Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence

Journal: PLoS ONE

Article Title: Cyclin-Dependent Kinase 4 Phosphorylates and Positively Regulates PAX3-FOXO1 in Human Alveolar Rhabdomyosarcoma Cells

doi: 10.1371/journal.pone.0058193

Figure Lengend Snippet: A ) In vitro kinase assay using GFP or GFP-PAX3-FOXO1 (GFP-PF; wild-type or S430A mutant) pulled down from transfected Rh30 cells. Top panel: 32 P Phospho-image. Bottom panel: protein substrates revealed by Coomassie blue staining. B ) Schematic diagram of three GST-PF fusion proteins (showing only the PAX3-FOXO1 portion). PF, full length PAX3-FOXO1; 1–423, the N-terminal 423 residues of PAX3-FOXO1; 1–459, N-terminal 459 residues of PAX3-FOXO1; S430, Serine 430; PAX3, the PAX3 portion of PAX3-FOXO1; FOXO1, the FOXO1 portion of PAX3-FOXO1. C ) In vitro kinase assay using bacterially expressed PAX3-FOXO1. From top to bottom panel: 32 P Phospho-image ( 32 P); protein substrates revealed by Coomassie blue staining (Coomassie); Western blot using phosphor-ser CDKs substrate antibody (IB: Phospho-Ser); and Western blot using anti-GST monoclonal antibody (IB: GST). D ) Detection of phosphorylation using GFP or GFP-PF (wild-type or S430A mutant) proteins pulled down from transfected Rh30 cells. Top panel: Western blot using anti-GFP monoclonal antibody; Bottom panel: Western blot using phosphor-ser CDKs substrate antibody. Rb, positive control for Cdk4 activity; M, protein marker indicating molecular weight (kD).

Article Snippet: In the in vitro kinase assays, 100 ng of Cdk4/cyclin D1 (SignalChem, Richmond, Canada) was used per reaction.

Techniques: In Vitro, Kinase Assay, Mutagenesis, Transfection, Staining, Western Blot, Positive Control, Activity Assay, Marker, Molecular Weight

Journal: PLoS ONE

Article Title: Cyclin-Dependent Kinase 4 Phosphorylates and Positively Regulates PAX3-FOXO1 in Human Alveolar Rhabdomyosarcoma Cells

doi: 10.1371/journal.pone.0058193

Figure Lengend Snippet: A ) NIH3T3 cells were co-transfected with wild-type GFP-PAX3-FOXO1 (left panel) or GFP-PAX3-FOXO1 S430A (right panel), 6 X PRS9 reporter, TK-renilla (PRL-TK), and one of the plasmids as indicated. Comparisons were made between other samples and a sample transfected with pcDNA3 (set as 100 relative luciferase units, or RLU). Cdk4, pCMV-Cdk4 (wild-type); Cdk4DN, pCMV-Cdk4DN (inactive Cdk4); cyclin D1, pCMV-cyclin D1. B ) Protein expression in A) was confirmed by Western blot analysis using anti-GFP antibody (IB: GFP), anti-Cdk4 antibody (IB: Cdk4), anti-cycin D1 (IB: cyclin D1), and anti-actin (IB: actin). Actin was used as a loading control. GFP-PF, GFP-PAX3-FOXO1. C ) NIH3T3 cells were transfected with 6 X PRS9 reporter, TK-renilla, and one of the plasmids indicated. PF WT, wild-type GFP-PAX3-FOXO1; PFS430A, GFP-PAX3-FOXO1S430A; PFS430D, GFP-PAX3-FOXO1S430D. Comparisons were made between other samples and a sample transfected with wild-type GFP-PAX3-FOXO1 (set as 100 RLUs). D ) Protein expression in B) was confirmed by Western blot analysis. Actin was used as a loading control. Luciferase activity was measured 24 h after transfection. Firefly luciferase activity was normalized using Renilla luciferase activity. * p <0.05; *** p <0.001.

Article Snippet: In the in vitro kinase assays, 100 ng of Cdk4/cyclin D1 (SignalChem, Richmond, Canada) was used per reaction.

Techniques: Transfection, Luciferase, Expressing, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Cyclin-Dependent Kinase 4 Phosphorylates and Positively Regulates PAX3-FOXO1 in Human Alveolar Rhabdomyosarcoma Cells

doi: 10.1371/journal.pone.0058193

Figure Lengend Snippet: A ) ARMS cells (Rh30 and Rh41) and ERMS cells (RD and JR1) were treated with 0.69 µM fascaplysin for indicated times before cell viability was determined. Fascaplysin at 0.69 µM started to inhibit cell viability of Rh30 cells as shown in . B ) Cdk4 levels in various cell lines.

