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: (A) Enhanced calcium influx was observed in FG4592 treated podocytes whereas 2-APB, a calcium channel blocker; and TRPC6 knockdown abrogated FG4592 induced calcium flux. Error bars indicate mean ± SE; n=3. **, p < 0.001; ***, p < 0.0005; ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison test. ( B ) TG2 activity was measured as a readout of hydroxamate formed in the invitro TG2 activvity in podocyte lysates from FG4592, 2-APB, and TRPC6 knockdown conditions and the activity represented as units per mg of protein. Error bars indicate mean ± SE; n=3. ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison test. (C) TRPC6 and TG2 expression was assessed in podocytes transfected with TRPC6 siRNA and treated with or without FG4592. (D) Densitometric analysis of western blots was normalized to β-actin. Data presented as mean ± SE; n=3. *, p < 0.01; ***, p < 0.0005; ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison 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: Knockdown, Comparison, Activity Assay, Expressing, Transfection, Western Blot

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) The promoter region of TG2 with ZEB2 binding E-box regions and region that was amplified following ChIP is indicated by arrows. (B) DNA from immunoprecipitated samples of podocytes treated with or without FG4592 was PCR amplified for ZEB2. E2-box in N-cadherin promoter serves as a positive control for ZEB2 interaction. Data presented as mean ± SE; n=3. ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison test. (C) Immunoblotting analysis showing protein levels of ZEB2, TRPC6, and TG2 in HEK293T cells which are ectopically express ZEB2 and ZEB2 knockdown conditions. (D) Intracellular calcium flux was observed in FG4592 treated podocytes and podocytes in which ZEB2 is ectopically expressed or knockdown. Data presented as mean ± SE; n=3. ***, p < 0.0005; ****, p < 0.0001 by one-way ANOVA after Tukey’s multiple comparison test. (E) TG2 activity was assessed in podocyte lysate treated with FG4592 and podocytes in which ZEB2 is ectopically expressed or knockdown. Data presented as ± SE; n=3. **, p < 0.001; ***, p < 0.0005; by one-way ANOVA after Tukey’s multiple comparison 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: Binding Assay, Amplification, Immunoprecipitation, Positive Control, Comparison, Western Blot, Knockdown, Activity Assay

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