Journal: bioRxiv

Article Title: Surface topography is a context-dependent activator of TGF-β signaling in mesenchymal stem cells

doi: 10.1101/2020.01.13.903195

Figure Lengend Snippet: A) In silico design of the PS-1018 surface used in subsequent experiments. Scale bar represent 10 μm. B) Similar as the PS-281 surface, the PS-1018 surface elicits smaller, elongated morphological characteristics and a reduction in F-actin stress fibers. F-actin was immunolabeled by phalloidin (yellow) and the nucleus counterstained with Hoechst33342 (magenta). Scale bar represent 50 μm. C) Immunolabeling of F-actin after 1h of cell culture reveals that MSCs on a flat surface exhibit a round morphology with a diffuse F-actin pattern. D-E) Immunolabeling of F-actin of MSCs cultured for 1h on the PS-1018 surface reveals an increase in F-actin stress fibers concentrated on the upper part of the micro-topographical structures, while a diffuse pattern is observed at the bottom of the PS-1018 structures. Scale bar represent 10 μm. F) EGR1 levels are elevated at 24h as measured through qPCR (* P<0.05), a similar observation as with the PS-281 microarray data. G-H) Immunolabeling of EGR1 demonstrates elevated intensities on the PS-1018 surface at 2h, with only a few cells on the PS-1018 surface showing elevated levels after 24h. Quantification of EGR1 fluorescent signal confirms the visual observation, with elevated levels measured at 2h (*** P<0.001), and no significant difference at 24h. Scale bar represent 100 μm. I-J) Immunolabeling of SRF demonstrates elevated levels on the PS-1018 surface at 2h (*** P<0.001), with no significant differences at 24h. Barplot represent the mean with error bars representing SEM. Scale bar represent 100 μm.

Article Snippet: Primary antibodies used in this study are: anti-SCX antibody (1:200; Abcam; ab58655), anti-EGR1 antibody (1:200; ThermoFisher; T.126.1), anti-Phospho-Smad2/3 (1:200; Cell Signaling Technologies; 8828S) and anti-SRF antibody (1:200; Santa Cruz; sc-335).

Techniques: In Silico, Immunolabeling, Cell Culture, Microarray

Journal: bioRxiv

Article Title: Surface topography is a context-dependent activator of TGF-β signaling in mesenchymal stem cells

doi: 10.1101/2020.01.13.903195

Figure Lengend Snippet: A) Volcano plot of the PS-281 microarray with probe targets associated with TGF-β signaling represented in blue. DEG cut-off is determined at a 1.5 fold change and an adjusted P value of 0.05. Increased levels of TGF- β R-II are observed (1.9 and 2.0 fold increase; *** P<0.001). B) qPCR of MSCs cultured on the PS-1018 surface validates the PS-281 observation regarding TGF- β R-II expression, with elevated levels observed at 8h and 24h. The ERK inhibitor U0216 inhibits the topography induced TGF- β R-II expression (* P<0.05). C) The PS-1018 surface induces elevated levels of the TGF-β target gene SCX. Significant elevated levels were detected at 8h, 24h and 48h (** P<0.01), with a maximum expression at 24h. In addition, the inhibitor U0216 abolishes topography-induced SCX expression. D) Schematic representation of the mechanism involving the activation of SRF and EGR1 leading to an upregulation of TGF- β R-II . Barplots represent the mean with error bars representing SEM.

Article Snippet: Primary antibodies used in this study are: anti-SCX antibody (1:200; Abcam; ab58655), anti-EGR1 antibody (1:200; ThermoFisher; T.126.1), anti-Phospho-Smad2/3 (1:200; Cell Signaling Technologies; 8828S) and anti-SRF antibody (1:200; Santa Cruz; sc-335).

Techniques: Microarray, Cell Culture, Expressing, Activation Assay

Journal: Journal of molecular and cellular cardiology

Article Title: Transcriptional Control of a Novel Long Noncoding RNA Mymsl in Smooth Muscle Cells by a Single Cis-Element and its Initial Functional Characterization in Vessels

doi: 10.1016/j.yjmcc.2019.11.148

Figure Lengend Snippet: A. The schematic of luciferase reporter of the −400 bp of Mymsl putative promoter (CArG WT) with the predicted CArG sequence. 10T1/2 cells were co-transfected with WT Mymsl or SM22 (positive control) and either SRF-VP16 or MYOCD expression plasmids versus their individual control vectors for 36 hrs before assessment of luciferase activity. Luciferase activity was normalized to the internal control reporter renilla. SRF and MYOCD-dependent activation of the Mymsl promoter was defined as fold increase over its individual vector control group (set to 1). Values are means± SEM from one experiment with 3 biological replicates. Two separate experiments were performed. B. The indicated Mymsl CArG WT or CArG mutant reporters were transfected for luciferase assay as in (A). Data are means ± SEM from 3 separate experiments. C. Chromatin Immunoprecipitation assays (ChIPs) were carried out in growing MASMCs for the analysis of SRF binding to the putative CArG box. Signal of amplified DNA was normalized to the input control. Relative enrichment of the CArG box containing fragment was expressed as fold increase over the IgG control (set to 1). Primers to CArG in the intron1 of mouse Cnn1 was used as positive control. Data are means ± SEM from 4 separate experiments. *P < 0.05.

