sphere represents the free space inside the molecular cage. For clarity, H atoms were omitted. Schematic (right panel) was created with Diamond software. B Experimental and simulated PXRD patterns of MOP-1. C TEM image of MOP-1 (left). Scale bar, 20 nm. The particle size of MOP-1 in H 2 O (right). Three experiments were repeated independently with similar results. D The schematic diagram showed that the SHP-2 mediated STAT3 inactivation. Schematic diagram was created with Microsoft Office PowerPoint. E , F An in-depth mechanism investigation of mESC pluripotency control by MOP-1. Binding model from a global view of a complex composed of SHP-2 and MOP-1 illustrated by electrostatic surface potential ( E ). Binding modes are illustrated by ribbon diagrams of a complex composed of SHP-2 and MOP-1 (the left panel), a complex composed of SHP-2 with MOP (the middle panel) and a complex composed of SHP-2 with ZrMOP (the right panel). The top panel is the global view of the catalytic PTP structure of SHP-2, the bottom panel is the focused view of binding modes illustrated by the Ribbon diagrams ( F ). G Binding kinetics of MOP-1 (top panel) and MOP-2 (bottom panel) with SHP-2 were measured by the SPR assay. H The ICP-MS analysis of the binding quantity between MOPs and SHP-2 (mean ± s.e.m, n = 6). I The inhibition efficiency of SHP-2, JAK2, JAK1, SHP-1, PTP1B, Cyt c , ACP and lipase by MOP-1 at a concentration of 2 μM using enzyme assay (mean ± s.e.m, n = 3). Data in ( H ) and ( I ) are analyzed by one-way ANOVA. **** P < 0.0001, the binding between MOP-1 and SHP-2 vs. the binding between MOP-2 and SHP-2, relative activity of SHP-2 vs. relative activity of JAK2, JAK1, PTP1B, Cyt c , ACP and lipase. *** P < 0.001, relative activity of SHP-2 vs. relative activity of SHP-1.

Article Snippet: The binding kinetics and affinity of MOP-1 or MOP-2 to SHP-2 (MCE, HY-P700618), SHP-1 (MCE, HY- P71141 ), JAK1 (MCE, HY-P700583), JAK2 (MCE, HY-P701102) and PTP1B (MCE, HY- P73685 ) were analyzed by SPR (Biacore 8 K, Cytiva).

Techniques: Software, Control, Binding Assay, SPR Assay, Inhibition, Concentration Assay, Enzymatic Assay, Activity Assay

Journal: Nature Communications

Article Title: Metal-organic polyhedra maintain the self-renewal of embryonic stem cells

doi: 10.1038/s41467-025-63811-6

Figure Lengend Snippet: A, B Cell viability assay of MOP-1 (top panel, mean ± s.e.m, n = 3), MOP-2 (second panel, mean ± s.e.m, n = 3), ZrMOP (third panel, mean ± s.e.m, n = 6) and ligand (bottom panel, mean ± s.e.m, n = 5). Schematics (left panel) were created with Diamond software. A MOPs treated mESC (J1 cell line) exhibited flat morphology compared with the mESC cultured in LIF addition medium (denoted as LIF mESC) and mESC cultured without LIF (denoted as Ctrl mESC), which grew in colonies and expressed positive alkaline phosphatase (ALP) staining ( B ). Scale bar, 100 μm. C Representative images of ALP staining (left panel) and quantification area of LIF and MOP-1 mESC colonies (right panel) (mean ± s.e.m, n = 3). Scale bars, 40 μm). D The cell cycle distribution of MOP-1 mESC was evaluated by flow cytometry. The percentage of G0/G1, S, G2/M phases of the cell cycle (mean ± s.e.m, n = 3). E Plots of FITC-annexin V/PI flow cytometry of mESC cultured with or without MOP-1 for 48 h. F Representative immunoblot images (left panel) and quantification (right panel) of p-SHP-2 in MOP-1 mESC under different concentrations (mean ± s.e.m, n = 3). G Representative immunoblot images of STAT3 in LIF mESC and MOP-1 mESC at different withdrawal times (mean ± s.e.m, n = 4). Data in ( C ) and ( D ) are analyzed by a two-tailed unpaired t -test. Data in ( F ) are analyzed by one-way ANOVA. # P < 0.05, 0 μM MOP-1 mESC vs. LIF mESC; * P < 0.05, 2 μM and 4 μM MOP-1 mESC vs. 0 μM MOP-1 mESC; no statistical significance (NS), LIF mESC vs. 2 μM and 4 μM MOP-1 mESC, MOP-1 mESC vs. LIF mESC or Ctrl mESC.