Article Snippet: In the in vitro kinase assays, 100 ng of Cdk4/cyclin D1 (SignalChem, Richmond, Canada) was used per reaction.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Conditional Neuronal Simian Virus 40 T Antigen Expression Induces Alzheimer-Like Tau and Amyloid Pathology in Mice

doi: 10.1523/JNEUROSCI.0186-07.2007

Figure Lengend Snippet: Antibodies

Article Snippet: , cdk4 (H303) , Full-length cdk4 , , 1:200 , Santa Cruz Biotechnology.

Techniques: Virus

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Approach for target identification using a combination of competitive photoaffinity labeling (PAL) and affinity purification in rat hippocampal homogenate followed by quantitative proteomics. (B) Representative anti-biotin Western blot of hippocampal homogenate after PAL and competition with 2 . (C) Identification of CaMKIIα from LC-MS/MS data as the best hit from non-linear regression analysis for all proteins, and (D) concentration-dependent competition of individual proteins by 2 during PAL. (E) Target validation by [ 3 H]- 1 autoradiography using brain slices from Camk2a and Camk2b wildtype (+/+) and knockout (-/-) mice (cresyl violet staining for tissue visualization). (F-H) Target validation by [ 3 H]- 1 binding to whole cell homogenate from transfected HEK293T cells. (F) [ 3 H]- 1 saturation binding to CaMKIIα ( n = 5), shown is one representative curve; means ± SD). (G) CaMKIIα competition with GHB ( n = 3), 1 ( n = 5) and 2 ( n = 3), pooled data (means ± SEM). (H) Subtype selectivity of [ 3 H]- 1 for CaMKIIα cf. CaMKIIβ/γ/δ. Data are pooled ( n = 3) for each subtype and depicted as specific binding (% of total).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Labeling, Affinity Purification, Western Blot, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Autoradiography, Knock-Out, Staining, Binding Assay, Transfection

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Schematic of a single CaMKIIα subunit comprised of a kinase domain (gray), regulatory segment (green), linker (yellow), and hub domain (lilac). 12-14 hub domains oligomerize into the holoenzyme, shown here in an activated form with kinase domains displaced from the hub. (B) Concentration-dependent binding of 2 to immobilized CaMKIIα 6x Hub measured by SPR (top), and Langmuir binding isotherm (bottom), representative data. (C) Ball and stick model of key binding residues (bold), nearby residues, and hydrogen bonds in green-dashed lines. (D) X-ray crystal structure of 2 bound to the CaMKIIα 6x (14-mer) Hub. (E) Close-up view of a single hub subunit showing the key molecular interactions, displacement (flip) of Trp403 with ligand bound highlighted. (F) Quenching of intrinsic fluorescence caused by Trp403 flip (6x Hub) with increasing concentrations of 2 ( n = 8), pooled data (means ± SEM). (G) Mutational analysis of key residues in the pocket using [ 3 H]- 1 equilibrium binding to CaMKIIα-HEK293T overexpressing cells. Compared to wildtype (WT), binding is completely obliterated in a construct lacking the hub as well as in R433Q, R453Q, R469Q, triple mutant R433/453/468/Q (RRR→QQQ) and H395A mutants ( n = 3).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Concentration Assay, Binding Assay, Fluorescence, Construct, Mutagenesis

Journal: bioRxiv

Article Title: GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

doi: 10.1101/2020.09.28.310474

Figure Lengend Snippet: (A) Right-shifted thermal shift assay melting curves of CaMKIIα WT hub upon binding of GHB, 1 and 2 ( left ) and 1 concentration-dependence (right), representative data. (B) No effect of 1 on Ca 2+ -stimulated Thr286 phosphorylation. Shown is quantification of mean band intensities of Ca 2+ -stimulated pThr286 levels ( left ) normalized to total CaMKIIα expression of cultured cortical neurons (DIV 18-20) incubated with 50-100 μM Ca 2+ alone or together with 3 mM of 1 for 1 h and representative Western blots ( right ). GAPDH was used as loading control (C) Time-( left ) and concentration-dependent effects of 1 ( right ) on cell survival at 24 h in cultured cortical neurons (DIV 16-18) stimulated with 100-200/20 μM Glu/Gly for 1 h (tat- 4 as control). Cell death was normalized to maximum cell death as measured by LDH release. (One-way ANOVA, post-hoc Dunnett’s test). (D) Quantification of GluN2B-CaMKIIα co-localization in hippocampal neurons (DIV 14-19) exposed to Glu 400 μM for 2 min and immediately fixed ( left ) and representative immunostained images ( righ t). For B-D: Number in bar diagrams indicates number of experiments/individual cultures. Box plots (boxes, 25–75%; whiskers, minimum and maximum; lines, median). (One-way ANOVA, post-hoc Dunnett’s test).

Article Snippet: 6x Hub human protein, WT hub or full-length human recombinant CaMKIIα (#02-109, Carna Biosciences) were immobilized on to a biosensor surfaces by amine coupling using a 20 mM NaAc pH 5 immobilization buffer.

Techniques: Thermal Shift Assay, Binding Assay, Concentration Assay, Expressing, Cell Culture, Incubation, Western Blot