rabbit anti phf6  (Bethyl)

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) PHF6 protein expression is reduced in cell lines derived from lymphocytes of individuals affected by BFLS. Schematic drawing of the protein domains and coding exon structure of the PHF6 gene with BFLS variants indicated and Western blot showing expression of PHF6 migrating at approximately 41 kDa in 8 males affected by BFLS, one female carrier and four unaffected males (control). Protein expression in individuals with the PHF6 p.C45Y, p.C99F and p.R342* variants was reduced, while the p.D333del and p.E337del variants are expressed at a similar or even higher level to the four unaffected individuals (control). Note the band migrating at approximately 38.5 kDa in the patients with the p.R342* variant indicating that the truncated protein is likely to be expressed at a low abundance. Details on individuals included in this figure are in . (B, C) RNA-sequencing reads as reads per kilobase per one million reads in the sequencing library (RPKM) mapping to Phf6 exons in Phf6 +/Y vs. Phf6 –/Y mouse E15.5 developing cortex (B) and Phf6 +/Y ;Nes-cre Tg/+ vs. Phf6 lox/Y ;Nes-cre Tg/+ mouse E16.5 cortical neurons (C). (D) Detection of PHF6 protein by western immunoblotting in E15.5 mouse brains of Phf6 genotypes as indicated. Each lane represents an individual foetus. Note the absence of PHF6 protein at ~41 kDa in foetuses with germline deletion ( Phf6 –/Y ;Nes-cre +/+ ) or nervous system-specific deletion ( Phf6 lox/Y ;Nes-cre Tg/+ ) of the single allele of Phf6 in males or germline deletion of one allele and nervous system-specific deletion of the second allele in the female ( Phf6 –/lox ;Nes-cre Tg/+ ). N = 8 individuals with BFLS, 1 BFLS carrier and 4 control individuals (A), 3 Phf6 +/Y and 5 Phf6 –/Y E15.5 mouse foetuses (B), 3 Phf6 +/Y ;Nes-cre Tg/+ and 4 Phf6 lox/Y ;Nes-cre Tg/+ E16.5 mouse foetuses (C) and 3 Phf6 deleted and 5 control E15.5 mouse foetuses, genotypes indicated above the blot (D). Data are displayed as mean ± sem and individual foetuses are represented by circles (B,C). RNA sequencing data (B,C) were analysed as described in the methods section; p values displayed above bars, two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Variant Assay, RNA Sequencing Assay, Sequencing

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) Time to first seizure in female heterozygous germline deleted Phf6 +/– mice compared to Phf6 +/+ wild-type control mice. Wild-type mice did not display seizures. Median time to seizure onset was 662 days. N = 70 Phf6 +/+ mice and 103 Phf6 +/– mice. (B) Time to first seizure in females with Phf6 heterozygous deletion in the nervous system compared to controls; Phf6 +/+ ;Nestin-cre transgenic control mice did not display seizures. N = 11 Phf6 +/+ ;Nes-cre Tg/+ and 11 Phf6 lox/+ ;Nes-cre Tg/+ mice. Time to seizure onset of a small number of Phf6 lox/lox ;Nes-cre Tg/+ mice (homozygous nervous system-specific deleted) is shown in . (C) Time to first seizure in males with Phf6 deletion in the nervous system compared to controls. Phf6 +/Y ;Nestin-cre transgenic control mice did not display seizures. N = 33 Phf6 +/Y ;Nes-cre Tg/+ and 13 Phf6 lox/Y ;Nes-cre Tg/+ mice. Checked points indicate mice that were sacrificed due to illness or other reasons. Data were analysed by a log-rank (Mantel-Cox) test. NS = nervous system. Survival data of mice with germline deletion of Phf6 are shown in .