Article Snippet: The binding kinetics and affinity of MOP-1 or MOP-2 to SHP-2 (MCE, HY-P700618), SHP-1 (MCE, HY- P71141 ), JAK1 (MCE, HY-P700583), JAK2 (MCE, HY-P701102) and PTP1B (MCE, HY- P73685 ) were analyzed by SPR (Biacore 8 K, Cytiva).

Techniques: Viability Assay, Software, Cell Culture, Staining, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Nature chemical biology

Article Title: Bidirectional modulation of HIF-2 activity through chemical ligands.

doi: 10.1038/s41589-019-0234-5

Figure Lengend Snippet: Fig. 2 | Allosteric mechanism of PT2385 revealed by the crystal structure and H/D-ex MS. a, Binding position for PT2385 (circled in black) within the entire HIF-2α–ARNT crystal structure. b, Close-up look at the location of PT2385 inside the HIF-2α PAS-B domain, with green mesh showing the FO– FC omit map contoured at 2.7σ. c, Interactions of PT2385 (yellow) with surrounding residues in the pocket. d, The overall arrangement of ARNT and HIF-2α PAS-B domains is displayed on the left, with the HIF-2α PAS-B from PT2385-bound (magenta) or apo (orange) complexes superimposed. On the right, an enlarged and rotated view shows the different side chain orientations of M252 in these two complexes. The side chain movement of HIF-2α M252 caused by antagonist binding disrupts the dimerization of the HIF-2α–ARNT complex at the PAS-B–PAS-B interface. e, H/D-ex MS results mapped on the crystal structure of the HIF-2α–ARNT complex. The regions showing dynamic changes upon PT2385 binding are colored according to the maximum differences of deuteration levels (red for 40–50%; pink for 25–40%) as compared to the apo form complex (detailed in Supplementary Fig. 4), on top of the background colors of HIF-2α (gray) and ARNT (pale yellow). Enlarged and rotated views of dimer interfaces between the PAS-B domains and among the PAS-A and bHLH domains are shown on the left and right, respectively.

Article Snippet: HIF-2α– ARNT protein complex was diluted in a two-fold series in binding buffer (20 mM Tris pH 8.0, 20 mM NaCl), and each sample was incubated with 0.1 μM, 1 μM or 10 μM PT2385 (MedChemExpress HY-12867, 99.48% purity) for 1 h (with the same amount of 0.1% DMSO as a control), before binding assays started by the addition of 2 nM DNA.

Techniques: Binding Assay

Journal: Nature chemical biology

Article Title: Bidirectional modulation of HIF-2 activity through chemical ligands.

doi: 10.1038/s41589-019-0234-5

Figure Lengend Snippet: Fig. 4 | Newly identified antagonist T1001 points to a common inhibitory mechanism. a, Chemical structure of T1001. b, Binding of T1001 to the HIF-2α PAS-B domain (Kd of about 246 nM) measured by MST. c, Inhibition on the expression of HIF-2α target genes in 786-O cells by PT2385 (1 µM) and T1001 (10 µM). d, Binding site of T1001 within the HIF-2α PAS-B pocket, with green mesh showing the FO– FC omit map contoured at 3.0σ. e, Comparison of the inhibitory effects of 10 µM T1001 on expression of HIF-2 target genes in HEK293T cells transfected with wild-type HIF-2α or M252A mutant. f, Comparison of HIF-2α residues surrounding antagonists in the PAS-B structures. The ligands and residues are in magenta, pink and brown, for PT2385, 0X3 and T1001, respectively. g, A mechanistic diagram showing how antagonist binding leads to the movement of M252 to disrupt HIF-2α–ARNT heterodimers. The proximate positions of M252 side chain in apo (orange) and antagonist-bound structures are compared and related to the disruption potency. Error bars, mean ± s.d.; n = 3 (distinct replicates for cell cultures).

Article Snippet: HIF-2α– ARNT protein complex was diluted in a two-fold series in binding buffer (20 mM Tris pH 8.0, 20 mM NaCl), and each sample was incubated with 0.1 μM, 1 μM or 10 μM PT2385 (MedChemExpress HY-12867, 99.48% purity) for 1 h (with the same amount of 0.1% DMSO as a control), before binding assays started by the addition of 2 nM DNA.

