Journal: PLoS ONE

Article Title: MicroRNA-20a Constrains p300-Driven Myocardial Angiogenic Transcription by Direct Targeting of p300

doi: 10.1371/journal.pone.0079133

Figure Lengend Snippet: Lentiviral-transduced cardiomyocytes expressing a control sequence ( A , ct-miR) or miR-20a ( B and C ) together with EGFP (green) were visualized with anti-GATA-4 (magenta) and pan-alpha-actinin (red) antibodies and nuclear DNA was stained with DAPI (blue) . Note that cells expressing the miR-20a-EGFP lentivirus ( B and C, arrows) have greatly reduced staining for actinin, compared with adjacent non-transduced cells ( B and C, asterixes), or cells taking up the scrambled sequence (compare EGFP+ cells in A ). Original magnification = 32x.

Article Snippet: For immunofluorescence imaging, cardiac myocytes cultured on Poly-L-Lysine-coated coverslips were washed and fixed in 4% paraformaldehyde × 15 min and then permeabilized with 0.2% Triton X-100 × 5 min. After 30 min blocking with goat serum, cells were incubated with primary and secondary antibodies, washed 3 times with PBS, and mounted using Anti-Fade DAPI (Invitrogen).

Techniques: Expressing, Sequencing, Staining

Journal: Scientific Reports

Article Title: SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

doi: 10.1038/s41598-019-42878-4

Figure Lengend Snippet: SMAD3 is predominantly expressed in GCs of primordial follicles. ( A ) Diagram showing the follicle composition of the prepubertal mouse ovary at 4, 8 and 12 days of age (d4, d8, d12). ( B ) Immunofluorescent localisation of SMAD2 and SMAD3 protein in the prepubertal mouse ovary. SMAD2 (green) was weak/undetectable in prepubertal ovaries, including primordial (yellow arrow) and early growing primary follicles (yellow asterisk), but was strong in corporal lutea from adult ovary (d16 inset). SMAD3 (red) localised to the nuclei and cytoplasm of GCs of primordial and transitional follicles (arrows), with nuclear expression appearing weaker in GCs of growing primary-plus follicles (arrowheads). Cell nuclei are counterstained with DAPI (blue). Rabbit IgG is shown as a negative control. ( C,D ) Relative expression of Smad2 and Smad3 mRNA by qPCR in d4, d8 and d16 ovaries. ( E–G ) Quantitative analyses of SMAD3 immunofluorescence. ( E ) Data represents total SMAD3 staining intensity in GCs by follicle stage. Each point represents the % positive pixels in the GC compartment for an individual follicle. ( F ) Nuclear SMAD3 intensity in GCs by follicle stage. Each point represents the % positive pixels in individual GC nuclei within each follicle stage. Points in red and blue represent measurements from d4 and d8 ovaries, respectively. ( G ) Nuclear/cytoplasmic ratio of SMAD3 in GCs by follicle stage. Each point represents the % positive pixels in all GC nuclei relative to % positive pixels in the GC compartment for an individual follicle obtained from d8 ovaries. Dashed lines ( E,F ) demarcate lower and upper quartiles to represent proportions of samples that were considered low, medium and high intensity staining, respectively. ( H ) Subcellular expression of SMAD3 by western blotting in protein lysates from d4 mouse ovaries. Samples were separated into cytosolic, membrane-bound organelle, and nuclear fractions. The cytoplasmic protein GAPDH is included as a control. Gel images have been cropped from originals provided in Fig. . PF, primordial; T, transitional; P, primary; P + , primary plus, S, secondary follicle. Data in C, D are means ± SEM (n = 5 ovaries/age) and differences are relative to d4. Data in E, F, G are medians ± interquartile ranges and differences are relative to the follicle stage in parentheses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Sections were mounted with a drop of Prolong® Gold anti-fade reagent with DAPI (ThermoFisher) and were imaged using an inverted Leica SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).