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Control, Transgenic Assay

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) Images of 10 μm brain serial sections stained with cresyl-violet from a representative Phf6 lox/Y ;Nes-cre Tg/+ and a Phf6 +/Y ;Nes-cre Tg/+ paired control. Adult brains from 3 Phf6 lox/Y ;Nes-cre Tg/+ mice were fixed 17 to 37 days after onset of seizures, each with an age-matched Phf6 +/Y ;Nes-cre Tg/+ control (394 to 483 days old). Scale bar = 1 mm. (B-D) Volumes of the brain, lateral ventricles and cortex in Phf6 lox/Y ;Nes-cre Tg/+ versus Phf6 +/Y ;Nes-cre Tg/+ mice showing data here corresponding to the brain region spanning the lateral ventricles. Volumes were measured for the brain, lateral ventricle and cortex area in seven matched sections per animal across sections spaced at approximately 480 μm intervals. N = 3 age-matched pairs of Phf6 lox/Y ;Nes-cre Tg/+ mouse and Phf6 +/Y ;Nes-cre Tg/+ mice. Bars in the graph show the mean ± sem of each genotype, while lines between individual data points join each Phf6 lox/Y ;Nes-cre Tg/+ animal to its paired control. Data were analysed by a two-tailed paired t-test. (E) Images showing representative cresyl-violet-stained sections of the cerebral cortex from the brains of Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ mice. The approximate region of the cortex area shown is indicated by the red rectangles on the coronal images on the left. Numbers next the sections indicate the cortical layers I to VI. Scale bar = 100 μm. Related data are shown in , and Figs; assessment of interneuron number, Purkinje cell number and incidence of hydrocephalus are displayed in , and Figs, respectively.

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Staining, Control, Two Tailed Test

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) Representative images of cortical layer staining using SATB2, CTIP2, and TBR1 showing the developing Phf6 +/Y and Phf6 –/Y E18.5 parietal cortex divided into 10 pial to ventricular bins. Scale bar = 50 μm. (B) Percentage of cells expressing the marker proteins in each bin for three mice per genotype. No significant differences between genotypes were observed. (C) Percentage of cells of each type across the entire thickness of the cortex. No significant differences between genotypes were observed. N = 3 Phf6 +/Y and 3 Phf6 –/Y foetuses. Data are presented as mean ± sem and were analysed by two-way ANOVA with Šídák’s multiple comparisons test. Circles (C) represent individual mouse foetuses. Related data are shown in and Figs; assessment of neurite outgrowths in cultured cortical neurons is displayed in .

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Staining, Expressing, Marker, Cell Culture

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) Representative bright field images of neural stem and progenitor cells (NSPCs) isolated from the dorsal telencephalon of E12.5 Phf6 +/Y ;Nes-cre Tg/+ and Phf6 lox/Y ;Nes-cre Tg/+ embryos and grown as neurospheres in proliferation medium with FGF2 and EGF. Scale bar equals 220 μm. (B) Secondary neurosphere formation assay of Phf6 +/Y ;Nes-cre Tg/+ and Phf6 lox/Y ;Nes-cre Tg/+ NSPCs plated at the cell numbers per 96-well plate well as indicated. (C) Representative immunofluorescence images of Phf6 +/Y ;Nes-cre Tg/+ and Phf6 lox/Y ;Nes-cre Tg/+ NSPCs plated on laminin coated slides and grown for 5 days in differentiation medium (without FGF2 or EGF and with FCS) stained for a neuronal marker (ßIII-tubulin, red) and an astrocyte marker (GFAP, green) and counterstained with DAPI. Scale bar equals 25 μm. (D) Flow cytometry assessment of the percentage of neuron (ßIII-tubulin + ), astrocytes (GFAP + and S100ß + ) and oligodendrocytes (O4 + ) in Phf6 +/Y ;Nes-cre Tg/+ and Phf6 lox/Y ;Nes-cre Tg/+ NSPCs plated on laminin coated slides and grown for 5 days in differentiation medium. N = NSPC cell isolates from 4 embryos per genotype cultured in triplicates (B) and NSPC cell isolates from 9 Phf6 +/Y ;Nes-cre Tg/+ and 5 Phf6 lox/Y ;Nes-cre Tg/+ embryos (D). Data are depicted as means ± sem. Data were analysed by two-factorial ANOVA (B) or unpaired two-tailed Student’s t-test (D). Related data are displayed in and Figs.

