Journal: Nature Communications

Article Title: Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation

doi: 10.1038/ncomms10830

Figure Lengend Snippet: ( a , b ) SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml −1 PDGF-BB for 24 h ( a ) or 48 h ( b ). ( a ) SMCs were fixed, immunofluorescently stained for PTEN (green) and analysed for PTEN localization using confocal microscopy; nuclei were stained for DAPI (blue). ( b ) PTEN was immunoprecipitated (IP) from cytoplasmic (cyto) and nuclear (nuc) fractions of vehicle- or PDGF-stimulated SMCs. Co-immunoprecipitating SRF was detected by immunoblotting (IB). Representative western blot from three separate experiments. ( c ) SMCs were transfected with a construct expressing SRF–GFP, maintained in serum-restricted conditions or stimulated with 20 ng ml −1 PDGF-BB, fixed and analysed for GFP localization; nuclei were stained for DAPI (blue). Shown are representative images (two serum-restricted and four PDGF-stimulated cells are shown); arrows indicate cytoplasmic localized SRF–GFP; nuclei are outlined with white lines. ( d ) SMCs were transfected with HA-tagged wild-type PTEN (WT), nuclear localized PTEN (NLS) or nuclear excluded PTEN (NES). SMCs were maintained in serum-restricted conditions or stimulated with 20 ng ml −1 PDGF-BB, fixed, immunofluorescently stained for HA (red) and analysed for PTEN localization; nuclei were stained for DAPI (blue). Arrowheads, HA–PTEN-transfected SMCs. ( e ) SMCs were transfected with GFP–SRF (ctrl) or co-transfected with GFP–SRF and WT PTEN or nuclear localized PTEN (NLS) then maintained in serum-restricted conditions or stimulated with 20 ng ml −1 PDGF-BB. Transfected SMCs exhibiting cytoplasmic GFP expression were scored as described in Methods. Data represent average per cent positive±s.e.m. N =3 independent experiments; * P <0.01 versus control 0.1% CS; ** P <0.01 versus control PDGF and WT PTEN PDGF; *** P <0.01 versus control PDGF. ( f ) Non-transfected (Ctrl) and HA–PTEN-transfected SMCs were co-transfected with an Acta2 promoter-Luciferase reporter construct. SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml −1 PDGF-BB for an additional 24 h. Luciferase activity normalized to β-galactosidase was determined; shown are fold changes from Ctrl serum-restricted (0.1% CS) SMCs. Data represent average fold changes±s.e.m. N =3 independent experiments; * P <0.01 versus Ctrl 0.1% CS; ** P <0.01 versus Ctrl PDGF. Molecular weight markers were cropped out for final SRF blot; please see . Scale bars for all images, 20 μm.

Article Snippet: Plasmid-encoding human SRF tagged with GFP (RG208596) was obtained from OriGene.

Techniques: Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Luciferase, Activity Assay, Molecular Weight

Journal: Frontiers in oncology

Article Title: Transcriptional Regulation of ING5 and its Suppressive Effects on Gastric Cancer.

doi: 10.3389/fonc.2022.918954

Figure Lengend Snippet: FIGURE 2 | The promoting effects of both SRF and YY1 proteins on ING5 expression The results of RT-PCR and Western blot revealed the successful silencing of SRF and YY1 expression in AGS and SGC-7901 cells, which showed low ING5 mRNA and protein expression (A). Co-IP was performed to explore whether SRF bind to YY1, p53 and ING5 or YY1 to SRF, p53 and ING5 in both gastric cancer cells, treated with SFR or YY1 siRNA (B). Double immunofluorescence was carried out to observe the co-localization of YY1, SRF or ING5, and p53 in gastric cancer cells (C). Ctr, control; RT-PCR, reverse-transcriptional polymerase chain reaction; WB, western blot; Co-IP, co-immunoprecipitation.

Article Snippet: SRF siRNA (sc-36563) and YY1 siRNA (sc-36863) were purchased from Santa Cruz and used for their expression silencing in SGC7901 and AGS.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Co-Immunoprecipitation Assay, Control, Polymerase Chain Reaction, Immunoprecipitation

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: To knock down endogenous SRF, iPSCs were treated with SRF shRNA lentiviral particles with the presence of 5 μ g/mL Polybrene (sc-134220, Santa Cruz) according to the recommended manual.

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