Journal: Cell Death and Differentiation

Article Title: Histone variant H3.3 orchestrates neural stem cell differentiation in the developing brain

doi: 10.1038/cdd.2017.77

Figure Lengend Snippet: Histone H3.3 Is expressed in embryonic neural progenitor cells. ( a ) H3.3 and different neurogenesis markers are present in cortical lysates collected at various time points (E10, E13, E15, E17, and P0). ( b ) A scatter diagram shows the change trend of H3.3 and different neurogenesis markers. ( n =3; bar represents mean±S.E.M). ( c ) E13 and E15 embryonic brain sections were co-stained with anti-H3.3 and anti-NESTIN antibodies. The scale bar represents 25 μ m. ( d ) E13 and E15 embryonic brain sections were co-stained with anti-H3.3 and anti-SOX2 antibodies. The scale bar represents 25 μ m. ( e , f ) In the NSCs, the level of H3.3 protein is reduced by shRNA-mediated interference (H3.3KD#1: h3f3a- shRNA-1 combine with h3f3b- shRNA-1; H3.3KD#2: h3f3a- shRNA-2 combine with h3f3b- shRNA-2). H3.3 protein is increased by h3f3b -WT overexpression ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01, *** P <0.001; β -ACTIN served as loading control). ( g ) Protein level of neural stem cell markers, including PAX6 and SOX2 are reduced in the H3.3 knockdown NSCs versus the control. However, neuronal differentiation marker TUJ1 is increased ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; β -ACTIN served as loading control). ( h ) Protein level changes of PAX6, SOX2, and TUJ1 caused by H3.3KD#2 ( h3f3a- shRNA-2 targeting to UTR and h3f3b- shRNA-2) knockdown can be rescue by H3.3 ( h3f3a ) overexpression in NSCs ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; β -ACTIN served as loading control)

Article Snippet: Anti- beta III Tubulin (MAB1637, Millipore), Anti- beta III Tubulin (T2200, Sigma), anti-NeuN (MAB377), anti-PCNA (SC7907, SANTA CRUZ), anti-PAX6 (AB2237, Millipore, Darmstadt, Germany), anti-H3.3 (MABE872, Millipore), anti-Tri-Methyl-Histone H3(Lys4) (05-1339, Millipore), anti-Histone H3 (4499, Cell Signaling Technology, Beverly, MA, USA), anti-H4K16ac (07-329, Millipore), anti- β -ACTIN (20536-1-Ap, Proteintech, Rosemont, IL, USA), anti-NESTIN (MAB353, Millipore), anti-SOX2 (3728, Cell Signaling Technology), anti-SOX2 (MAB2018, R&D, Minneapolis, MN, USA), anti-Flag (F1804, Sigma), anti-phospho-Histone H3(Ser10) (3377, Cell Signaling Technology), anti-HA (M20003, Abmart, Shanghai, China), anti-Tri-Methyl-Histone H3(Lys36) (9763, Cell Signaling Technology), anti-BrdU (AB6326, Abcam, Cambridge, UK), anti-GLI1 (SC20687, SANTA CRUZ, Delaware Avenue, CA, USA), IgG (bs0295P, Bioss, Beijing, China), and anti-Caspase3 (9662, Cell Signaling Technology) anti-Lamin A/C (bs1446, bioworld, Nanjing, China).

Techniques: Staining, shRNA, Over Expression, Control, Knockdown, Marker

Journal: Cell Death and Differentiation

Article Title: Histone variant H3.3 orchestrates neural stem cell differentiation in the developing brain

doi: 10.1038/cdd.2017.77

Figure Lengend Snippet: H3.3 Knockdown decreases the acetylation of H4K16. ( a , b ) Protein levels of different transcriptional activation markers are detected in the H3.3 knockdown NSCs versus the control. Among them, H4K16 is selectively downregulated ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; Lamin A/C served as loading control). ( c , d ) H4K16ac, H3.3, are detected at various time points (E10, E13, E15, E17, and P0) in cortical lysates. Samples for western blot analysis are shown. A scatter diagram shows the change trend of H4K16ac, H3.3, and different neurogenesis markers ( n =3 independent experiments; bar represents mean±S.E.M; β -ACTIN served as loading control). ( e ) NSCs were isolated from the E12.5 mice brains and cultured in the proliferative medium for 24 h. The cells were co-stained with anti-H4K16ac and anti-H3.3 antibodies; anti-H4K16ac anti-SOX2 antibodies; anti-H4K16ac and anti-NESTIN antibodies. Scale bar represents 25 μ m. ( f , g ) Protein level change of H4K16ac can be rescued by H3.3 overexpression in NSCs ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; Lamin A/C served as loading control). ( h , i ) The level of H4K16ac was gradually reduced by gradually increased dose of H3.3KD#2 shRNA-mediated interference in NSC cells ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; Lamin A/C served as loading control)

