Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Cross-sectional profiles of regenerating fibres. Three serial frozen cross sections of a biopsy obtained from regenerating vastus lateralis skeletal muscle 7 days after injury induced by electrical stimulation-elicited eccentric contractions (right). Three serial cross sections from the control (uninjured) leg of the same individual are shown (left) for comparison. The sections have been stained with alpha-sarcoglycan, beta-dystroglycan or dystrophin to label the sarcolemma, along with a basement membrane protein (laminin or collagen IV) and a myogenic marker (desmin or neonatal/embryonic myosin; MHCn/e). Each column of images contains single channel and combined images for each staining. In the injured muscle, dystrophin staining is completely absent in several fibres, while the basement membrane (laminin) is preserved. MHCn/e staining is evident in some small dystrophin-negative fibres. A similar pattern is evident from the alpha-sarcoglycan and beta-dystroglycan staining, with negative fibres, together with desmin+ cells, contained within a preserved basement membrane (collagen IV). Asterisk indicates some of the necrotic fibres. Note the different profiles of the injured fibres, such as varying fibre size, and infiltrating cells either confined to the fibre periphery or dispersed throughout the fibre. Scale bar, 100 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Control, Comparison, Staining, Membrane, Marker

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Early sarcomere formation. Confocal images of regenerating zones of human single muscle fibres at 7 days post injury, where immature actin striations (phalloidin, red) were clearly visible within a desmin+ (green) filamentous sheath ( a ) or within a CD56+ membrane ( b ). The nuclei (blue) are likely to be myonuclei. Note part of the adjacent undamaged fibre at the bottom of the image in a , where the mature striation pattern of the sarcomere can be seen. The dashed line indicates the location of the YZ orthogonal view. Scale bars, 20 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Membrane

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Myotube formation in vivo. a Single slice from a confocal stack of images of a regenerating zone of human skeletal muscle (day 7), capturing 6–7 adjoining nuclei (green) enclosed in a CD56+ (magenta) structure (arrow), likely a myotube, which is aligned along the fibre axis. Note the faintly visible adjoining uninjured fibre underneath, and part of another regenerating fibre at the bottom left of the image. The dashed line indicates the location of the YZ orthogonal view, where the fine CD56 staining can be seen entirely surrounding the myotube and where 2 peripherally located myonuclei are visible in the undamaged fibre. b Confocal images of 2 biopsy cross sections, from the same individual as in a , stained for CD56 and desmin, where CD56 can be seen encasing desmin+ structures, in 3 regenerating fibres (arrows) in each series of images. Scale bars, 20 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: In Vivo, Staining

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Myogenic and inflammatory cell activity viewed in serial cross sections. Microscope images of serial sections of one biopsy collected from regenerating human muscle 7 days after an injury protocol. Sixty serial sections (12 μm thick) were cut and stained for various markers. Shown here is a selection of these sections, where the section number (number sign) and proteins stained are indicated. Note the change in fibre diameter of the injured fibres, which are most clearly seen in section #19 (asterisk) by their lack of dystrophin (dys) staining and preserved basement membrane (collagen IV, col4). As observed in the single fibre analysis, these fibres are characterised by infiltration of macrophages (CD68+), the presence of neonatal/embryonic myosin (MHCne) and differentiating myogenic cells (desmin+, CD56+, myogenin+, nestin+). Scale bar 100 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Activity Assay, Microscopy, Staining, Selection, Membrane

Journal: Skeletal Muscle

Article Title: The breaking and making of healthy adult human skeletal muscle in vivo

doi: 10.1186/s13395-017-0142-x

Figure Lengend Snippet: Satellite cells on single fibres. Confocal images of satellite cells on the surface of regenerating human muscle fibres 30 days post injury. Three desmin+ (nestin-negative) satellite cells are visible in a , where nestin demonstrates a striated staining pattern of the myofibre, indicating ongoing regeneration. Scale bar 100 μm. Proliferating satellite cells are shown in b , evident from the expression of Ki67 and CD56, or desmin and nestin together with a nucleus undergoing mitosis. Scale bars, 20 μm

Article Snippet: Sections were stained with various combinations of antibodies against laminin, CD56, desmin and embryonic myosin (F1.652; Developmental Studies Hybridoma Bank); neonatal myosin (NCL-MHCn; Novocastra, Leica Microsystems A/S, Ballerup, Denmark); alpha-sarcoglycan (NCL-L-a-SARC, Novocastra); beta-dystroglycan (NCL-L-a-SARC, Novocastra); myogenin (F5d, Developmental Studies Hybridoma Bank); nestin, CD68, collagen IV and dystrophin (cat. no. D8168, Sigma-Aldrich Denmark A/S, Copenhagen, Denmark); myosin type I (BA.D5, Developmental Studies Hybridoma Bank) and myosin type II (A4.74, Developmental Studies Hybridoma Bank).

