Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Human T-lymphocytes were reprogrammed into iPSCs. WT and CHD8 KD karyotypes were normal.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: CRISPR/Cas9-mediated CHD8 KD . (a) CRISPR/Cas9 target of 20 nucleotides (green box) and PAM site (green letters) adjacent to the CHD8 Ser62 codon was used for the single guide RNA. To facilitate homologous recombination, a donor DNA molecule was used consisting of left (HAL) and right (HAL) homology arms and loxP sites (blue arrows) flanking the EGFP cassette (EGFP cDNA with upstream splice acceptor sequence (SA), puromycin resistance gene (Puro), and CAG promoter). (b) EGFP facilitated manual selection of successfully targeted iPSC colonies under fluorescence microscopy. (c) This was confirmed by fluorescence-activated cell sorting.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques: CRISPR, Homologous Recombination, Sequencing, Selection, Fluorescence, Microscopy, FACS

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Gene expression analysis. (a) qRT-PCR showed that, compared to WT, CHD8 mRNA levels were reduced in KD cell lines by an average of 41% in iPSCs, 52% in NPCs, and 59% in neural cells. (b and c) Western blot analysis of NPCs showed that CHD8 protein was reduced by an average of 58%.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Effects of CHD8 KD on proliferation and differentiation. CHD8 KD caused (a) increased iPSC colony numbers, (b) increased NPC growth, and (c) suppression of spontaneous differentiation around the edges of iPSC colonies.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: NPC rosette formation. During the differentiation of iPSCs to NPCs, cells typically organize into rosettes by approximately two weeks. CHD8 KD delayed rosette formation by one week, or by approximately a third.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Neurite outgrowth. 12 days after NPCs were plated, CHD8 KD resulted in a significantly decreased neurite area (white arrows) per cell (nuclear) area.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Effect of CHD8 KD on the cell cycle. A significantly smaller percentage of CHD8 KD NPCs (58.84 ± 4.59) is found in the G0/G1 phase of the cell cycle compared to WT NPCs (72.16 ± 6.8, P = 0.007, t-test), and a larger percentage of CHD8 KD NPCs (21.90 ± 7.08) is found in the G2/M phases of the cell cycle compared to WT NPCs (12.10 ± 6.82, P = 0.057, t-test).

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: bioRxiv

Article Title: CHD8 regulates the balance between proliferation and differentiation of human iPSCs in neural development

doi: 10.1101/732693

Figure Lengend Snippet: Effects of CHD8 KD on cell composition. CHD KD resulted in: (a) decreased MAP2/GFAP ratio, (b) decreased fraction of VGLUT1+ cells, and (c) decreased percentage of SYN1+ cells.

Article Snippet: The membranes were incubated with gentle shaking overnight at 4°C with rabbit anti-CHD8 (N-terminal) primary antibody (Cell Signaling Technology, Table S2) diluted 1:2000 in 5% (w/v) BSA, 1X TBS, and 0.1% Tween-20.

Techniques:

Journal: Molecular Autism

Article Title: Distinct, dosage-sensitive requirements for the autism-associated factor CHD8 during cortical development

doi: 10.1186/s13229-020-00409-3

Figure Lengend Snippet: Increased proliferation of basal neural progenitors in Chd8 neo/neo embryos. a Immunohistochemistry to detect PH3B+ (blue) and TBR2+ (green) nuclei in coronal sections through the telencephalon of E14.5 embryos. White arrowheads indicate PH3B+/TBR2+nuclei. White box is shown as zoomed inset containing a double positive cell. b Quantification of TBR2+/PH3B+cells per 100 µm of neocortex in E14.5 embryos (+/+, n = 20; neo/+, n = 9; +/−, n = 7; neo/neo, n = 6; neo/−, n = 8; Mean ± SEM, * p < 0.05, *** p < 0.001, ANOVA followed by Tukey’s multiple comparisons test). c – e Cleaved caspase 3 (CC3) immunostaining of dorsal neocortex of E14.5 embryos. Black arrows indicate CC3+ cells and black box in E highlights area shown in zoomed inset containing a CC3+ cell. Scale bar = 100 μm. f Relative quantification of CC3+ cells in neocortex of E14.5 Chd8 mutant embryos, normalised to respective wildtype littermates (+/+, n = 17; neo/ + , n = 7; neo/neo, n = 6; neo/−, n = 11; Mean ± SEM ** p < 0.01). Embryos used in these experiments were from 8 different litters

Article Snippet: The following antibodies were used: mouse anti-BrdU (BD Biosciences, 1/100), rabbit anti-phosphohistone 3B (Cell Signaling, 1/100), mouse anti-phosphohistone 3B (Abcam, 1/200), chicken anti-TBR2 (Merck Millipore, 1/200), rabbit anti-cleaved-caspase 3 (Cell Signaling, 1/200), rabbit anti-doublecortin (Abcam, 1/400) or rabbit anti-CHD8 (Bethyl, 225A, 1/400).

Techniques: Immunohistochemistry, Immunostaining, Mutagenesis