Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: Flow chart depicting steps to assemble the filtration units and the methodology to be followed for isolating gDNA using the PES membrane. Note that the methodology described requires no centrifugation steps. RT, room temperature; TE, Tris-EDTA.

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Filtration, Membrane, Centrifugation

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Agarose Gel Electrophoresis, Extraction, Membrane, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (A) or QIAamp DNA Blood Mini Kit from Qiagen (B). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter. PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Membrane, Extraction, Amplification, Biomarker Discovery, Isolation, Agarose Gel Electrophoresis, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared using PES membranes from different suppliers. Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers: EMD Millipore (A), Pall (B), and Sterlitech (C). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter. PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lane 1: HLA-A Exon 2; lane 2: HLA-A Exon 3; lane 3: HLA-B Exon 2; lane 4: HLA-B Exon 3; lane 5: HLA-C Exon 2; lane 6: HLA-C Exon 3; lane 7: HLA-DPB1 Exon 3; lane 8: HLA-DQB1 Exon 3; lane 9: HLA-DRB1 Exon 3; lane 10: HLA-DPB1 Exon 2; lane 11: HLA-DQB1 Exon 2; lane 12: HLA-DRB1 Exon 2. M, DNA MW marker (250 bp–10 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Extraction, Membrane, Amplification, Biomarker Discovery, Isolation, Agarose Gel Electrophoresis, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared by PES membranes with different pore sizes. Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (A) or 0.45 µm (B) PES membrane from EMD Millipore. Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter. PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1–6: HLA-A Exon 2, HLA-A Exon 3, HLA-B Exon 2, HLA-B Exon 3, HLA-C Exon 2, and HLA-C Exon 3; lanes 7–9: Exon 3 genes of HLA-DPB1, DQB1, and DRB1; lanes 10–12: Exon 2 genes of DPB1, DQB1, and DRB1. M, DNA MW marker (250 bp–10 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Extraction, Membrane, Amplification, Biomarker Discovery, Isolation, Agarose Gel Electrophoresis, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: Agarose gel electrophoresis of gDNA samples digested with either HindIII or EcoRI. DNAs digested with HindIII (A–C); EcoR1 digested gDNAs (D–F). A, D) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B, E) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C, F) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Agarose Gel Electrophoresis, Extraction, Membrane, Marker

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: TGF-β1 expression and exosome production are increased in UUO-induced renal fibrosis models. (A-C) Representative micrographs of immunohistochemical staining (A) and quantitative data (B,C) show TGF-β1 and Collagen I expression at different time points after UUO. Scales bars = 50μm. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d (n = 6). (D-F) Representative micrographs of Masson staining and Sirius red staining (D) and quantitative data (E, F) show collagen fiber accumulation after UUO. Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d (n = 6). (G, H) Representative western blot (G) and quantitative data (H) on TGF-β1, fibronectin and exosomal-specific proteins CD63 and TSG101 after UUO. Numbers (1 to 3) indicate each individual animal in the given group. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d (n = 6). (I) Transmission electron microscopy (TEM) shows the exosomes and microvesicles released by renal tubular epithelial cells after UUO. Scales bars = 300 nm. (J) Double immunofluorescence staining (green for CD63 and red for TSG101) demonstrates the generation of exosomes predominantly in tubular epithelial cells after UUO. Scales bars = 5 μm. (K, L) Quantitative determination of CD63- and TSG101-positive renal tubules. Data were obtained from 3 images per mouse, with 6 mice per group. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Transmission Assay, Electron Microscopy, Double Immunofluorescence Staining

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: TGF-β1 promotes the secretion of exosomes by renal tubular epithelial cells and activates fibroblasts in vitro . (A) Schematic diagram of experimental process. Exosomes from NRK-52E cells treated without (Ctrl-Exos) or with TGF-β1 (TGFβ1-Exos) were extracted and incubated with NRK-49F cells. (B,C) DLS and NTA of exosomes from NRK-52E cells. (D) TEM image of exosomes isolated from NRK-52E cells. Scale bar = 100 nm. (E, F) Representative western blot (E) and quantitative data (F) of CD63 as an exosome marker in exosomes from TGF-β1- or GW4869-treated NRK-52E cells. Numbers (1 to 3) indicate each independent treatment in the given group. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/ml TGFβ1-Exos (n = 3). (G) Fluorescent staining image of PKH-67-labeled NRK-52E cells. Scales bars=50 μm. (H) Fluorescent staining image of NRK-52E cell-derived exosomes taken up by NRK-49F cells. Scales bars=10 μm. (I, K) Representative western blot (I) and quantitative data (K) of α-SMA and PCNA in NRK-49F cells incubated with exosomes from TGF-β1- or GW4869-treated NRK-52E cells. Numbers (1 to 3) indicate each independent treatment in the giving group. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/ml TGFβ1-Exos (n = 3). (J) Proliferation rate of NRK-49F cells incubated with NRK-52E cell-derived exosomes measured by CCK-8. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/mL TGFβ1-Exos (n = 3). (L-N) Double immunofluorescence staining (green for Col-I and red for fibronectin) demonstrates the expression of Col-I and fibronectin in NRK-49F cells incubated with NRK-52E cell-derived exosomes. Scales bars=50 μm. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/mL TGFβ1-Exos.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: In Vitro, Incubation, Isolation, Western Blot, Marker, Staining, Labeling, Derivative Assay, CCK-8 Assay, Double Immunofluorescence Staining, Expressing

