Journal: Cell Death & Disease

Article Title: MicroRNA-27a promotes podocyte injury via PPAR γ -mediated β -catenin activation in diabetic nephropathy

doi: 10.1038/cddis.2017.74

Figure Lengend Snippet: High glucose induces miR-27a expression in cultured podocytes. ( a ) qRT-PCR analysis shows the level of miR-27a in various conditions as indicated. ( b ) Representative western blotting shows the expression of PPAR γ and β -catenin target genes in various conditions as indicated. Cell lysates were immunoblotted with specific antibodies against PPAR γ , active β -catenin, snail1, α -SMA, podocin and β -actin. qRT-PCR shows that miR-27a was increased in a ( c ) time and ( d ) dose-dependent manner. ( e-f ) qRT-PCR and ( g-h ) western blot analyses show the expression level of PPAR γ and β -catenin target genes in a time- and dose-dependent manner. ( i ) PPAR γ gene transcription was amplified by miR-27ai and diminished by miR-27am. Mouse podocytes were co-transfected with miR-27ai, miR-27am, and control or wild-type or mutant 3′-UTR of PPAR γ and transfection efficiency was evaluated by luciferase reporter assay. * P <0.05; # P <0.001. Active β -cat, active β -catenin; CTNNB1, catenin beta-1; HG, high glucose; miR-iNC: miRNA inhibitor negative control; mt: mutant type; NG, normal glucose; wt: wild type

Article Snippet: Lysates from podocytes and microdissected glomeruli from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris-buffered saline with Tween-20 (TBST) and probed using primary antibodies against PPAR γ (phospho S112) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR γ (1:100, ab45036, Abcam, Cambridge, UK), non-phospho (active) β -catenin (Ser45) (D2U8Y, 1:50, #19807, Cell Signaling Technology, Beverly, MA, USA), active β -catenin (05-665; EMD Millipore, Billerica, MA, USA), snail1 (1:100, ab53519, Abcam), α -SMA (1:100, ab5694, Abcam), NPHS2 (1:100, ab50339, Abcam), synaptopodin (1:50, 39067, GeneTex, San Antonio, TX, USA) and β -actin (1:200, ab6276, Abcam) at 4 °C overnight.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Amplification, Transfection, Control, Mutagenesis, Luciferase, Reporter Assay, Negative Control

Journal: Cell Death & Disease

Article Title: MicroRNA-27a promotes podocyte injury via PPAR γ -mediated β -catenin activation in diabetic nephropathy

doi: 10.1038/cddis.2017.74

Figure Lengend Snippet: MiR-27ai attenuates podocyte injury via PPAR γ -mediated β -catenin inactivation in high glucose. ( a ) qRT-PCR analysis shows miR-27ai reduced miR-27a expression in HG cultured podocytes. ( b ) Representative western blotting shows the expression of phosphorylated and total PPAR γ and β -catenin target genes in various conditions as indicated. ( c ) qRT-PCR analysis shows miR-27ai upregulated the level of PPAR γ and podocin but downregulated β -catenin target genes. ( d ) HG enhanced the interaction of phosphorylated PPAR γ and active β -catenin by co-immunoprecipitation. ( e ) Transwell migration assay and quantitative data show decreased migration of HG cultured podocytes. Scale bar, 100 μ m. ( f ) Wound-healing assay and quantitative data show decreased invasion of HG cultured podocytes. ( g ) Flow cytometric analysis shows decreased podocyte apoptosis. * P <0.05; # P <0.001. Active β -cat, active β -catenin; 7-AAD, 7-aminoactinomycin D; CTNNB1, catenin beta-1; HG, high glucose; IB, immunoblotting; IP, immunoprecipitation; miR-iNC: miRNA inhibitor negative control; miR-NC: miRNA negative control; NG, normal glucose; PE, phycoerythrin; pPPAR γ , phosphorylated peroxisome proliferator-activated receptor γ

Article Snippet: Lysates from podocytes and microdissected glomeruli from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris-buffered saline with Tween-20 (TBST) and probed using primary antibodies against PPAR γ (phospho S112) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR γ (1:100, ab45036, Abcam, Cambridge, UK), non-phospho (active) β -catenin (Ser45) (D2U8Y, 1:50, #19807, Cell Signaling Technology, Beverly, MA, USA), active β -catenin (05-665; EMD Millipore, Billerica, MA, USA), snail1 (1:100, ab53519, Abcam), α -SMA (1:100, ab5694, Abcam), NPHS2 (1:100, ab50339, Abcam), synaptopodin (1:50, 39067, GeneTex, San Antonio, TX, USA) and β -actin (1:200, ab6276, Abcam) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Immunoprecipitation, Transwell Migration Assay, Migration, Wound Healing Assay, Negative Control