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Isolation, Tube Formation Assay, Immunofluorescence, Staining, Marker, Flow Cytometry, Cell Culture, Two Tailed Test

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) Heatmap showing differentially expressed genes between N = 3 Phf6 +/Y and 5 Phf6 –/Y E15.5 cortices. Values are row-wise z-scores. (B) Heatmap showing differentially expressed genes between N = 3 Phf6 lox/Y ;Nes-cre Tg/+ and 4 Phf6 +/Y ;Nes-cre Tg/+ E16.5 cortical neuron isolates. Values are row-wise z-scores. (C) Barcode plot showing correlation between the genes differentially expressed in the Phf6 –/Y vs . Phf6 +/Y E15.5 cortex and in the Phf6 lox/Y ;Nes-cre Tg/+ vs. Phf6 +/Y ;Nes-cre Tg/+ E16.5 cultured cortical neurons. The horizontal axis shows the t-statistic for all genes in the Phf6 -deleted E15.5 cortex and the blue and red shaded areas are genes that tend towards down- or upregulation respectively in the Phf6 deleted E15.5 cortex. The vertical lines represent the genes differentially expressed in the E16.5 Phf6 deleted cultured cortical neurons. Worms show the relative enrichment of differentially expressed genes in the E16.5 dataset among the differentially expressed genes in the E15.5 dataset. The data sets are positively correlated (genes tend to be similarly up- or downregulated in the same direction). ROAST p-value shown. (D) Log 2 -fold change in RNA levels of the top 10 differentially expressed genes by FDR in Phf6 –/Y vs. Phf6 +/Y E15.5 cortex and Phf6 lox/Y ;Nes-cre Tg/+ vs. Phf6 +/Y ;Nes-cre Tg/+ E16.5 cortical neurons. FDR shown above bars. (E,F) Barcode plots showing correlation between differentially expressed genes for indicated datasets. The horizontal axis shows moderated t-statistics for all genes in either the Phf6 -deleted E15.5 cortex (E) or the E16.5 cultured cortical neuron dataset (F). The blue and red shaded areas represent genes that tend towards down or upregulation. The vertical lines represent the genes differentially expressed in the Phf6 -deleted PN0 cortex samples described by Cheng and colleagues . Worms show the relative enrichment of differentially expressed genes of PN0 genes among the differentially expressed genes of the E15.5 and E16.5 datasets. In each case, the data sets are positively correlated, i.e., genes are generally differentially expressed in the same direction. P-values are from ROAST gene set tests. (G,H) Top 10 enriched GO terms in the Phf6 -deleted E15.5 cerebral cortex (G) and E16.5 cultured cortical neurons (H). BP = biological process, MF = molecular function. The analysis of RNA sequencing data is described in the methods section. FDR < 0.05 was considered significant. Related data are shown in and Figs and in and Tables.

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Cell Culture, RNA Sequencing Assay

Journal: PLOS Genetics

Article Title: Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson–Forssman–Lehmann intellectual disability syndrome

doi: 10.1371/journal.pgen.1011428

Figure Lengend Snippet: (A) Representative immunofluorescence images of cortical neurons isolated from E16.5 Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ foetuses detecting the excitatory neuron marker vesicular glutamate transporter 1 (VGLUT1; green) counterstained with DAPI (blue) after 5 days in culture. (B) Percentage of Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ cells positive for VGLUT1 staining in cultures of cortical neurons. (C) Representative fluorescence images of cortical neurons isolated from E16.5 Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ foetuses loaded with a Ca 2+ responsive dye (Fluo-4, AM) to reveal high intracellular calcium concentrations (green). (D) Representative calcium imaging tracks of individual neurons isolated from E16.5 Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ cortices assessed after 7 days of culture in vitro. (E,F) Percentage of Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ cortical neurons displaying minor (1% to <10% of the maximal amplitude; E) or major calcium signalling peaks (≥10% of the maximal amplitude; F) assessed after 5 and 7 days of culture in vitro. (G) Number of calcium signalling peaks per neuron in neurons displaying peaks above background (≥1% of maximal amplitude) in Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ cortical neuron cultures assessed after 5 to 7 days in vitro. (H) Maximum peak height per neuron in neurons displaying peaks above background (≥1% of maximal amplitude) in Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ cortical neuron cultures assessed after 5 to 7 days in vitro. (I,J) Height of minor (1% to <10% of the maximal amplitude; I) and major peaks (≥10% of the maximal amplitude; J) in Phf6 lox/Y ;Nes-cre Tg/+ and Phf6 +/Y ;Nes-cre Tg/+ cortical neuron cultures assessed after 5 to 7 days in vitro. N = cortical neurons isolated from 3 Phf6 +/Y ;Nes-cre Tg/+ and 3 Phf6 lox/Y ;Nes-cre Tg/+ foetuses (A,B) and 906 Phf6 +/Y ;Nes-cre Tg/+ and 932 Phf6 lox/Y ;Nes-cre Tg/+ cortical neurons from 8 Phf6 +/Y ;Nes-cre Tg/+ and 4 Phf6 lox/Y ;Nes-cre Tg/+ foetuses (C-J). Data are displayed as mean ± sem (B,G,H), percentage of cells (E,F) or median and quartiles (I,J). Circles represent individual foetuses (B) or individual neurons (G,H). Data were analysed by unpaired Student’s t-test (B,G,H), Chi-squared test (E,F) and two-way ANOVA with cell and genotype as the independent factors (I,J). Related data are displayed in .