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubular cell-derived exosomes promote renal fibrosis in vivo . (A) Experimental design. TGFβ1-Exos or Ctrl-Exos from NRK-52E cells were injected into UUO mice by tail vein injection at 1, 3 and 5 days. (B) Representative images show the presence of PKH-67-labeled TGFβ1-Exos isolated from NRK-52E cells in mouse kidneys after intravenous injection. Kidney sections were examined at 24 h after injection. Arrows indicate PKH-67 labeled exosomes (green). Scales bars=50 μm. (C, D) Representative western blot (C) and quantitative data (D) of TSG101, CD63 and fibronectin in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Numbers (1 to 3) indicate each individual animal in the given group. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo (n = 6). (E, F) Representative western blot (E) and quantitative data (F) of fibronectin, Fsp-1 and PCNA in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Numbers (1 to 3) indicate each individual animal in the given group. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo (n = 6). (G, J) Treble immunofluorescence staining (G) and quantitative data (J) demonstrate the expression of E-cad, α-SMA and fibronectin in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Scales bars=50μm. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo. (H, I, K, L) Representative micrographs of Col-I immunohistochemical staining, Masson staining, Sirius red staining (H) and quantitative data (I, K, L) show fibronectin and collagen deposition in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, In Vivo, Injection, Labeling, Isolation, Western Blot, Immunofluorescence, Staining, Expressing, Immunohistochemical staining

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Rab27a knockout inhibits exosome secretion and alleviates UUO-induced renal fibrosis in vivo . (A) Rab27a -/- mouse. (B, C) Rab27a knockout was confirmed by PCR screening (targeted allele: 476 bp) (B) and sequencing confirmation (C) . (D, E) Representative western blot (D) and quantitative data (E) of CD63 in Rab27a knockout kidneys after UUO (n = 6). * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d. (F, I) Representative western blot (F) and quantitative data (I) of Fsp-1, PCNA and α-SMA in Rab27a knockout kidneys after UUO (n = 6). * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d. (H, J) Representative immunofluorescence micrographs (J) and quantitative data (H) show Fsp-1 expression (n = 6). Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d. (G, K-M) Masson staining, Col-I immunohistochemical staining and fibronectin immunofluorescence staining indicated the deposition of collagens and fibronectin. Representative micrographs (G) and quantitative data (K-M) are presented (n = 6). Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Knock-Out, In Vivo, Sequencing, Western Blot, Immunofluorescence, Expressing, Staining, Immunohistochemical staining

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubule cell-derived exosomal miR-21 promotes fibroblast activation in vitro . (A) NRK-52E-delivered exosomal miRNA sequencing after TGF-β1 (15 ng/ml) treatment. (B) Experimental design. Concentration of TGF-β1, 15 ng/ml. (C, G-I) Representative immunofluorescence micrographs (C) and quantitative data (G-I) show Col-I, fibronectin and α-SMA expression in NRK-49F cells after stimulation with NRK-52E-delivered exosomes. Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus Ctrl. (D) PCR detection of miR-21 in exosomes and NRK-52E cells after TGF-β1 treatment (n = 3). * p < 0.05 versus sham in cells, # p < 0.05 versus sham in exosomes. (E) PCR detection of miR-21 in NRK-52E-delivered exosomes after miR-21 mimic or inhibitor transfection (n = 3). * p < 0.05 versus sham, # p < 0.05 versus Ctrl. (F) Proliferation of NRK-49F cells after stimulation with exosomes from NRK-52E cells (n = 3). * p < 0.05 versus sham, # p < 0.05 versus Ctrl.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, Activation Assay, In Vitro, Sequencing, Concentration Assay, Immunofluorescence, Expressing, Transfection

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubule cell-derived exosomal miR-21 mediates fibroblast activation through the PTEN/AKT pathway in vitro . (A) Experimental design. Concentration of TGF-β1, 15 ng/ml. (B) PTEN mRNA level after siPTEN treatment. NS, no significant difference versus Ctrl, # p < 0.05 versus Ctrl. (C) Proliferation of NRK-49F cells after miR-21 inhibitor or siPTEN transfection (n = 3). # p < 0.05. (D-G) Representative immunofluorescence micrographs (G) and quantitative data (D-F) show Col-I, fibronectin and α-SMA expression in NRK-49F cells after miR-21 inhibitor or siPTEN transfection (n = 3). Scales bars=50 μm. # p < 0.05. (H-K) Representative western blot (H) and quantitative data (I-K) show PTEN, Akt, p-Akt and PCNA expression in NRK-49F cells after miR-21 inhibitor or siPTEN transfection (n = 3). # p < 0.05.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, Activation Assay, In Vitro, Concentration Assay, Transfection, Immunofluorescence, Expressing, Western Blot

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubular cell-derived exosomal miR-21 promotes renal fibrosis through the PTEN/AKT pathway in vivo . (A-D) Representative immunofluorescence micrographs (A) and quantitative data (B-D) show Col-I, fibronectin and α-SMA expression in mouse kidneys after intravenous exosome injection (n = 6). Scales bars=50 μm. * p < 0.05 versus Ctrl-Exo, # p < 0.05 versus TGFβ1-Exo. (E-I) Representative western blot (F) and quantitative data (E, G-I) show Fsp-1, PCNA, PTEN, Akt and p-Akt expression in mouse kidneys after intravenous exosome injection (n = 6). * p < 0.05 versus Ctrl-Exo, # p < 0.05 versus TGFβ1-Exo.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, In Vivo, Immunofluorescence, Expressing, Injection, Western Blot