Journal: Cell Death & Disease

Article Title: MicroRNA-27a promotes podocyte injury via PPAR γ -mediated β -catenin activation in diabetic nephropathy

doi: 10.1038/cddis.2017.74

Figure Lengend Snippet: PPAR γ -mediated β -catenin activation induces podocyte injury in high glucose. ( a ) Representative western blotting shows the expression of phosphorylated and total PPAR γ and β -catenin target genes in various conditions as indicated. ( b ) qRT-PCR analysis shows PPAR γ siRNA decreased PPAR γ and podocin but increased β -catenin target genes. ( c ) Transwell migration assay and quantitative data show increased migration. Scale bar, 100 μ m. ( d ) Wound-healing assay and quantitative data show increased invasion. ( e ) Summarized data showing increased podocyte apoptosis by flow cytometric analysis. ( f ) Immunofluorescence microscopy and quantitative data show PPAR γ siRNA-induced β -catenin activation was attenuated by co-transfection with miR-27ai. Scale bar, 20 μ m. Representative ( g ) qRT-PCR and ( h ) western blotting show the expression of PPAR γ and β -catenin target genes in various conditions as indicated. Representative ( i ) transwell migration assay and ( j ) wound-healing assay show the increased ability of migration and invasion caused by PPAR γ abolishment were mitigated by co-transfection with miR-27ai. * P <0.05; # P <0.001. Active β -cat, active β -catenin; CTNNB1, catenin beta-1; NT, non-targeting; pPPAR γ , phosphorylated peroxisome proliferator-activated receptor γ

Article Snippet: Lysates from podocytes and microdissected glomeruli from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris-buffered saline with Tween-20 (TBST) and probed using primary antibodies against PPAR γ (phospho S112) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR γ (1:100, ab45036, Abcam, Cambridge, UK), non-phospho (active) β -catenin (Ser45) (D2U8Y, 1:50, #19807, Cell Signaling Technology, Beverly, MA, USA), active β -catenin (05-665; EMD Millipore, Billerica, MA, USA), snail1 (1:100, ab53519, Abcam), α -SMA (1:100, ab5694, Abcam), NPHS2 (1:100, ab50339, Abcam), synaptopodin (1:50, 39067, GeneTex, San Antonio, TX, USA) and β -actin (1:200, ab6276, Abcam) at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Transwell Migration Assay, Migration, Wound Healing Assay, Immunofluorescence, Microscopy, Cotransfection

Journal: Cell Death & Disease

Article Title: MicroRNA-27a promotes podocyte injury via PPAR γ -mediated β -catenin activation in diabetic nephropathy

doi: 10.1038/cddis.2017.74

Figure Lengend Snippet: MiR-27a promotes PPAR γ -mediated β -catenin activation in diabetic rats. Representative ( a ) micrographs of ISH and ( b ) quantitative data illustrate the expression of miR-27a in podocytes in various groups as indicated. Representative ( c ) immunohistochemical staining and ( d ) quantitative data show the expression of PPAR γ and β -catenin target genes in various groups as indicated. The level of miR-27a in ( e ) glomeruli and ( f ) plasma samples of diabetic rats in various groups as indicated. Frozen rat kidney sections were hybridized with digoxygenin (DIG)-labeled miRCURY Locked Nucleic Acid (LNA) microRNA Detection Probes. Paraffin-embedded sections were immunostained for PPAR γ (total and phosphorylated), active β -catenin, snail1, α -SMA and synaptopodin. Scale bar, 50 μ m. Glomeruli were dissected using laser microdissection and detected with qRT-PCR. * P <0.05; # P <0.001. Active β -cat, active β -catenin; DM, diabetes mellitus; DM_miR-iNC, diabetic rats treated with miRNA inhibitor negative control; DM_miR-27ai, diabetic rats treated with miR-27ai; NC, normal control; pPPAR γ , phosphorylated peroxisome proliferator-activated receptor γ

Article Snippet: Lysates from podocytes and microdissected glomeruli from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris-buffered saline with Tween-20 (TBST) and probed using primary antibodies against PPAR γ (phospho S112) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR γ (1:100, ab45036, Abcam, Cambridge, UK), non-phospho (active) β -catenin (Ser45) (D2U8Y, 1:50, #19807, Cell Signaling Technology, Beverly, MA, USA), active β -catenin (05-665; EMD Millipore, Billerica, MA, USA), snail1 (1:100, ab53519, Abcam), α -SMA (1:100, ab5694, Abcam), NPHS2 (1:100, ab50339, Abcam), synaptopodin (1:50, 39067, GeneTex, San Antonio, TX, USA) and β -actin (1:200, ab6276, Abcam) at 4 °C overnight.

