Journal: Communications Biology

Article Title: The chromatin remodelling factor Chd7 protects auditory neurons and sensory hair cells from stress-induced degeneration

doi: 10.1038/s42003-021-02788-6

Figure Lengend Snippet: a Immunohistochemistry in wildtype cochlea showing Chd7 expression in hair cells at E16.5 and P7. Expression is weaker at P7 compared to E16.5. Hair cells are stained with F-actin and Calbindin 1. Tectorial membrane is stained with Satb2. b In-situ hybridisation showing loss of Chd7 expression in Atoh1 Cre/+ ;Chd7 f/f mutant hair cells within the middle region of the cochlea at E16.5. Atoh1 Cre/+ or Chd7 floxed siblings were used as controls (indicated at the top of the panel). IHC inner hair cells; OHC outer hair cells; TM tectorial membrane. Scale bars 25 µm.

Article Snippet: Primary antibodies used were: rabbit Myo7a (1:1000, Proteus, 25-6790); rabbit NeuN (1:1000, Abcam, ab177487); mouse NF-M (1:200, ThermoFisher Scientific, 13-0700); rabbit Sptbn1 (1:500, Bethyl Laboratories, A300-936A); rabbit Lmx1a (1:100, Abcam, ab139726); rabbit Epha3 (1:100, St John’s Laboratory, STJ110712); mouse Calbindin (1:50, Abcam, ab82812); mouse Parvalbumin (1:100, Sigma, P3088); mouse Satb2 (1:100, Abcam, ab51502); rabbit Chd7 (1:100, ThermoFisher Scientific, PA5-72964); mouse CtBP2 (1:100, BD Biosciences, 612044).

Techniques: Immunohistochemistry, Expressing, Staining, In Situ, Hybridization, Mutagenesis

Journal: Communications Biology

Article Title: The chromatin remodelling factor Chd7 protects auditory neurons and sensory hair cells from stress-induced degeneration

doi: 10.1038/s42003-021-02788-6

Figure Lengend Snippet: a Cochlear explants of control and Chd7 homozygous mutants were treated with FM1-43 for 30 s to assess mechanotransduction. b CtBP2 immunohistochemistry of control and Chd7 homozygous mutants at P7 shows that ribbon synapses at the inner hair cells are unaffected in mutants. c Quantification of CtBP2 puncta (ribbon synapses) per inner hair cell. CTRL = control; HOM = homozygote.

Article Snippet: Primary antibodies used were: rabbit Myo7a (1:1000, Proteus, 25-6790); rabbit NeuN (1:1000, Abcam, ab177487); mouse NF-M (1:200, ThermoFisher Scientific, 13-0700); rabbit Sptbn1 (1:500, Bethyl Laboratories, A300-936A); rabbit Lmx1a (1:100, Abcam, ab139726); rabbit Epha3 (1:100, St John’s Laboratory, STJ110712); mouse Calbindin (1:50, Abcam, ab82812); mouse Parvalbumin (1:100, Sigma, P3088); mouse Satb2 (1:100, Abcam, ab51502); rabbit Chd7 (1:100, ThermoFisher Scientific, PA5-72964); mouse CtBP2 (1:100, BD Biosciences, 612044).

Techniques: Immunohistochemistry

Journal: Communications Biology

Article Title: The chromatin remodelling factor Chd7 protects auditory neurons and sensory hair cells from stress-induced degeneration

doi: 10.1038/s42003-021-02788-6

Figure Lengend Snippet: a – l F-actin-stained hair cells in the cochlea of control ( a – f ) and Chd7 homozygous mutants ( g – l ) at P14. Dashed boxes in a – c and h , i indicate the zoomed regions shown in ( d – f ) and ( j – l ). Scale bars = 20 µm. m Inner hair cells showing pyknotic, fragmented (arrow) and missing (arrow) nuclei in Chd7 mutants at P21. Scale bars = 25 µm. n Average number of hair cells per 200 µm regions in base, middle and apical turns of each cochlea per animal ( n = 8 per genotype; each animal is represented by one circle, triangle or square). Separate inner and outer hair cell quantification is provided in Fig. . Statistical significance was obtained by performing a nested one-way ANOVA and Dunnett’s multiple comparison test. ** P -value = 0.005. o Average number of neurons in the spiral ganglion per section at different postnatal stages in control (CTRL) and Chd7 homozygous mutants (HOM). Student’s t test, P -values: *= < 0.05, **< 0.005, ***= < 0.0005, ****= < 0.00005). p Spiral ganglia neurons stained with NeuN and neurofilament (NF) at P7 in control and mutant animals. Scale bars = 50 µm.