Techniques: Staining, Expressing

Journal: Regenerative Therapy

Article Title: miR-145-enriched BMSCs-derived exosomes ameliorate neurogenic erectile dysfunction in aged rats via TGFBR2 inhibition

doi: 10.1016/j.reth.2025.04.004

Figure Lengend Snippet: Characterization of CCSMCs and their interaction with exosomes . (A) Primary CCSMCs migrating from cavernous tissue explants. (B) Immunofluorescence staining with anti-α-SMA and anti-Desmin antibodies confirming the identity of CCSMCs. (C) Internalization of PKH67-labeled Exo and Exo-145 by CCSMCs, showing green fluorescence within the cytoplasm. (D) Elevated miR-145 expression levels in CCSMCs after co-culture with Exo or Exo-145. Data are presented as mean ± SEM. ∗∗∗ p < 0.001, ns: no significant difference.

Article Snippet: Third-generation CCSMCs were identified through immunofluorescence staining for desmin (1:200, 16520-1-AP, Proteintech) and α-smooth muscle actin (1:200, 14395-1-AP, Proteintech).

Techniques: Immunofluorescence, Staining, Labeling, Fluorescence, Expressing, Co-Culture Assay

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: MiR-29b promotes podocyte injury by targeting PGC-1α. (A) Bioinformatics analysis shows the predicted binding sites of miR-29b in the PGC-1α 3′-untranslated region (UTR) using the TargetScan software. (B) Sequence validation of the wild type or mutant PGC-1α 3′-UTR for the luciferase reporter construction. The wild-type miR-29b binding site in PGC-1α 3′-UTR (upper) and the mutated one (bottom) in the region corresponding to the miR-29b seed sequence are shown. (C) Luciferase reporter assay show that miR-29b decreased the luciferase activity in 293T cells co-transfected with wild-type PGC-1α 3′ UTR, but not with mutant PGC-1α 3′ UTR. *** P < 0.001 versus control group (n=4). (D) Mouse podocytes (MPC5) were transfected with miR-29b mimic or negative control (miR-Ctrl, NC) for 24h. qRT-PCR analysis shows the relative levels of miR-29b. ** P < 0.01 versus control group (n=3). (E) Immunostaining of ZO-1 were presented. Arrows indicate positive staining. Bar = 25μm. (F) Representative western blot showing expression of ZO-1, podocalyxin and Desmin in two groups. Numbers (1-3) indicate each individual culture in each given group. (G) Representative western blot showing expression of PGC-1α, TFAM, TOMM20 and COX1 in two groups. Numbers (1-3) indicate each individual culture in each given group. (H) Representative micrographs show mitotracker staining, immunostaining of TOMM20 and mitoSOX probe staining. Arrows indicate positive staining. Bar = 20μm or 50μm. (I-J) Graphical representations of basal OCR, maximal OCR, ATP-linked OCR, spare respiratory capacity and FAO-linked OCR in different groups. * P < 0.05, ** P < 0.01 versus control group (n=3). (K) MPC5 cells were transfected with miR-29b inhibitor (anti-miR-29b) or control (anti-miR-Ctrl, NC) for 24 h. qRT-PCR analysis shows the relative levels of miR-29b.*** P < 0.01 versus control group (n=3). (L) Representative western blot showing expression of PGC-1α, Zo-1 and podocalyxin in two groups. Numbers (1-3) indicate each individual culture in each given group. (M) MPC5 cells were transfected with pDel-β-catenin or co-transfected with miR-29b inhibitor for 24 h. Representative western blot showing expression of PGC-1α, Zo-1 and Desmin among three groups. Numbers (1-3) indicate each individual culture in each given group.

