Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: ( A ) 5mC-immunostaining (im) is intensively present in the nucleus (size >12μm) of postmitotic Purkinje Cells (PC) at P7 (red, crossed arrows) at PC layer (PCL). 5mC is also distinctively present only in the non-dividing granule cells of the inner portion of the External Granule Layer (EGLi, blue crossed arrows) but not outer portion of EGL (EGLo). ( B) By P28, the waning of 5mC-im was evident in PCs (red, dashed circles; ~20μm diameter). Basket cells surrounding the PCs (purple dots, <8μm) were intensively immunostained by 5mC (as well as all other interneurons). Mature granule cells inhabiting the Inner Granule Layer (IGL) retained the acquired 5mc-im throughout the remainder of the time-course. (C) By P45 re-methylation of PCs occurred as the 5mC-im returned to some but not to all de-methylated PCs (red, crossed arrows denote re-methylated PCs). ( D ) At P7, 5hmC immunostaining (im) is intensively present in PCs (red, crossed arrows), though distributed distinctly from 5mC. Some granule cells of the inner EGL express 5hmC-im though not at the upper surface of the EGL, where granule cell progenitors reside. (E) At P28, a clear de-methylation of 5hmC occurs in the PCs (red, dashed circles) as occurs with 5mC. Granule cells which have migrated to the IGL continue to acquire 5hmC (blue, crossed arrows) as do the emerging basket interneurons surrounding the perimeter of PCs (purple dots). ( F ) At P45, re-methylation of 5hmC occurs in line with 5mC re-methylation at PCs (red, crossed arrows denote re-methylated PCs). Interneurons and granule cells appear to refrain from de-methylation throughout their developmental time-course. Scale bars: A-F = 20μm; Methyl Green Nissl counterstain. Dashed red circles depict the approximate boundaries of the PC cell body. Dashed black lines depict approximate boundaries of the PCL.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Immunostaining, Methylation

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: The calcium-binding protein Calbindin D-28k (green) is a characteristic Purkinje cell protein and appears markedly immunoreactive at P7 in the PCL. At P7, 5hmC (red) is also abundant in PC nuclei and granule cells of the inner EGL and IGL. At P21, even while an abundance of DNA methylation markers undergo dramatic loss of immunoreactivity, Purkinje cells retain calbindin expression. In contrast, interneurons emerging in the ML (as well as basket cells surrounding the large Purkinje bodies) abundantly express 5hmC at P21. EGL: external granule layer, IGL: internal granule cell layer, PCL: Purkinje cell layer, ML: molecular layer. Dashed borders represent the approximate cytological borders of the PCL. Scale bar = 20μm.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Binding Assay, DNA Methylation Assay, Expressing

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: Purified gDNA obtained from laser micro-dissected Purkinje and Granule cells was quantitatively analyzed for 5-methylcytosine and 5-hydroxymethylcytosine content (%) via antibody-based colorimetric assay. ( A-B ) Purkinje cells undergo remarkable loss of both 5mC and 5hmC between the first and fourth post-natal weeks, coincident with the Purkinje cell morphological and transcriptional transformation. ( C-D ) Granule cells of the external granule surface, as they undergo radial migration into the internal granule layer and become post-mitotic, acquire 5mC as indicated by earlier immunohistochemical analysis . Further, granule cells of the internal granule layer (IGL) continue to acquire methylation between the first and fourth postnatal week as granule cells settle into their mature state. All values represented as mean ± SEM ( A .) **P-value = 0.0078; ( B .) ***P-value = 0.0001; N = 4 per age. ( C .) *P-value = 0.0186; ( D .)** P-value = 0.0036; N = 8 per age.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Purification, Colorimetric Assay, Transformation Assay, Migration, Immunohistochemical staining, Methylation

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: While distributed throughout the nucleus at the embryonic stage (not shown), at P7, major 5mC-immunostaining (im) (red) is packed into large punctates in the DAPI dense (blue), heterochromatic regions of the PCs located in the Purkinje Cell Layer (PCL) ( A, white arrows ). (B) 5hmC (green) was detected primarily in the euchromatic DAPI sparse regions ( B, white arrows ). At P7, as the granule cells in the external granular layer (EGL) migrate into the internal granular layer (IGL), 5mC appears to precede 5hmC expression, as no overlap was observed at P7 ( D, crossed arrows ). De-methylation of 5mC and 5hmC in PCs progresses through P14 and peaks between P21 and P28. Notice the loss of 5mC and 5hmC in most of the PCs in the PCL ( E, F white arrows ). In contrast, DNA methylation in granule cells is independent of the PC program. At P21, when migration from the EGL has ceased and cells have permanently settled in the IGL, there is significant overlap between 5mC and 5hmC as denoted by yellow fluorescence ( H, crossed arrows ). Furthermore, by P21 the characteristic punctate staining of 5mC observed during the first postnatal week has been replaced by a more homogenous distribution in the matured, rounded nuclei of the granule cells. ML: molecular layer. Scale bar: A-H = 20μm.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Immunostaining, Expressing, Methylation, DNA Methylation Assay, Migration, Fluorescence, Staining