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Staining, Clinical Proteomics, Labeling, Laser Capture Microdissection, Quantitative RT-PCR, Negative Control, Control

Journal: Cell Death & Disease

Article Title: MicroRNA-27a promotes podocyte injury via PPAR γ -mediated β -catenin activation in diabetic nephropathy

doi: 10.1038/cddis.2017.74

Figure Lengend Snippet: miR-27a contributes to podocyte depletion and disrupts podocyte architectural integrity in diabetic rats. ( a ) Immunofluorescence staining shows glomerular synaptopodin and WT1 expression in different groups. Paraffin-embedded rat kidney sections were co-immunostained for synaptopodin (red) and WT1 (green). Nuclei were visualized by DAPI. Scale bar, 20 μ m. ( b ) Transmission electron microscopic analysis shows morphological changes in the podocyte foot process in different groups as indicated. Scale bar, 1.0 μ m. Red asterisks specify podocyte foot processes. ( c ) Level of synaptopodin, ( d ) podocyte number, ( e ) level of WT1 and ( f ) percentage of podocyte foot process effacement in different groups were shown. ( g ) Representative immunofluorescence micrographs show glomerular pPPAR γ and active β -catenin expression in diabetic rats with magnified white insets on the lower right quadrant. Paraffin-embedded rat kidney sections were co-immunostained for pPPAR γ (green) and active β -catenin (red). Nuclei were visualized by DAPI. Arrows indicate the colocalization (yellow). Scale bar, 20 μ m. ( h ) Representative co-immunoprecipitation analysis shows the interaction of pPPAR γ and active β -catenin in laser capture microdissected glomeruli in different groups as indicated. * P <0.05; # P <0.001. Active β -cat, active β -catenin; DM, diabetes mellitus; DM_miR-iNC, diabetic rats treated with miRNA inhibitor negative control; DM_miR-27ai, diabetic rats treated with miR-27ai; IB, immunoblotting; IP, immunoprecipitation; NC, normal control; pPPAR γ , phosphorylated peroxisome proliferator-activated receptor γ ; WT1, Wilm's tumor 1

Article Snippet: Lysates from podocytes and microdissected glomeruli from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris-buffered saline with Tween-20 (TBST) and probed using primary antibodies against PPAR γ (phospho S112) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR γ (1:100, ab45036, Abcam, Cambridge, UK), non-phospho (active) β -catenin (Ser45) (D2U8Y, 1:50, #19807, Cell Signaling Technology, Beverly, MA, USA), active β -catenin (05-665; EMD Millipore, Billerica, MA, USA), snail1 (1:100, ab53519, Abcam), α -SMA (1:100, ab5694, Abcam), NPHS2 (1:100, ab50339, Abcam), synaptopodin (1:50, 39067, GeneTex, San Antonio, TX, USA) and β -actin (1:200, ab6276, Abcam) at 4 °C overnight.

Techniques: Immunofluorescence, Staining, Expressing, Transmission Assay, Immunoprecipitation, Negative Control, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MicroRNA-27a promotes podocyte injury via PPAR γ -mediated β -catenin activation in diabetic nephropathy