Article Snippet: The membrane was blocked with 5% skim-milk/PBST, probed with rabbit anti-PHF6 (Bethyl Laboratories, A301-452A), mouse anti-α Tubulin (Invitrogen, A11126) and subsequently HRP conjugated anti-rabbit IgG (Sigma, NA934) and HRP conjugated anti-mouse IgG (Sigma, NA931).

Techniques: Immunofluorescence, Isolation, Marker, Staining, Fluorescence, Imaging, In Vitro

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Overexpression analysis of variants in PHF6 from females vs males. ( a ) Protein scheme of PHF6 (Q8IWS0) with all missense variants previously published in male and female individuals , , , , , as well as three selected truncating variants , analysed via immunofluorescence coloured in red. Analysed variants (immunofluorescence) are printed in bold. Coding domains (according to uniprot ) are displayed in colour. NLS nuclear localization domain, NoLS nucleolar localization domain. ( b ) Table including all published missense variants occurring in male and female individuals, their VIPUR score, their localization in regard to ePHD and their origin (inherited/de novo). Variants p.(Met1Tyr) and p.(Ser138Cys) do not reside in a domain and cannot be modelled. Analysed variants (immunofluorescence) are printed in bold. ePHD extended atypical PHD-like zinc finger domain, 1 = N-terminal, 2 = C-terminal. ( c ) Immunofluorescence of HEK-293 cells transiently transfected with HA-tagged wildtype or mutant PHF6 co-stained with nucleolin (NCL). While wildtype PHF6 was expressed in the nucleoplasm as well as the nucleolus co-localizing with NCL, all three variants occurring in females led to an accumulation of PHF6 into aggregates, partly co-localizing with NCL. The truncating variant c.677delG (p.(Gly226Glufs*53)) resulted in small and more aggregates in the nucleus, and the two variants in the C-terminal ePHD resulted in large PHF6 aggregates while the expression of aberrant PHF6 in the nucleoplasm is reduced. In contrast, the three variants occurring in males resembled the wildtype with PHF6 expression in both the nucleoplasm and the nucleolus, co-localizing with NCL. Per variant, at least 40 cells were analyzed. Scale bar depicts 20 µm. Cells were stained with rabbit anti-HA (H6908, Sigma-Aldrich, 1:75) and mouse anti-nucleolin (39–6400, Thermo Fisher Scientific, 1:200). ( d ) Quantification of nuclear aggregates. The number of cells with or without aberrant large or small PHF6 aggregates in the nucleus was quantified for each variant and the wildtype. At least 40 cells were evaluated for each variant. ♀, variant identified in a female individual; ♂, variant identified in a male individual.

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: Over Expression, Immunofluorescence, Transfection, Mutagenesis, Staining, Variant Assay, Expressing