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: The peak heterochromatic appearance of 5mC-im occurs at the same time as peak Dnmt1-im ( A , red crossed arrows). Similarly, the peak euchromatic staining of 5hmC-im occurs at the same time as peak Tet1-im ( D , red crossed-arrows) in PCs at P7. De-methylation follows progressively, as by P21 many PCs lacked DNMT1 ( B , red arrows), and subsequently were devoid of Tet1 ( E , red arrows). The methyl green counterstaining reveals 5hmC negative and Tet1-negative PC cell bodies (red arrows). Meanwhile, surrounding basket cells (and other interneurons) acquire Tet1 ( E , purple dots). By P45, as re-methylation of 5mC is occurring, DNMT1-im is notably returned to the PC nuclei ( C , red crossed-arrows). Similarly, Tet1 is observed parallel with the resumed observation of DNMT1-im expression ( F , red crossed arrows) in PCs. PCL: Purkinje layer; EGL: external granule layer; IGL: internal granule layer. Scale bars: A-C = 20μm; D-F = 20μm.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Staining, Methylation, Expressing

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: ( A ) Marked MeCP2 was found in the nuclei of PCs at P7 (red, crossed arrows) where its granular distribution within the nuclei is co-localized at this time point with 5mC . A noticeable waning of MeCP2-immunostainning in the PCs is observed by P21 ( B , red arrows), though not to the extent observed in 5mC at the same time point. Basket interneurons (purple dots), on the other hand, acquire MeCP2 as they for a perimeter around the PCs. A similar de-methylation phenomenon is observed with MBD1-im at P21 in some but not all PCs observed ( D , red arrows). PCs aligned in the deeper regions of the cerebellar foliae were more susceptible to this loss. IGL: internal granule cell layer, PCL: Purkinje cell layer, ML: molecular layer, Counterstaining: Nissl green. Scale bar: A-D = 20 μm.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Methylation

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: 5fC and 5caC are downstream metabolites of 5hmC (catalyzed by Tet enzymes) and prevail throughout cerebellar neurons including PCs (red, crossed arrow) and post-mitotic granule cells at P7 ( A,C ). As 5hmC is de-methylated at P21 , 5fC and 5caC are also greatly reduced ( B, D , red arrows). On the other hand, as PCs undergo de-methylation of the 5fC and 5caC derivatives, surrounding basket cells acquire immunoreactivity. IGL: internal granule cell layer, PCL: Purkinje cell layer, ML: molecular layer, Nissl counterstaining: methyl green. Scale bar: A-B = 50μm, C-D = 20μm.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: Methylation

Journal: PLoS ONE

Article Title: Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

doi: 10.1371/journal.pone.0162063

Figure Lengend Snippet: This scheme illustrates the cell-specific epigenetic distribution of the DNA methylation marks 5mC (green dots) and 5hmC (red dots) in the nuclei of neurons during development of the cerebellum. As cerebellar granule cells (light green) occupy the outer external granule layer (EGL o ), they are still mitotic and devoid of 5mC and 5hmC. Immediately after completing mitosis, these granule cells exit the cell cycle and begin radial migration through the inner EGL, PCL, and finally into the internal granule layer (IGL). As soon as granule cells break through the EGL o , they strongly acquire 5mC and 5hmC, though these two methylation marks are independently distributed in the nuclei of granule cells. From P7 forward, mature granule cells of the IGL maintain their methylation, though these become homogenously distributed in contrast to pre-migration distribution. Independently, Purkinje cells (PC) of the cerebellum exhibit a unique epigenetic program. Post-mitotic Purkinje cells generated in the dorsal rhomboid lip at E14 and arrived at the Purkinje cell layer (PCL) at E17 and already express 5mC quite prominently (and to a lesser extent 5hmC). As the PC grow in size, it becomes clear that 5mC are distributed in a granular fashion in heterochromatin, while 5hmC are distributed as fine particles in euchromatin. Remarkably, just prior to PC’s undergoing characteristic dendritogenesis and synaptogenesis (P14-30), a dramatic loss of 5mC and 5hmC occurs in their nuclei. As Purkinje cells settle into synaptic maturity, 5mC and 5hmC reappear in the nuclei though diminished from peak levels observed at P7. In contrast, the basket interneurons closely associated with PCs appear to have acquired a rich expression of 5mC and 5hmC while PCs undergo de-methylation and re-methylation.