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Binding Assay, Software, Sequencing, Biomarker Discovery, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Transfection, Control, Negative Control, Quantitative RT-PCR, Immunostaining, Staining, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Inhibition of miR-29b mitigates high glucose-induced podocyte injury by increasing PGC-1α. (A) qRT-PCR analysis shows the relative levels of miR-29b after high glucose treatment for 48h. ** P < 0.01 versus control group (n=3), ††† P < 0.001 versus high glucose group (n=3). (B-E) Representative western blot and quantitative data showing expression of podocalyxin, WT1 and Desmin. Numbers (1-3) indicate each individual culture in each given group. * P < 0.05, ** P < 0.01 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3). (F) Representative micrographs showing immunostaining of F-actin. Arrows indicate actin skeleton rearrangement. Bar = 15μm. Representative TEM micrographs showing mitochondrial ultrastructure following different treatments. Arrows indicate injured mitochondria. Bar = 1μm. (G-K) Representative western blot and quantitative data showing expression of PGC-1α, TFAM, TOMM20 and COX1. Numbers (1-3) indicate each individual culture in each given group. * P < 0.05, ** P < 0.01 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3). (L) Graphical representation of mitochondrial membrane potential (MMP). MMP was detected by JC-1 staining and analyzed by flow cytometry. The data is shown as the ratio of the fluorescence intensity at absorbance of 590 nm (JC-1 aggregate) to 520 nm (JC-1 monomer). *** P < 0.001 versus control group; † P < 0.05 versus high glucose group (n=3). (M) Representative micrographs show immunostaining of TOMM20 staining. Arrows indicate positive staining. Bar = 25μm. (N) Representative micrographs show mitoSOX probe staining. Arrows indicate positive staining. Bar = 25μm. (O-P) Graphical representations of basal OCR, maximal OCR, ATP-linked OCR, spare respiratory and FAO-linked OCR in different groups. * P < 0.05, ** P < 0.01, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3). (Q) Graphical representations of the relative mRNA abundance of PPARα, CPT1a, CPT2 and ACOX1 in different groups. ** P < 0.01, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3).

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Inhibition, Quantitative RT-PCR, Control, Western Blot, Expressing, Immunostaining, Membrane, Staining, Flow Cytometry, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Ectopic expression of miR-29b aggravates podocyte injury in ADR nephropathy. (A) Schematic diagram shows the experimental procedure. (B) qPCR analyses show renal miR-29b level in two groups as indicated. *** P < 0.001 versus ADR mice group (n=5). (C) Ectopic expression of miR-29b augmented proteinuria in ADR mice. Urinary albumin was expressed as mg/mg urinary creatinine. * P < 0.05 versus ADR mice group (n=5). (D) Representative micrographs show PAS staining in different groups. Arrows indicate positive staining. Bar = 50μm. (E-J) Representative western blot and quantitative data showing expression of fibronectin, Podocalyxin, nephrin and Desmin in two groups. Numbers (1-5) indicate each individual animal in each given group. * P < 0.05, ** P < 0.01 versus ADR mice group (n=5). (K) Representative micrographs show immunostaining of fibronectin and nephrin. Arrows indicate positive staining. Bar = 50μm and 25μm. (L-M) Representative western blot and quantitative data showing expression of PGC-1α in two groups. Numbers (1-5) indicate each individual animal in each given group. *** P < 0.001 versus ADR mice group (n=5). (N) Representative micrographs show immunostaining of PGC-1α. Arrows indicate positive staining. Bar = 50μm. (O) Representative TEM micrographs showing podocytes foot process in two groups. Bar = 1μm.

