Journal: JCI Insight

Article Title: HIPK2 C-terminal domain inhibits NF- κ B signaling and renal inflammation in kidney injury

doi: 10.1172/jci.insight.175153

Figure Lengend Snippet: ( A ) The schematics shows WT full-length human HIPK2 structure and FLAG-tagged HIPK-CT deletion mutant. Nuclear localization signals (NLS) and caspase-6 cleavage sites on D923 and D984 are indicated. Amino acids 1–1,198 and 985–1,198 are shown. ( B ) The effects of HIPK2-CT were examined on transcriptional activities of p65 NF-κB, Smad3, or p53 by luciferase reporters in HEK293T cells. Cells transiently transfected with luciferase vectors were treated with 10 ng/mL TNF-α, 10 ng/mL TGF-β1, or 0.5 μg/mL adriamycin for 16 hours. Vehicle-treated cells served as negative controls. Values represent mean ± SD from 3 independent experiments. **** P < 0.0001 between indicated groups by 2-way ANOVA with Tukey’s correction. ( C ) HEK293T cells were transiently transfected with control (Ctrl) or FLAG-tagged HIPK2-CT plasmid, and lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted using anti-p65 and anti-FLAG antibodies. ( D ) The same lysates were used for immunoprecipitation with anti-p65 antibody and immunoblotted with anti-p65 and anti-FLAG antibodies. Input lysates were also immunoblotted to show the expression of FLAG-tagged proteins. ( E ) HEK293T cells cotransfected with mCherry-p65 and GFP-HIPK2 or GFP-HIPK2-CT were imaged with confocal microscopy. The top row shows representative examples of GFP proteins, and the bottom row shows both mCherry-p65 and GFP proteins. DNA was counterstained with DAPI. Scale bar: 10 μm.

Article Snippet: HEK293T cells were cotransfected with pcDNA4B or HIPK2-CT, as well as Renilla expression vector to normalize transfection efficiency, and p65 luciferase reporter construct or Smad3-binding element luciferase reporter construct or p53 luciferase reporter construct (p65-luc construct was a gift from the laboratory of Huabao Xiong [Department of Medicine, Precision Immunology Institute, Icahn School of Medicine at Mount Sinai], ref. , and SBE-luc and p53-luc were obtained from Addgene).

Techniques: Mutagenesis, Luciferase, Transfection, Control, Plasmid Preparation, Immunoprecipitation, Expressing, Confocal Microscopy

Journal: JCI Insight

Article Title: HIPK2 C-terminal domain inhibits NF- κ B signaling and renal inflammation in kidney injury

doi: 10.1172/jci.insight.175153

Figure Lengend Snippet: ( A ) HEK293T cells transfected with control (Ctrl) or FLAG-tagged HIPK2-CT plasmids (1 μg) were stimulated with 10 ng/mL TNF-α for indicated times (0–30 minutes). Cell lysates were probed for phospho-p65 (S536), total p65, phospho-IκB (S32), and total IκB. GAPDH was used as an internal control. ( B ) Densitometric analysis of p-p65 (S536) and phospho-IκB (S32) levels (normalized to GAPDH). Values represent mean ± SD from 3 independent experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001 between indicated groups by 2-way ANOVA with Bonferroni’s correction. ( C ) HEK293T cells transfected with 0, 0.1, or 1 μg HIPK2-CT plasmid were treated with 10 ng/mL TNF-α for 15 minutes. Cell lysates were probed for phospho-IκB (S32) and total IκB. ( D ) Densitometric analysis of normalized phospho-IκB (S32) and total IκB levels. Values represent mean ± SD from 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 between indicated groups by 1-way ANOVA with Tukey’s correction. ( E ) Lysates of HEK293T cells transfected with FLAG-tagged HIPK2-CT were immunoprecipitated with anti-IκBα antibody and immunoblotted with anti-FLAG antibody. Input lysates were also immunoblotted to show the expression of FLAG-tagged proteins.