Techniques: Binding Assay, Inhibition, Expressing, Comparison, Transfection, Mutagenesis, Disruption

Journal: Nature chemical biology

Article Title: Bidirectional modulation of HIF-2 activity through chemical ligands.

doi: 10.1038/s41589-019-0234-5

Figure Lengend Snippet: Fig. 5 | The allosteric mechanism of agonist M1001. a, Chemical structure of M1001. b, Binding of M1001 to the HIF-2α PAS-B domain (Kd of about 667 nM) measured by MST. c, Opposite effects of PT2385 (1 µM) and M1001 (10 µM) on the expression of HIF-2 target genes in 786-O cells. d, Binding position for M1001 (circled in black) within the entire HIF-2α–ARNT crystal structure. The visible loop region between ARNT PAS-A and PAS-B domains (A/B loop) is also indicated. e, Close-up look at the location of M1001 inside the HIF-2α PAS-B domain, with green mesh showing the FO– FC omit map contoured at 2.7σ. f, Interactions of M1001 (magenta) with surrounding residues in the pocket. g, The overall arrangement of ARNT and HIF-2α PAS-B domains is displayed in the middle, with the HIF-2α PAS-B domains from M1001-bound (cyan) or apo (orange) complexes superimposed; enlarged or rotated views on both sides show the conformational changes due to M1001 binding. The side chain movement of HIF-2α Y281 not only promotes an additional hydrogen bond between HIF-2α Y281 and ARNT Y456 (right), but also alters the conformation of HIF-2α E287 and enables its interaction with ARNT N350 on the PAS-A/B loop (left). h, H/D-ex MS results mapped on the structure of the HIF-2α–ARNT complex. The regions showing dynamic changes upon M1001 binding are colored according to the maximum differences of deuteration levels (dark blue for −50 to −40%, pale purple for −40 to −25%, and pink for 25 to 40%) as compared to the apo form complex (detailed in Supplementary Fig. 11), on top of the background colors of HIF-2α (gray) and ARNT (pale yellow). Enlarged and rotated views of dimer interfaces between the two PAS-B domains and between the HIF-2α PAS-A and PAS-B domains are shown on the left and right, respectively. Error bars, mean ± s.d.; n = 3 (distinct replicates for cell cultures).

Article Snippet: HIF-2α– ARNT protein complex was diluted in a two-fold series in binding buffer (20 mM Tris pH 8.0, 20 mM NaCl), and each sample was incubated with 0.1 μM, 1 μM or 10 μM PT2385 (MedChemExpress HY-12867, 99.48% purity) for 1 h (with the same amount of 0.1% DMSO as a control), before binding assays started by the addition of 2 nM DNA.

Techniques: Binding Assay, Expressing

Journal: Nature chemical biology

Article Title: Bidirectional modulation of HIF-2 activity through chemical ligands.

doi: 10.1038/s41589-019-0234-5

Figure Lengend Snippet: Fig. 6 | The agonistic effects of M1002. a, Chemical structure of M1002. b, Clear agonistic effects on the expression of HIF-2 target genes in 786-O cells by M1002 (10 µM) as compared to the antagonist PT2385 (1 µM). c, TR-FRET-based protein-binding assays testing the effects of M1002 on the associations between ARNT and its partners HIF-2α, HIF-1α and NPAS3. d, Comparison of the antagonistic effects of 10 µM on HIF-2 target genes in HEK293T cells transfected with wild-type HIF-2α or Y281A mutant. For b–d, error bars, mean ± s.d.; n = 3 (distinct replicates for cell cultures). e, Co-IP results showing the effects of HIF-2α ligands (PT2385, T1001, M1001 and M1002 at various concentrations) on the association between full-length HIF-2α and VHL proteins. This experiment was independently repeated twice with similar results.

Article Snippet: HIF-2α– ARNT protein complex was diluted in a two-fold series in binding buffer (20 mM Tris pH 8.0, 20 mM NaCl), and each sample was incubated with 0.1 μM, 1 μM or 10 μM PT2385 (MedChemExpress HY-12867, 99.48% purity) for 1 h (with the same amount of 0.1% DMSO as a control), before binding assays started by the addition of 2 nM DNA.

Techniques: Expressing, Protein Binding, Comparison, Transfection, Mutagenesis, Co-Immunoprecipitation Assay