doi: 10.1038/cddis.2017.74

Figure Lengend Snippet: Expression patterns of miR-27a and PPAR γ/β -catenin signaling molecules in human renal biopsy samples. Representative ( a ) ISH staining and ( b ) quantitative data illustrate the expression of miR-27a in human renal biopsy tissues from DN patients ( n =62) and control healthy donor transplant kidney tissues ( n =13). Representative ( c ) immunohistochemical staining and ( d ) quantitative data illustrate the expression of pPPAR γ and β -catenin target genes in different groups as indicated. Fresh snap-frozen human renal biopsies were sectioned and hybridized with digoxygenin (DIG)-labeled miRCURY Locked Nucleic Acid (LNA) microRNA Detection Probes. Paraffin-embedded human renal biopsy sections were immunostained for pPPAR γ , active β -catenin, snail1, α -SMA and synaptopodin. Red insets in the left side images of each panel were magnified and shown on the right side. Black arrows indicate podocytes. Representative ( e ) immunofluorescence images and quantitative data show ( f ) glomerular synaptopodin (green) and WT1 (red) expression and ( g ) podocyte number in human renal biopsy samples. ( h ) Representative immunofluorescence micrographs show glomerular pPPAR γ (green) and active β -catenin (red) expression in human renal biopsy samples. Scale bar, 50 μ m. ( i ) Magnified white insets show the colocalization of pPPAR γ and active β -catenin (yellow). Scale bar, 20 μ m. ( j ) Quantitative data show the level of pPPAR γ (green) and active β -catenin (red). Nuclei were visualized by DAPI. White arrow indicates colocalization of pPPAR γ and active β -catenin. Glomeruli were demarcated with white dashed lines. ( k ) A hypothetical model illustrating that miR-27a, via PPAR γ -mediated β -catenin activation, promotes podocyte injury in DN. MiR-27a inhibits PPAR γ gene transcription whereas indirectly stimulates PPAR γ phosphorylation, which activates β -catenin signaling and triggers β -catenin-dependent reprogramming and target gene expression levels. These events promote podocyte injuries as demonstrated by increased migration, invasion, and apoptosis, and decreased adhesion abilities. # P <0.001. Active β -cat, active β -catenin; LEF1, lymphoid-enhancer factor 1; P, phosphorylation; pPPAR γ , phosphorylated peroxisome proliferator-activated receptor γ ; WT1, Wilm's tumor 1

Article Snippet: Lysates from podocytes and microdissected glomeruli from each experimental group were separated in parallel on two 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5% nonfat milk in 0.1% tris-buffered saline with Tween-20 (TBST) and probed using primary antibodies against PPAR γ (phospho S112) (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PPAR γ (1:100, ab45036, Abcam, Cambridge, UK), non-phospho (active) β -catenin (Ser45) (D2U8Y, 1:50, #19807, Cell Signaling Technology, Beverly, MA, USA), active β -catenin (05-665; EMD Millipore, Billerica, MA, USA), snail1 (1:100, ab53519, Abcam), α -SMA (1:100, ab5694, Abcam), NPHS2 (1:100, ab50339, Abcam), synaptopodin (1:50, 39067, GeneTex, San Antonio, TX, USA) and β -actin (1:200, ab6276, Abcam) at 4 °C overnight.

Techniques: Expressing, Staining, Control, Immunohistochemical staining, Labeling, Immunofluorescence, Activation Assay, Phospho-proteomics, Targeted Gene Expression, Migration

Journal: Science Advances

Article Title: Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling

doi: 10.1126/sciadv.adg8118

Figure Lengend Snippet: ( A ) Diagram of syncytiotrophoblast layer within human placenta. ( B ) Laser capture microdissection was performed to collect syncytiotrophoblast-enriched cellular fractions. Scale bars, 100 μm. ( C ) PLCβ1/PLCβ3 protein expression within syncytiotrophoblast. ( D ) Correlation between villous placental PLC activity and syncytiotrophoblast PLCβ1/PLCβ3 protein levels. ( E ) ITPR3 mRNA within syncytiotrophoblast. ( F ) Overview of superoxide buffering by SOD2 in the mitochondrial matrix and the cellular effects of excess superoxide, adapted from . ( G ) SOD2 mRNA and protein expression within syncytiotrophoblast. ( H ) MDA protein expression within syncytiotrophoblast; * P < 0.05, independent samples t test (two-tailed) (C, G, and H) and Mann-Whitney U test (E). Each datapoint represents a biological replicate. STB, syncytiotrophoblast. Images (A and F) were created using BioRender ( www.biorender.com ).

Article Snippet: Briefly, TaqMan gene expression assay for human GAPDH (Thermo Fisher Scientific, Hs02786624_g1, 4453320, FAM-MGB), SOD2 (Thermo Fisher Scientific, Hs00167309_m1, 4453320, FAM-MGB), and ITPR3 (Thermo Fisher Scientific, Hs01573539_m1, 4448892, FAM-MGB) were pooled into a reaction containing TaqMan PreAmp Master Mix and cDNA and then preamplified on a thermal cycler for 14 cycles.

Techniques: Laser Capture Microdissection, Expressing, Activity Assay, Two Tailed Test, MANN-WHITNEY