Article Snippet: Primary antibodies used were: rabbit Myo7a (1:1000, Proteus, 25-6790); rabbit NeuN (1:1000, Abcam, ab177487); mouse NF-M (1:200, ThermoFisher Scientific, 13-0700); rabbit Sptbn1 (1:500, Bethyl Laboratories, A300-936A); rabbit Lmx1a (1:100, Abcam, ab139726); rabbit Epha3 (1:100, St John’s Laboratory, STJ110712); mouse Calbindin (1:50, Abcam, ab82812); mouse Parvalbumin (1:100, Sigma, P3088); mouse Satb2 (1:100, Abcam, ab51502); rabbit Chd7 (1:100, ThermoFisher Scientific, PA5-72964); mouse CtBP2 (1:100, BD Biosciences, 612044).

Techniques: Staining, Mutagenesis

Journal: Communications Biology

Article Title: The chromatin remodelling factor Chd7 protects auditory neurons and sensory hair cells from stress-induced degeneration

doi: 10.1038/s42003-021-02788-6

Figure Lengend Snippet: a Auditory brainstem response (ABR) tests of Atoh1 Cre/+ ;Chd7 flox mutants and controls at 4 weeks and 8 weeks reveals profound hearing loss in homozygous and mild-moderate hearing loss in heterozygous Chd7 mutants across all frequencies. b ABR tests of NeuroD1 Cre/+ ;Chd7 flox mutants and controls at 4 weeks and 8 weeks reveals mild-moderate hearing loss in homozygous mutants. Frequencies where significant threshold elevations were observed are indicated by asterisks ( P -values: *< 0.05, **< 0.005, ***< 0.0005, ****< 0.000005). See Figs. and for ABR profiles of each mouse. Error bars represent the standard error of mean (see Figs. and ). CTRL = control; HET = heterozygote; HOM = homozygote.

Article Snippet: Primary antibodies used were: rabbit Myo7a (1:1000, Proteus, 25-6790); rabbit NeuN (1:1000, Abcam, ab177487); mouse NF-M (1:200, ThermoFisher Scientific, 13-0700); rabbit Sptbn1 (1:500, Bethyl Laboratories, A300-936A); rabbit Lmx1a (1:100, Abcam, ab139726); rabbit Epha3 (1:100, St John’s Laboratory, STJ110712); mouse Calbindin (1:50, Abcam, ab82812); mouse Parvalbumin (1:100, Sigma, P3088); mouse Satb2 (1:100, Abcam, ab51502); rabbit Chd7 (1:100, ThermoFisher Scientific, PA5-72964); mouse CtBP2 (1:100, BD Biosciences, 612044).

Techniques:

Journal: Communications Biology

Article Title: The chromatin remodelling factor Chd7 protects auditory neurons and sensory hair cells from stress-induced degeneration

doi: 10.1038/s42003-021-02788-6

Figure Lengend Snippet: a Cochlear explants of control and Chd7 homozygous mutants were treated with gentamicin to induce oxidative stress. Rapid hair cell death is observed in mutants within 5 h whereas control hair cells and untreated mutant hair cells survive. Green = Cre recombined cells expressing membrane GFP, magenta = all hair cells stained with Myo7a, blue = DAPI stained nuclei. b Quantification of Myo7a+ and GFP+ hair cells per 200 µm region in untreated and treated explants in both controls (CTRL) and mutants (HOM). Two-tailed unpaired t test shows the significant difference between mutant and control. P -value = 0.0025. Scale bars = 25 µm.

Article Snippet: Primary antibodies used were: rabbit Myo7a (1:1000, Proteus, 25-6790); rabbit NeuN (1:1000, Abcam, ab177487); mouse NF-M (1:200, ThermoFisher Scientific, 13-0700); rabbit Sptbn1 (1:500, Bethyl Laboratories, A300-936A); rabbit Lmx1a (1:100, Abcam, ab139726); rabbit Epha3 (1:100, St John’s Laboratory, STJ110712); mouse Calbindin (1:50, Abcam, ab82812); mouse Parvalbumin (1:100, Sigma, P3088); mouse Satb2 (1:100, Abcam, ab51502); rabbit Chd7 (1:100, ThermoFisher Scientific, PA5-72964); mouse CtBP2 (1:100, BD Biosciences, 612044).