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Expressing, Staining, Western Blot, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Inhibition to miR-29b mitigates mitochondrial dysfunction and podocyte injury in db/db mice. (A) Schematic diagram shows the experimental procedure. (B) qPCR analyses show renal miR-29b level in two groups as indicated. * P < 0.05 versus db/db mice group (n=5). (C) Inhibition of miR-29b by antagomiR mitigated proteinuria in db/db mice. Urinary albumin was expressed as mg/mg urinary creatinine. * P < 0.05 versus db/db mice group (n=5). (D, F-G) Representative western blot and quantitative data showing expression of fibronectin, Desmin, Podocalyxin and nephrin in two groups. Numbers (1-4) indicate each individual animal in each given group. * P < 0.05, ** P < 0.01, *** P < 0.001 versus db/db mice group (n=5). (E) Representative micrographs show immunostaining of fibronectin, podocalyxin and Zo-1. Arrows indicate positive staining. Bar = 25μm. (H, J-M) Representative western blot and quantitative data showing expression of PGC-1α, TFAM, TOMM20 and Cytb in two groups. Numbers (1-4) indicate each individual animal in each given group. * P < 0.05, *** P < 0.001 versus db/db mice group (n=5). (I) Co-staining of nephrin and TOMM20 in glomerular podocytes in different groups. Arrows indicate the co-localization of nephrin and TOMM20. Bar = 25μm. (N) Graph showing the mtDNA level in 2 groups. *** P < 0.001 versus db/db mice group (n=5). (O) Graphical representations of the relative mRNA abundance of PPARα, CPT1a, CPT2 and ACOX1 in different groups. * P < 0.05 versus db/db mice group (n=5). (P) Co-staining of PGC-1α (red) and WT1 (green), ADRP (red) and synaptopodin (green) in glomerular podocytes in different groups. Arrows indicate positive staining. Bar = 25μm.

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Inhibition, Western Blot, Expressing, Immunostaining, Staining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Activation of PGC-1α inhibits miR-29b-induced mitochondrial dysfunction and cell injury in podocytes. (A) Schematic diagram shows the experimental procedure. (B) qPCR analyses show renal miR-29b level in different groups as indicated. *** P < 0.001 versus control group (n=5). (C) Representative micrographs show PAS staining in different groups. Arrows indicate positive staining. Bar = 20μm. (D-E) Representative micrographs confirming the specific expression of miR-29b in podocytes through immunofluorescence staining with anti-Flag and nephrin antibodies. Arrows indicate positive staining. Bar = 250μm or 25μm. (F-I) Representative western blot and quantitative data showing expression of podocalyxin, nephrin and Desmin in different groups. Numbers (1-3) indicate each individual animal in each given group. * P < 0.05, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01, ††† P < 0.001 versus miR-29b plasmid group (n=5). (J) Representative micrographs show immunostaining of fibronectin, nephrin and PGC-1α. Arrows indicate positive staining. Bar = 20μm or 50 μm. (K-O) Representative western blot and quantitative data showing expression of PGC-1α, TFAM, TOMM20 and COX1 in different groups. ** P < 0.01, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01, ††† P < 0.001 versus miR-29b plasmid group (n=5).

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Activation Assay, Control, Staining, Expressing, Immunofluorescence, Western Blot, Plasmid Preparation, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Specific ablation of miR-29b in podocytes mitigates high glucose-induced podocyte injury and mitochondrial dysfunction in glomerular mini-organ culture. (A) Representative image showing the isolated miR-29b flox/flox mice glomeruli under microscopy. (B) Graph showing the miR-29b level in Adv-NC group, Adv-NC+HG group and Adv-NPHS2-Cre+HG group. *** P <0.001 versus negative control group (Adv-NC); ††† P <0.001 versus high glucose group (Adv-NC+HG) (n=3). (C-D) Representative western blot and quantitative data showing expression of PGC-1α. Numbers (1-3) indicate each individual culture in each given group. ** P < 0.01 versus negative control group (Adv-NC); † P < 0.05 versus high glucose group (Adv-NC+HG) (n=3). (E) Graphical representations of the relative mRNA abundance of TFAM, COX1, Cytb and TOMM20 in different groups. * P < 0.05 versus negative control group (Adv-NC); † P < 0.05, †† P < 0.01 versus high glucose group (Adv-NC+HG) (n=3). (F-J) Representative western blot and quantitative data showing expression of podocalyxin, nephrin, WT1 and Desmin. Numbers (1-3) indicate each individual culture in each given group. * P < 0.05, ** P < 0.01, *** P < 0.001 versus negative control group (Adv-NC); † P < 0.05, †† P < 0.01 versus high glucose group (Adv-NC+HG) (n=3). (K-N) Graphical representations of the relative mRNA abundance of PPARα, CPT1a, CPT2 and ACOX1 in different groups. * P < 0.05, ** P < 0.01 versus negative control group (Adv-NC); † P < 0.05, †† P < 0.01 versus high glucose group (Adv-NC+HG) (n=3).

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Organ Culture, Isolation, Microscopy, Negative Control, Western Blot, Expressing