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Variants in PHF6 in female individuals lead to a loss of protein. In fibroblasts of female individuals with variants in PHF6 , only the wildtype PHF6 allele was expressed. ( a ) Immunofluorescence of fibroblasts of three individuals with either a duplication of exons 4 and 5, a missense variant c.914G>T (p.(Cys305Phe), or a truncating variant c.955C>T (p.(Arg319*) and one control individual. In the control, PHF6 expression could be observed in all fibroblast cells. In affected individuals, expression of PHF6 was present in only 50% of cells. Scale bar represents 20 µm. Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls. Affected individuals showed expression of PHF6 in 30–50% of cells, respectively. For each individual, at least 63 cells were evaluated. ( c ) Sanger sequencing of genomic DNA (gDNA) and of cDNA from RNA of affected individuals. In the individuals with a missense or a truncating variant respectively, both wildtype and mutant allele could be observed on gDNA level. On cDNA level, only the wildtype allele was present, thus indicating nonsense mediated mRNA decay. ( d ) Exemplary western blot from fibroblasts of control and affected individuals stained for PHF6 (Santa Cruz Biotechnology, sc-365237, 1:500) and Tubulin (ab7291, abcam, 1:10,000) as a housekeeping gene. PHF6 levels in patient fibroblasts were decreased. Grouped blots were cropped from different blots. Uncropped blots can be found in Supplementary Fig. . ( e ) Graphical analysis of three western blot replicates indicating 50%-70% of remaining PHF6 protein in patient fibroblasts. Error bars depict the standard deviation. Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: Immunofluorescence, Variant Assay, Expressing, Staining, Sequencing, Mutagenesis, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Transcriptome analysis of neuron-like PHF6 KO cells. ( a ) Heatmap displaying 1,338 deregulated genes between differentiated SK-N-BE (2) PHF6 KO and control cells with adjusted p value < 0.05 (FDR corrected). 626 genes were upregulated, and 712 were downregulated in PHF6 KO cells. Heatmap was created using the pheatmap package v.1.0.12 applying the standard settings (complete linkage method for hierarchical clustering for both columns and rows, scaling for rows). Blue = downregulated; red = upregulated. ( b , c ) Gene Ontology (GO) term analysis of ( b ) upregulated and ( c) downregulated genes depicting the top five GO terms for biological processes. ( d ) Comparison of deregulated genes after KO/KD of common chromatin remodelers and transcription factors with deregulated genes after PHF6 KO divided in up- and downregulated genes. Deregulated genes after KO/KD of either KDM5C ( Kdm5c KO mouse model, RNA sequencing of amygdala and frontal cortex) , the BAF complex (dKO Baf155/Baf170 mouse model, RNA sequencing of the pallium of E17.5 embryos) , BMI1 ( Bmi1 KO mouse model, RNA sequencing of whole brain) or TCF4 ( TCF4 KD SH-SY5Y neuroblastoma cells) expressed a highly significant overlap with up- and downregulated genes after PHF6 KO. Darkness of color correlates with significance. ( e ) Significant GO terms involved in neuron development and axonogenesis (p < 0.005). ( f ) Venn diagram depicting that deregulated genes were significantly enriched for genes implicated in neurodevelopmental disorders (NDDs). Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: Overlap of deregulated genes in PHF6 KO SK-N-BE (2) cells with other deregulated gene sets.

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques:

Journal: Scientific Reports

Article Title: Loss of PHF6 leads to aberrant development of human neuron-like cells

doi: 10.1038/s41598-020-75999-2

Figure Lengend Snippet: PHF6 KO in cells reveals impaired neurite outgrowth, proliferation and migration. ( a ) Example of differentiating control and PHF6 KO cells transfected with mCD8-GFP. The cell with PHF6 KO had a longer primary neurite (axon) than the control cell. Scale bar depicts 20 µm. ( b ) Quantitative analysis of total neurite length, number of primary neurites and axon length (longest primary neurite) of day 10 differentiated PHF6 KO and control cells. PHF6 KO cells had significantly longer protrusions (total neurite length) and significantly more primary neurites. Additionally, axon length was also significantly increased in PHF6 KO cells. Of note, while control cells developed 1–2 neurites, some of the PHF6 KO cells had up to five neurites. For each cell line, at least 15 cells were evaluated per biological replicate. ( c ) After 8 days of differentiation, PHF6 KO cells showed significantly impaired proliferation in comparison to control cells. ( d ) Scratch assay showing that undifferentiated PHF6 KO cells migrated significantly slower than control cells. All experiments were performed in biological triplicates, except for the proliferation assay, which was performed in biological duplicates. Error bars depict the standard deviation. Asterisks indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Cells were stained with rabbit polyclonal anti-PHF6 antibody (HPA001023, Sigma-Aldrich, 1:50). ( b ) Numerical analysis of fibroblasts with variants in PHF6 and controls.

Techniques: Migration, Transfection, Wound Healing Assay, Proliferation Assay, Standard Deviation