Article Snippet: Chromatin complexes were precipitated with either antibody to SRF (Cat. # D7A9, Cell Signaling for bladder; Santa Cruz, G-20, sc-335 X for MASMC), H3K79me2 (Cat. # ab3594, Abcam) or rabbit negative IgG control (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab171870","term_id":"90078505","term_text":"AB171870"}} Ab171870 ).

Techniques: Luciferase, Sequencing, Transfection, Positive Control, Expressing, Control, Activity Assay, Activation Assay, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Binding Assay, Amplification

Journal: Journal of molecular and cellular cardiology

Article Title: Transcriptional Control of a Novel Long Noncoding RNA Mymsl in Smooth Muscle Cells by a Single Cis-Element and its Initial Functional Characterization in Vessels

doi: 10.1016/j.yjmcc.2019.11.148

Figure Lengend Snippet: A. qRT-PCR analysis of Mymsl and Myh11 in cultured MASMCs isolated from WT versus CArG mutants transduced with Ad-MYOCD or Ad-empty control virus. Experiments were done with 2 MASMC isolates. Values are means± SEM from one experiment with 3 biological replicates. *P < 0.05. B. Mymsl RNA levels in medial SMC layers of aortas (differentiated/contractile phenotype) versus cultured MASMCs (dedifferentiated/synthetic phenotype) from WT and CArG mutant mice. Two separate experiments were performed. Values are means± SEM from one experiment with 3 biological replicates. *P < 0.05. C. RNA levels of Mymsl and the indicated SMC-marker genes in uninjured versus injured carotid arteries from WT and CArG mutants at 1 week post injury. Values are shown as means ± SEM (n ≥ 5). *P < 0.05. D. qPCR for in vivo ChIPs in bladders from WT versus CArG mutants for SRF binding to the promoter region of Mymsl flanking the above CArG element. The enrichment of intronic CArG1 of Cnn1 was included as a negative control. Values are presented as enrichment ratio of mutant to WT (set to 1) (n=3). E. qPCR analysis of the enrichment of H3K79Me2 to CArG chromatin in Mymsl promoter in bladders from both WT and CArG mutants. Values are presented as enrichment ratio of mutant to WT (set to 1) (n=5).

Article Snippet: Chromatin complexes were precipitated with either antibody to SRF (Cat. # D7A9, Cell Signaling for bladder; Santa Cruz, G-20, sc-335 X for MASMC), H3K79me2 (Cat. # ab3594, Abcam) or rabbit negative IgG control (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab171870","term_id":"90078505","term_text":"AB171870"}} Ab171870 ).

Techniques: Quantitative RT-PCR, Cell Culture, Isolation, Transduction, Control, Virus, Mutagenesis, Marker, In Vivo, Binding Assay, Negative Control

Journal: Nature Communications

Article Title: MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation

doi: 10.1038/s41467-019-09636-6

Figure Lengend Snippet: MKL1 activity results in reduced chromatin accessibility. a Quantification of the size distribution of DNase I digested DNA fragments from control iPS cells and caMKL1-blocked cells. n = 3 for each genotype. b FRAP analysis of the dynamic movement of histone H1 0 -mCherry in control iPS cell nuclei or caMKL1-blocked cell nuclei. **** P < 0.0001. n denotes the number of analyzed nuclei. Statistics were performed using two-tailed paired t -test. Error bars denote standard deviation. c , d Quantification of the size distribution of DNase I digested DNA fragments from WT and caMKL1-overexpressing ES cells ( c ) and SRF f/f iPS cells and SRF Δ/Δ iPS cells ( d ). n = 3 for each genotype. e , f MA plots of differential ATAC-seq peaks in WT vs caMKL1-expressing ES cells ( e ) and SRF f/f vs SRF Δ/Δ iPS cells ( f ). Pink dots indicate regions of significant differences in chromatin accessibility (left). The width of boxplots (right) is proportional to the number of regions of differential chromatin accessibility, showing overall lower accessibility in caMKL1-expressing ESCs ( e ), and overall increased accessibility in SRF Δ/Δ iPS cells ( f ). The center line of boxplots indicate the median value; the edges of the boxplots are at the 25th and 75th percentile; the whiskers extend to 1.5 times the interquartile range past the box. Points that are outside whiskers are shown individually. g Quantification of the size distribution of DNase I digested DNA fragments from caMKL1-blocked cells treated with or without UNC0642. n = 3 for each genotype. h Percentage of Oct4:GFP+ cells emerging from blocked cells following treatment with UNC0642. GFP+ cells were determined by FACS. All data shown are representative of three independent experiments

Article Snippet: For immunoprecipitation, Oct4 antibody (Cell Signaling, 5677S) and SRF antibody (Active Motif, 61385, Santa Cruz, sc-335) were used at 6 μg/ChIP sample, H3K4me3 antibody (Millipore, 07-473), H3K27ac antibody (Abcam, Ab4729), and H3K27me3 antibody (Millipore, 07-449) were used at 1 μg/ChIP sample.

Techniques: Activity Assay, Two Tailed Test, Standard Deviation, Expressing