Techniques: Expressing, Negative Control, Immunofluorescence, Staining, Western Blot, Membrane, Control

Journal: Scientific Reports

Article Title: SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

doi: 10.1038/s41598-019-42878-4

Figure Lengend Snippet: CCND2 and P27 are maintained in GCs of primordial follicles but decrease as follicles initiate growth. ( A ) Immunofluorescent localisation of CCND2 (red) in the prepubertal mouse ovary. High power images show strong (persistent) nuclear CCND2 staining in GCs of primordial follicles (PF; arrows). Transitional follicles (T) show nuclei with variable CCND2 (arrow heads) whereas larger primary plus staged follicles (P+) show weak CCND2 expression. ( B ) Nuclear CCND2 protein expression in GCs by follicle stage. Each point represents the % positive pixels for all GC nuclei in an individual follicle. (C) Relative expression of Ccnd2 mRNA by qPCR in d4, d8 and d16 ovaries. (D) Immunofluorescent localisation of P27 (green) in the prepubertal mouse ovary. High power images (lower panels) show strong nuclear P27 staining in GCs of primordial follicles (PF; arrows). Transitional follicles (T) show nuclei with variable P27 whereas larger secondary staged follicles (S) show weak P27 expression. (E) Nuclear P27 protein expression in GCs by follicle stage. Each point represents the % positive pixels for all GC nuclei in an individual follicle. (F) Relative expression of p27 mRNA by qPCR in d4, d8 and d16 ovaries. (G) Co-localisation of CCND2 and P27 in the prepubertal mouse ovary. After image processing (d4 and d8; lower panels), only positive pixels in the green and red channels are shown, highlighting co-localisation in white. A negative IgG control (d16; lower right) is shown. (H) P27/CCND2 ratio in individual GCs by follicle stage. Each point represents the % positive pixels for P27 relative to % positive pixels for CCND2 an individual nucleus. Numbers in parentheses refer to the GC number range for each follicle stage. (I) Western blot of P27 and CCND2 following co-immunoprecipitation from d4 and d16 ovaries. Protein samples (12.5 µg) were immunoprecipitated using anti- P27 (IP) or non-specific rabbit IgG (IgG; control). Non-immunoprecipitated protein (5 µg) was used as positive control (IN). Gel images have been cropped from originals provided in Fig. . PF, primordial; T, transitional; P, primary; P+, primary plus, S, secondary follicle. Nuclei in A, D, G are labelled with DAPI (blue). Data in B, E, H are medians ± interquartile ranges and differences are relative to the follicle stage in parentheses. Points in red and blue represent measurements from d4 and d8 ovaries, respectively. Data in C, F are means ± SEM (n = 5 ovaries/age) and differences are relative to d4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Sections were mounted with a drop of Prolong® Gold anti-fade reagent with DAPI (ThermoFisher) and were imaged using an inverted Leica SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).

Techniques: Staining, Expressing, Control, Western Blot, Immunoprecipitation, Positive Control

Journal: Scientific Reports

Article Title: SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

doi: 10.1038/s41598-019-42878-4

Figure Lengend Snippet: Acute modulation of TGFβ signalling causes stage-specific effects on follicle development. Whole neonatal mouse ovaries (d4) were maintained in vitro and exposed for 2 hours with either 10 ng/ml of TGFβ1 ligand (T), 1 µM A83-01 inhibitor (I), or 1 µM DMSO (C). Ovaries were then placed in basic culture media for two additional days prior to morphological analysis. (A) Confocal images of sections of cultured ovaries immunolabelled with DDX4 (red) and SMAD3 (green) to highlight oocytes and GCs, respectively. Cell nuclei are counterstained with DAPI (blue). Low power images (upper panels) showing general distribution of follicles in each group. Scale bars = 100 µm. High power images (lower panels) showing examples of early growing primary (P) and primary plus (P+) follicles in each group as classified by oocyte size. Note the smaller size of the GC-layer in follicles exposed to the inhibitor. Scale bars = 20 µm. (B) Proportion of follicles classified by follicle stage. (C) Distribution of oocyte size by treatment. (D) Mean GC number per follicle. Quantitative data from cultured ovaries ( B–D ) was obtained from six different ovary sections per treatment group (n = 6 ovaries/group). Classification was based on oocyte size (Fig. ). Data show means ± 95% CI. *P < 0.05 and ****P < 0.0001 vs control. PF, primordial; T, transitional; P, primary; P+, primary plus follicle.

Article Snippet: Sections were mounted with a drop of Prolong® Gold anti-fade reagent with DAPI (ThermoFisher) and were imaged using an inverted Leica SP5 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).

Techniques: In Vitro, Cell Culture, Control