human srf sirna  (Sangon Biotech)

Journal: Stem Cells International

Article Title: Resveratrol Enhances Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells through Inhibiting Canonical WNT Signal Pathway and Enhancing Serum Response Factor-miR-1 Axis

doi: 10.1155/2016/2524092

Figure Lengend Snippet: Serum response factor- (SRF-) miRNA-1 axis also involves the CMs differentiation enhanced by RSV. (a) Transcription levels of SRF in cultured EBs treated with RSV (50 μ M) were detected by qRT-PCR on day 4. (b, c) Western Blot was further carried out to determine the protein levels of RSV (50 μ M) treated EBs after 4 days. (d) miRNA-1 expression level was also detected using qRT-PCR 4 days after RSV (50 μ M) administration. (e, f) Knockdown of SRF using produced Lentivirus expressing shRNA targeting SRF was confirmed by Western Blot analysis. (g) SRF-miRNA-1 signal axis was confirmed in EBs treated with RSV (50 μ M) on day 4. (h) Inhibition of endogenous miRNA-1 in floating cultured EBs 4 days after transfection of Lentivirus within anti-miRNA-1. (i) The effects of modulation of SRF or/and miRNA-1 on the ratio of beating EBs were evaluated on day 24 ( n = 100). Data represent the mean ± s.d. of three biological replicates. ∗ P < 0.05 compared with control; # P < 0.05 compared with RSV alone.

Article Snippet: Human SRF shRNA lentiviral particles and the control shRNA lentiviral particles were purchased from OriGene (TL320530).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Produced, shRNA, Inhibition, Transfection, Control

Journal: PLoS ONE

Article Title: Serum Response Factor Accelerates the High Glucose-Induced Epithelial-to-Mesenchymal Transition (EMT) via Snail Signaling in Human Peritoneal Mesothelial Cells

doi: 10.1371/journal.pone.0108593

Figure Lengend Snippet: (a) Morphological changes in HPMCs induced by HG stimulation for 96 h compared to the control. Magnification is 200×. (b) Fluorescence microscopy showed the altered location and expression of SRF and EMT markers E-cadherin and α-SMA in HPMCs induced by 0 h, 96 h and 7 d of treatment. Magnification is 200×. (c1) Western blot analysis showing the induction of SRF, p-SRF and EMT marker proteins E-cadherin and α-SMA expression by HG at 0 h, 24 h, 48 h, 72 h, 96 h, and 7 d in immortal HPMCs. (c2) Western blot analysis of SRF in HPMCs which were exposed with HG for 96 h and then transfected with SRF-siRNA or control vector. (d1-3) Induction of E-cadherin, α-SMA and SRF mRNA expression at 0 h, 24 h, 48 h, 72 h, 96 h, and 7 d in immortal HPMCs. Bars in A represent the fold induction over untreated cells and are depicted as the mean +/− S.E. of three independent experiments conducted in duplicate(*P<0.05 vs. control HPMCs). (e) Real-time PCR showing mRNA of SRF, E-cadherin and α-SMA in HPMCs transfected with SRF-siRNA or control vector (*P<0.05 vs. HG-HPMCs-control). (f) Fluorescence microscopy showed the location and expression of the EMT markers E-cadherin, α-SMA and SRF in HG-induced SRF-siRNA-treated HPMCs and control cells. Magnification, 200×.

Article Snippet: The siRNA plasmids that recognize human SRF (sc-36563) were purchased from Santa Cruz Biotechnology for the transient transfections.

Techniques: Control, Fluorescence, Microscopy, Expressing, Western Blot, Marker, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Serum Response Factor Accelerates the High Glucose-Induced Epithelial-to-Mesenchymal Transition (EMT) via Snail Signaling in Human Peritoneal Mesothelial Cells

doi: 10.1371/journal.pone.0108593

Figure Lengend Snippet: (a) Western blot analysis of SRF in HPMCs transfected with a SRF plasmid or control plasmid. (b) Fluorescence microscopy showed the location and expression of the EMT markers E-cadherin, α-SMA and SRF in HPMCs transfected with a SRF plasmid or control plasmid. Magnification is 100×. (c) Real-time PCR showing mRNA of SRF, E-cadherin and α-SMA in HPMCs transfected with a SRF plasmid or control plasmid (*P<0.05 vs. control).

Article Snippet: The siRNA plasmids that recognize human SRF (sc-36563) were purchased from Santa Cruz Biotechnology for the transient transfections.