Article Snippet: HEK293T cells were cotransfected with pcDNA4B or HIPK2-CT, as well as Renilla expression vector to normalize transfection efficiency, and p65 luciferase reporter construct or Smad3-binding element luciferase reporter construct or p53 luciferase reporter construct (p65-luc construct was a gift from the laboratory of Huabao Xiong [Department of Medicine, Precision Immunology Institute, Icahn School of Medicine at Mount Sinai], ref. , and SBE-luc and p53-luc were obtained from Addgene).

Techniques: Transfection, Control, Plasmid Preparation, Immunoprecipitation, Expressing

Journal: JCI Insight

Article Title: HIPK2 C-terminal domain inhibits NF- κ B signaling and renal inflammation in kidney injury

doi: 10.1172/jci.insight.175153

Figure Lengend Snippet: ( A ) Representative immunofluorescence image of p65 expression in kidneys of mice 3 days after UUO. Scale bar: 20 μm. ( B ) Western blot analysis of phospho-p65 (S536) and total p65 in kidney cortices of mice in each group ( n = 2 per group are shown). ( C ) Densitometric analysis of p-p65 to total p65 is shown for each mouse ( n = 5). **** P < 0.0001 between indicated groups by 1-tailed t test with Welch’s correction. ( D ) Representative immunohistochemical images of F4/80 expression in mouse kidneys. Scale bar: 100 μm. ( E ) Quantification of F4/80 + area per mouse kidney ( n = 5 mice per group, 20 fields per kidney analyzed). **** P < 0.0001 between indicated groups by 2-way ANOVA with Tukey’s correction. ( F ) Quantitative PCR analysis of Ccl2 , Tnfa, and Il6 transcripts in kidney cortices ( n = 5 mice per group). * P < 0.05, ** P < 0.01, **** P < 0.0001 between indicated groups by 1-way ANOVA with Dunnett’s correction.

Article Snippet: HEK293T cells were cotransfected with pcDNA4B or HIPK2-CT, as well as Renilla expression vector to normalize transfection efficiency, and p65 luciferase reporter construct or Smad3-binding element luciferase reporter construct or p53 luciferase reporter construct (p65-luc construct was a gift from the laboratory of Huabao Xiong [Department of Medicine, Precision Immunology Institute, Icahn School of Medicine at Mount Sinai], ref. , and SBE-luc and p53-luc were obtained from Addgene).

Techniques: Immunofluorescence, Expressing, Western Blot, Immunohistochemical staining, Real-time Polymerase Chain Reaction

Journal: JCI Insight

Article Title: HIPK2 C-terminal domain inhibits NF- κ B signaling and renal inflammation in kidney injury

doi: 10.1172/jci.insight.175153

Figure Lengend Snippet: ( A ) Blood urea nitrogen (BUN) levels of mice after 24 hours of LPS or vehicle (Ctrl) injection ( n = 5 mice per group). ** P < 0.01, **** P < 0.0001 between indicated groups by 1-way ANOVA with Dunnett’s correction. ( B ) Representative immunofluorescence images of p65 (green) and F4/80 (red). DNA was counterstained with DAPI. ( C ) Quantitative PCR analysis of Ccl2 , Tnfa, and Il6 transcripts in kidney cortices ( n = 5 mice per group). *** P < 0.001, **** P < 0.0001 between indicated groups by 1-way ANOVA with Dunnett’s correction.

Article Snippet: HEK293T cells were cotransfected with pcDNA4B or HIPK2-CT, as well as Renilla expression vector to normalize transfection efficiency, and p65 luciferase reporter construct or Smad3-binding element luciferase reporter construct or p53 luciferase reporter construct (p65-luc construct was a gift from the laboratory of Huabao Xiong [Department of Medicine, Precision Immunology Institute, Icahn School of Medicine at Mount Sinai], ref. , and SBE-luc and p53-luc were obtained from Addgene).

Techniques: Injection, Immunofluorescence, Real-time Polymerase Chain Reaction