Techniques: Mutagenesis, Expressing, Staining, Two Tailed Test

Journal: Cell stem cell

Article Title: Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome

doi: 10.1016/j.stem.2020.07.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CD144 (VE-Cadherin) microBeads Miltenyi Biotec Cat#130-097-857 Anti-mouse IgG microbeads Miltenyi Biotec Cat# 130-048-401 Rabbit polyclonal anti-ETS1 Active Motif Cat#39580; RRID: AB_2793266 Mouse monoclonal anti-RNA pol II (Clone: 4H8) Active Motif Cat#39097; RRID: AB_2732926; Rabbit monoclonal anti-CHD7 (Clone: D3F5) Cell Signaling Technology Cat#6505; RRID: AB_11220431 Mouse monoclonal anti-NPR3 (Clone: OTI11B5) OriGene Cat# TA501079; RRID: AB_11127026 Rabbit polyclonal anti-α-SMA Abcam Cat#ab5694; RRID: AB_2223021 Rabbit polyclonal anti-Ki67 Abcam Cat# ab15580; RRID: AB_443209 Mouse monoclonal anti-TNNT2 (Clone: 13- 11) Thermo Scientific Cat#MS-295-P; RRID: AB_61806; Rabbit polyclonal anti-TNNT2 Abcam Cat#ab45932; RRID: AB_956386 Mouse monoclonal anti-α-actinin (Clone: EA- 53) Sigma-Aldrich Cat#A7811; RRID: AB_476766 Mouse monoclonal anti-CDH11 (Clone: 5B2H5) Invitrogen Cat#321700; RRID: AB_2533068 Mouse monoclonal anti-CD31 (Clone: JC/70A) Abcam Cat#ab9498; RRID: AB_307284 Mouse monoclonal anti-FN1 (Clone: IST-9) Abcam Cat#ab6328; RRID: AB_305428 Mouse monoclonal anti-FN1- Xenopus laevis Developmental studies hybridoma bank Cat#4H2S; RRID: AB_2721949; Donkey anti-Mouse secondary antibody, Alexa 488 Invitrogen Cat#A21202; RRID: AB_141607 Donkey anti-Rabbit secondary antibody, Alexa 488 Invitrogen Cat#A21206; RRID: AB_2535792 Donkey anti-Mouse secondary antibody, Alexa 594 Invitrogen Cat#A21203; RRID: AB_2535789 Donkey anti-Rabbit secondary antibody, Alexa 594 Invitrogen Cat#A21207; RRID: AB_141637 Bacterial and Virus Strains GFP lentivirus ( Chen et al., 2017 ) NA ETS1 lentivirus ( Chen et al., 2017 ) NA Biological Samples Human fetal heart samples University of Washington Birth Defects Research Lab, USA.

Techniques: Virus, Recombinant, Knock-Out, Red Blood Cell Lysis, Blocking Assay, Membrane, Clinical Proteomics, Plasmid Preparation, Cell Culture, SYBR Green Assay, RNA Sequencing, Control, Software, Quantitative Proteomics, Sonication

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: The high expression of CHD7 in hDFCs after osteoinduction. (a) Immunohistochemical staining images unraveled that CHD7 is present in human dental follicle. Scale bar, 20 μ m. (b), (c) qRT-PCR and Western blot unraveled that the expression of CHD7 increased after 3-day and 7-day osteoinduction.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: Depletion of CHD7 decreases osteogenic differentiation of hDFCs. (a), (b) qRT-PCR and Western blot verified the knockdown efficiency of siCHD7. (c) Representative images and quantitative analyses of ALP and ARS staining of hDFCs in the siCHD7 and siCTRL group. (d) qRT-PCR analyses of the expression of RUNX2 , SP7 , BGLAP , DLX5 , BMP2 , and COL1A1 under osteogenic condition.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Expressing

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: The overexpression of CHD7 promotes osteogenic differentiation of hDFCs. (a), (b) qRT-PCR and Western blot verified the overexpression efficiency of CHD7 . (c) Representative images and quantitative analyses of ALP and ARS staining of hDFCs in the LV-CHD7 and LV-GFP group. (d) qRT-PCR analyses of the expression of RUNX2 , SP7 , BGLAP , DLX5 , BMP2 , and COL1A1 under osteogenic condition.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Staining, Expressing

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: RNA-seq revealed the downregulated enrichment of the PTH-related pathway after CHD7 depletion. (a) GO enrichment unraveled that skeletal system development and ossification were suppressed after CHD7 depletion. (b) Heatmap of representative osteogenesis associated genes. (c) KEGG enrichment unraveled that the PTH-related pathway was significantly suppressed after CHD7 depletion. (d) GSEA showed decreased enrichment of PTH-regulated genes in CHD7-deficient hDFCs.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: RNA Sequencing Assay

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: CHD7 regulates the expression of PTH1R. (a) qRT-PCR and Western blot of the PTH1R expression after CHD7 depletion. (b) qRT-PCR and Western blot of the PTH1R expression after the CHD7 overexpression. (c) Anti-CHD7 ChIP assay. CHD7 can bound to the promoter region of PTH1R, and the ChIP signaling was significantly suppressed after CHD7 depletion.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: The overexpression of PTH1R partially rescues the osteogenic differentiation of CHD7-deficiency hDFCs. (a), (b) qRT-PCR and Western blot verified the overexpression efficiency of PTH1R . (c) Representative images and quantitative analyses of ALP and ARS staining of hDFCs in the siCTRL, siCHD7, and rescue group. (d) qRT-PCR analyses of the expression of RUNX2 , SP7 , BGLAP , DLX5 , BMP2 , and COL1A1 under osteogenic condition.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Staining, Expressing