Journal: Scientific Reports

Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

doi: 10.1038/s41598-019-51998-w

Figure Lengend Snippet: Knockdown of cGAS or IFI16 partly inhibits nsDNA-mediated APOL1 expression in human immortalized AB8/13 podocytes. The cells were transfected for 48 h with control siRNA (Co) or siRNA pool targeting cGAS ( a – c ) or IFI16 ( d – f ) and subsequently transfected with 1 μg ml −1 nsDNA for 18 h. ( a , d ) Expression of indicated proteins was analyzed by immunoblotting. Protein size markers (kDa) are shown. Intensities of cGAS, STING, IFI16, APOL1, and GAPDH protein bands were quantified by densitometric scanning. ( a ) Expression levels of cGAS, STING, and APOL1 were normalized against GAPDH levels and presented as cGAS/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. In cells transfected with control siRNA only, the cGAS/GAPDH and STING/GAPDH ratios were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA was set as 1.0. The blot images were obtained from different individually probed gels. ( d ) Expression levels of IFI16, STING, and APOL1 are presented as IFI16/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. In cells transfected with control siRNA only, the IFI16/GAPDH and STING/GAPDH ratios were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA was set as 1.0. The blot images were obtained from different gels. The blot probed for APOL1 was re-probed for IRF3. Full images of the blots in (a) and (d) are shown in Supplementary Fig. . Expression of cGAS ( b ), IFI16 ( e ), and APOL1 ( c , f ) mRNA was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

Article Snippet: AB8/13 podocytes were cultured in six-well plates (2.5 × 10 5 cells/well) and transfected with the cGAS CRISPR double nickase plasmid (1–3 μg/ml, Santa Cruz Biotechnology) using the jetPrime reagent (VWR) following manufacturer’s protocol.

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

doi: 10.1038/s41598-019-51998-w

Figure Lengend Snippet: cGAS and IFI16 are required for maximal APOL1 expression in human immortalized AB8/13 podocytes in response to nsDNA. ( a ) AB8/13 podocytes were transfected for 48 h with control siRNA (Co) or siRNA pool targeting either cGAS or IFI16 or both. The cells were subsequently transfected with 1 μg ml −1 nsDNA for the times indicated. The blot images were obtained from different gels. Left panel: The blot probed for cGAS was re-probed for GAPDH. The blot probed for APOL1 was re-probed for TBK1. Right panel: The blot probed for cGAS was re-probed for GAPDH. The blot probed for APOL1 was re-probed for TBK1. Expression levels of IFI16, STING, and APOL1 are presented as IFI16/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. The IFI16/GAPDH and STING/GAPDH ratios in cells transfected with control siRNA only were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA (18 h) was set as 1.0. ( b ) cGAS −/− cells generated from parental AB8/13 podocytes (cGAS +/+ ) were transfected for 48 h with control siRNA (Co) or siRNA pool targeting IFI16 and subsequently transfected with 1 μg ml −1 nsDNA for the times indicated. Expression of indicated proteins was analyzed by immunoblotting. Protein size markers (kDa) are shown. The blot images were obtained from different gels. The blot probed for cGAS was re-probed for GAPDH. The other blot images were cropped from individually probed blots. The IFI16/GAPDH and STING/GAPDH ratios in cGAS +/+ cells exposed to transfection reagent only were both set as 1.0. The APOL1/GAPDH ratio in cGAS +/+ cells transfected with nsDNA (18 h) was set as 1.0. Full images of the blots are shown in Supplementary Fig. .

Article Snippet: AB8/13 podocytes were cultured in six-well plates (2.5 × 10 5 cells/well) and transfected with the cGAS CRISPR double nickase plasmid (1–3 μg/ml, Santa Cruz Biotechnology) using the jetPrime reagent (VWR) following manufacturer’s protocol.