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Serum Response Factor Accelerates the High Glucose-Induced Epithelial-to-Mesenchymal Transition (EMT) via Snail Signaling in Human Peritoneal Mesothelial Cells

doi: 10.1371/journal.pone.0108593

Figure Lengend Snippet: (a1) Western blot analysis of expression of Snail in HG-induced HPMCs in vitro compared to control HPMCs at 0 h, 24 h, 48 h, 72 h, 96 h, and 7 d. (a2) Western blot analysis of Snail in HPMCs transfected with SRF-siRNA or control vector. (a3) Western blot analysis of Snail in HPMCs transfected with a SRF plasmid or control plasmid. (b1) Real-time PCR showing the expression of Snail in HG-induced HPMCs at 0 h, 24 h, 48 h, 72 h, 96 h, and 7 d. (b2) Real-time PCR showing the expression level of Snail in HG-induced SRF-siRNA-treated HPMCs compared to HG-induced HPMCs. (b3) Real-time PCR showing the expression level of Snail in HPMCs infected with SRF plasmid compared with control HPMCs (*P<0.05 vs. control). (c) The results from immunohistochemistry showed Snail staining in the peritoneal samples of normal rats treated with HG or HG+SRF inhibitor (CCG-1423). Magnification is 200×. (d) Location and expression of Snail in ex vivo HPMCs from patients with different phenotypes from Groups 1, 2 or 3. (e) Fluorescence microscopy showed the location and expression of Snail in HG-induced in vitro HPMCs transfected with SRF-siRNA vector compared with the control vector. Magnification is 200×. (f) Fluorescence microscopy showed the location and expression of Snail in HPMCs transfected with a SRF plasmid or the control plasmid in vitro. Magnification is 200×.

Article Snippet: The siRNA plasmids that recognize human SRF (sc-36563) were purchased from Santa Cruz Biotechnology for the transient transfections.

Techniques: Western Blot, Expressing, In Vitro, Control, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Infection, Immunohistochemistry, Staining, Ex Vivo, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Serum Response Factor Accelerates the High Glucose-Induced Epithelial-to-Mesenchymal Transition (EMT) via Snail Signaling in Human Peritoneal Mesothelial Cells

doi: 10.1371/journal.pone.0108593

Figure Lengend Snippet: (a) Identification of SRE1 and SRE2 in the SNAIL promoter. Chromatin immunoprecipitation was used to examine SRF binding to the SNAIL promoter in HG-induced HPMCs and the control. Reaction controls included immunoprecipitations performed using a nonspecific IgG monoclonal antibody; PCR was performed using whole cell genomic DNA (Input). A representative example of three independent experiments is shown. (b) EMSA was used to analyze the SRF-binding site induced by HG. (b1) Samples 1 to 7 were induced by HG for 0 h, 5 h, 18 h, 72 h, and 96 h and compared with a positive control and negative control. SRE was bound after inducing for 72 h. (b2) Analysis of the SRF-binding site in the SNAIL promoter by EMSA. Biotin-labeled SRE1 and SRE2 oligonucleotides were used as a probe. Of the two examined putative SRE-binding sites within the SNAIL gene promoter, only SRE2 displayed specific SRF binding in HPMCs induced by HG. Samples 1 to 7 were 72 h+SRE, 72h+SRE + cold-SRE, 72h+SRE + cold SER Mut, 72h+SRE1, 72 h+SRE1+ cold-SRE1, 72h+SRE1+ cold-SRE1 Mut, 72h+SER2, 72h+SER2 +cold-SRE2, and 72h+SER2 +cold-SRE2 Mut. Cold probe, unlabeled SRE; SRE mut, mutated SRE. (c) For the competition assays with SRE1 and SRE2, 200-fold molar excess of the Snail plasmid and mutant SRE1, deleted SRE1, mutant SRE2, deleted SRE2 and mutant SRE1 and SRE2 were used in HPMCs (c1) and HG-induced HPMCs (c2). All results shown are representative of at least three independent experiments. * P<0.05 vs. Snail (SRE1+SRE2) plasmid; ** P<0.05 vs. control plasmid.

Article Snippet: The siRNA plasmids that recognize human SRF (sc-36563) were purchased from Santa Cruz Biotechnology for the transient transfections.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Control, Positive Control, Negative Control, Labeling, Plasmid Preparation, Mutagenesis