Article Snippet: To address issues including staining penetration and inherent reduction of PC immunoreactivity, we performed an immunostain using the Purkinje cell marker calbindin-D28K (1:1000, Bioss Antibodies, Woburn, MA).

Techniques: DNA Methylation Assay, Migration, Methylation, Generated, Expressing

Journal: Scientific Reports

Article Title: The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6b rd1/rd1 mouse model of retinal dystrophy

doi: 10.1038/s41598-024-52391-y

Figure Lengend Snippet: Antibodies utilized for immunohistochemical and immunofluorescent staining.

Article Snippet: Calbindin D28K , Novus , 1:1000/1:1000 , Chicken , NBP2-50028.

Techniques: Immunohistochemical staining, Staining

Journal: Journal of Animal Science and Biotechnology

Article Title: Adjusting phosphate feeding regimen according to daily rhythm increases eggshell quality via enhancing medullary bone remodeling in laying hens

doi: 10.1186/s40104-023-00829-0

Figure Lengend Snippet: Diurnal rhythms of serum calcium and phosphorus levels and uterine protein expressions in 40-week-old Hy-Line Brown laying hens during the egg laying cycle. A ) Serum calcium ( n = 9 per group); B ) serum phosphorus ( n = 9 per group); C ) protein expression of TRPV6 and CaBP-D28k ( n = 4 per group). White and black bars represent the light and dark. Data are presented as the mean ± SEM. P < 0.05 by one-way ANOVA followed by Duncan's multiple range tests and the letters (a–d) indicate significant differences among all treatment groups ( P < 0.05). ACTB, β-actin; CaBP-D28k, Calbindin D‐28k; TRPV6, Transient receptor potential vanilloid 6

Article Snippet: Primary antibodies (rabbit) to type 2a sodium-phosphate co-transporter (NPt2a, catalogue no. A9460), type III sodium-dependent phosphate transporter 1 (PiT1, catalogue no. A4117), type III sodium-dependent phosphate transporter 2 (PiT2, catalogue no. A6739), transient receptor potential vanilloid 6 (TRPV6, catalogue no. A16128), and calbindin D‐28k (CaBP-D28k, catalogue no. A0802) were purchased from ABclonal Technology (Wuhan, Hubei, China).

Techniques: Expressing

Journal: Journal of Animal Science and Biotechnology

Article Title: Adjusting phosphate feeding regimen according to daily rhythm increases eggshell quality via enhancing medullary bone remodeling in laying hens

doi: 10.1186/s40104-023-00829-0

Figure Lengend Snippet: Daily dynamic phosphorus feeding regimen increased uterine calcium transportation and eggshell quality in 70-week-old Hy-Line Brown laying hens for 12 weeks. A ) Eggshell thickness ( n = 62−66 per group); B ) eggshell strength ( n = 62−66 per group); C ) egg specific gravity ( n = 61−66 per group); D ) shell index ( n = 62−63 per group); D ) western blot analysis and statistical analysis of protein abundances of ACTB, TRPV6 and CaBP-D28k in the uterus collected from 18 h post-oviposition ( n = 3 per group), all samples were normalized to their respective ACTB levels of each sample. Data are presented as the mean ± SEM. P < 0.05 by one-way ANOVA followed by Duncan's multiple range tests and the letters (a−c) indicate significant differences among all treatment groups ( P < 0.05). RR, provided with regular phosphorus diet at both 09:00 and 17:00 for 12 weeks; RL, provided with regular phosphorus diet at 09:00 and low phosphorus diet at 17:00 for 12 weeks; LR, provided with low phosphorus diet at 09:00 and regular phosphorus diet at 17:00 for 12 weeks; LL, provided with low phosphorus diet at both 09:00 and 17:00 for 12 weeks. ACTB, β-actin; CaBP-D28k, Calbindin D‐28k; TRPV6, Transient receptor potential vanilloid 6

Article Snippet: Primary antibodies (rabbit) to type 2a sodium-phosphate co-transporter (NPt2a, catalogue no. A9460), type III sodium-dependent phosphate transporter 1 (PiT1, catalogue no. A4117), type III sodium-dependent phosphate transporter 2 (PiT2, catalogue no. A6739), transient receptor potential vanilloid 6 (TRPV6, catalogue no. A16128), and calbindin D‐28k (CaBP-D28k, catalogue no. A0802) were purchased from ABclonal Technology (Wuhan, Hubei, China).

Techniques: Western Blot