Article Snippet: Anti- beta III Tubulin (MAB1637, Millipore), Anti- beta III Tubulin (T2200, Sigma), anti-NeuN (MAB377), anti-PCNA (SC7907, SANTA CRUZ), anti-PAX6 (AB2237, Millipore, Darmstadt, Germany), anti-H3.3 (MABE872, Millipore), anti-Tri-Methyl-Histone H3(Lys4) (05-1339, Millipore), anti-Histone H3 (4499, Cell Signaling Technology, Beverly, MA, USA), anti-H4K16ac (07-329, Millipore), anti- β -ACTIN (20536-1-Ap, Proteintech, Rosemont, IL, USA), anti-NESTIN (MAB353, Millipore), anti-SOX2 (3728, Cell Signaling Technology), anti-SOX2 (MAB2018, R&D, Minneapolis, MN, USA), anti-Flag (F1804, Sigma), anti-phospho-Histone H3(Ser10) (3377, Cell Signaling Technology), anti-HA (M20003, Abmart, Shanghai, China), anti-Tri-Methyl-Histone H3(Lys36) (9763, Cell Signaling Technology), anti-BrdU (AB6326, Abcam, Cambridge, UK), anti-GLI1 (SC20687, SANTA CRUZ, Delaware Avenue, CA, USA), IgG (bs0295P, Bioss, Beijing, China), and anti-Caspase3 (9662, Cell Signaling Technology) anti-Lamin A/C (bs1446, bioworld, Nanjing, China).

Techniques: Knockdown, Activation Assay, Control, Western Blot, Isolation, Cell Culture, Staining, Over Expression, shRNA

Journal: Cell Death and Differentiation

Article Title: Histone variant H3.3 orchestrates neural stem cell differentiation in the developing brain

doi: 10.1038/cdd.2017.77

Figure Lengend Snippet: H3.3 regulates the expression of the GLI1. ( a , b ) Protein level of GLI1 is decreased in the H3.3 knockdown NSCs versus the control ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; β -ACTIN served as loading control). ( c , d ) Protein level change of GLI1 can be rescue by H3.3 overexpression ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; β -ACTIN served as loading control). ( e , f ) The level of GLI1 protein was gradually reduced by gradually increased dose of H3.3KD shRNA-mediated interference in NSC ( n =3 independent experiments; bar represents mean±S.E.M; * P <0.05, ** P <0.01; β -ACTIN served as loading control). ( g , h ) H3.3 and MOF promote the expression of the GLI1 in the collaborative role in NSCs. The indicated viruses were delivered into NSC cells, and the protein level of GLI1 was detected ( n =3 independent experiments; bar represents mean±S.E.M; * P <0.05, ** P <0.01; β -ACTIN served as loading control). ( i , j ) H3.3 and MOF promote the expression of the GLI1 in the collaborative dose-response role. The indicated viruses were delivered into NSC cells, and the protein level of GLI1 was detected ( n =3 independent experiments; bar represents mean±S.E.M; * P <0.05, ** P <0.01; β -ACTIN served as loading control). ( k , l ) H3.3K36R mutant overexpression failed to promote expression of the GLI1. The indicated viruses were delivered into NSCs, and the protein level of GLI1 was detected ( n =3 independent experiments; bar represents mean±S.E.M; ** P <0.01; β -ACTIN served as loading control)

Article Snippet: Anti- beta III Tubulin (MAB1637, Millipore), Anti- beta III Tubulin (T2200, Sigma), anti-NeuN (MAB377), anti-PCNA (SC7907, SANTA CRUZ), anti-PAX6 (AB2237, Millipore, Darmstadt, Germany), anti-H3.3 (MABE872, Millipore), anti-Tri-Methyl-Histone H3(Lys4) (05-1339, Millipore), anti-Histone H3 (4499, Cell Signaling Technology, Beverly, MA, USA), anti-H4K16ac (07-329, Millipore), anti- β -ACTIN (20536-1-Ap, Proteintech, Rosemont, IL, USA), anti-NESTIN (MAB353, Millipore), anti-SOX2 (3728, Cell Signaling Technology), anti-SOX2 (MAB2018, R&D, Minneapolis, MN, USA), anti-Flag (F1804, Sigma), anti-phospho-Histone H3(Ser10) (3377, Cell Signaling Technology), anti-HA (M20003, Abmart, Shanghai, China), anti-Tri-Methyl-Histone H3(Lys36) (9763, Cell Signaling Technology), anti-BrdU (AB6326, Abcam, Cambridge, UK), anti-GLI1 (SC20687, SANTA CRUZ, Delaware Avenue, CA, USA), IgG (bs0295P, Bioss, Beijing, China), and anti-Caspase3 (9662, Cell Signaling Technology) anti-Lamin A/C (bs1446, bioworld, Nanjing, China).

Techniques: Expressing, Knockdown, Control, Over Expression, shRNA, Mutagenesis