Techniques: Expressing, Transfection, Control, Generated, Western Blot

Journal: Scientific Reports

Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

doi: 10.1038/s41598-019-51998-w

Figure Lengend Snippet: nsDNA-induced APOL1 expression is partly attenuated by JAK1/JAK2 inhibitor Ruxolitinib. ( a ) Ruxolitinib (Ruxo) inhibited IFNβ-mediated phosphorylation of STAT1. Sets of AB8/13 podocytes were treated with DMSO (solvent) only (Control), treated for 2 h with 5 μM Ruxo, treated for 15 min with 10 ng ml −1 IFNβ, or pretreated with Ruxo for 2 h followed by IFNβ stimulation for 15 min (Ruxo/IFNβ). The blot images were obtained from different gels. The blot probed for P-STAT1 was re-probed for GAPDH. ( b , c ) AB8/13 podocytes were treated with Ruxo and IFNβ as indicated and subsequently transfected with 1 μg ml −1 nsDNA for 2 h ( b ) or 18 h ( c ). The IFI16/GAPDH and APOL1/GAPDH ratios in unstimulated cells (Control) were both set as 1.0. The blot images in ( b ) were obtained from different gels. The blot probed for P-IRF3 was re-probed for P-STING. Other blot images were cropped from individually probed blots. The blot images in ( c ) were obtained from different gels. The blot probed for IFI16 was re-probed for cGAS. Other blot images were cropped from individually probed blots. Full images of all blots are shown in Supplementary Fig. . ( d , e ) Ruxo abolished expression of APOL1 ( d ) and IFI16 mRNA ( e ) induced by exogenous IFNβ. ( f , g ) Ruxo partially inhibited nsDNA-induced APOL1 mRNA expression ( f ) but abolished nsDNA-induced IFI16 mRNA expression ( g ). Expression of APOL1 and IFNβ mRNA was analyzed by qRT-PCR 18 h after transfection with 1 μg ml −1 nsDNA. mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

Article Snippet: AB8/13 podocytes were cultured in six-well plates (2.5 × 10 5 cells/well) and transfected with the cGAS CRISPR double nickase plasmid (1–3 μg/ml, Santa Cruz Biotechnology) using the jetPrime reagent (VWR) following manufacturer’s protocol.

Techniques: Expressing, Phospho-proteomics, Solvent, Control, Transfection, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

doi: 10.1038/s41598-019-51998-w

Figure Lengend Snippet: Our proposed model of nsDNA-induced APOL1 expression through engagement of the cGAS/IFI16-STING pathway in human immortalized AB8/13 podocytes. Binding of cytosolic nsDNA by cGAS and IFI16 activates STING, which subsequently activates TBK1. Activated TBK1 phosphorylates IRF3, which promotes transcription of APOL1 and IFNβ . IFNβ released from the cells (or exogenous IFNβ) binds to IFNAR. IFNAR-associated JAK1 and Tyk2 kinases then phosphorylate STAT1, which promotes transcription of APOL1 and IFI16 . A putative IFI16-mediated activation of STING is indicated by a dashed arrow. A potential cooperation between cGAS and IFI16 is indicated by a double-headed arrow. Deficient STING phosphorylation observed in cGAS- or IFI16-knockdown cells (Fig. ) suggests that nsDNA-induced APOL1 expression may be mediated by a phospho-STING-independent pathway, marked by a green arrow. A dual JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) suppresses STAT1 activation and thereby inhibits IFI16 expression and STAT1-mediated APOL1 expression. Since IFNAR-mediated signaling involves JAK1 but not JAK2, our model only depicts the inhibition of JAK1 by Ruxo. Ruxo does not affect IRF3-mediated APOL1 expression.

Article Snippet: AB8/13 podocytes were cultured in six-well plates (2.5 × 10 5 cells/well) and transfected with the cGAS CRISPR double nickase plasmid (1–3 μg/ml, Santa Cruz Biotechnology) using the jetPrime reagent (VWR) following manufacturer’s protocol.

Techniques: Expressing, Binding Assay, Activation Assay, Phospho-proteomics, Knockdown, Inhibition