Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: ( A ) EG5 ( KIF11 ) protein domain structure indicating the position of the MLC variants p.L347Efs*8 and p.R387*. ( B ) qRT-PCR analysis of blood using allele-specific primers (mut: patient variant allele) showing strong reduction but not a complete absence of the mutant mRNA in all samples (F2-I.2, F2-II.1, F1-I.2 and F1-II.1), suggesting nonsense-mediated RNA decay. WT KIF11 mRNA levels decreased by up to 50% in patients compared with controls. Data represent average relative expression ( n = 3 experiments) ± SD. ( C ) Western blot analysis of lymphoblastoid cell line lysates indicate approximately 50% reduction in the levels of WT EG5 protein (119 kDa) in the patient samples (F2-I.2, F2-II.1, and F1-II.1) using C-terminal binding EG5 antibody (NB500-181, Novus Biologicals). Average of 2 technical repeats of identical biological materials is shown with β-actin as loading control. ( D ) Western blot analysis with an N-terminal binding anti-EG5 antibody (CC10014, Cell Applications) showed the presence of a truncated protein of the expected size in patient F1-I.2 (arrow). The position of molecular mass markers (in kDa) is indicated on the left of the gel. ( E ) Left panel: Sanger sequencing of gDNA from proband F5-II.1. PCR product shows a compound peak in exon 20, which is the synonymous KIF11 c.2922G>A; p.(P974=) variant. Right panel: Sanger sequencing of cDNA from F5-II.1. Black line indicates the boundary between the last base of exon 20 and first base of exon 21. Notice the heteroduplex in exon 20 indicating exon skipping. Analysis of the full trace identified a loss of the last 108 bases in exon 20 (r.2815_2922del), which is predicted to lead to a shorter EG5 protein similar to that of the synonymous variant c.2922G>T as shown by others .

Article Snippet: Eg5 monoclonal antibodies against the N-terminal (CC10014, Cell Applications or NB500-181, Novus Biologicals) were used in the Western blots, and β-actin was used as an internal control.

Techniques: Quantitative RT-PCR, Variant Assay, Mutagenesis, Expressing, Western Blot, Binding Assay, Control, Sequencing

Journal: JCI Insight

Article Title: Insights into KIF11 pathogenesis in microcephaly-lymphedema-chorioretinopathy syndrome from a lymphatic perspective

doi: 10.1172/jci.insight.177656

Figure Lengend Snippet: Mouse embryonic sections from developmental stages ( A ) E10.5 and ( B ) E12.5 were subjected to immunofluorescence staining. Selected magnified (framed) areas highlight regions containing dermal lymphatics and the thoracic duct, which were imaged for HOECHST (blue; channel 1), Ki67 (green; channel 2), EG5 (red; channel 3), and VEGFR3 (white; channel 4) protein expression. Additionally, whole-mount immunofluorescence staining was performed on ( C ) intestinal and ( D ) ear samples. Selected magnifications highlight regions with lacteals ( C ) and initial lymphatic vessels ( D ), also imaged for Ki67 (green; channel 2), EG5 (red; channel 3), and VEGFR3 (white; channel 4); merged images are also shown. Arrowheads indicate areas with coexpression of EG5 and VEGFR3. Scale bars: 100 μm. Additional labeling was added to facilitate the understanding of the embryonic anatomy: A, artery; LEC, lymphatic endothelial cell; NC, notochord; NT, neural tube; PLV, peripheral lymphatic vessel; PTD, primordial thoracic duct; V, vein.

Article Snippet: Eg5 monoclonal antibodies against the N-terminal (CC10014, Cell Applications or NB500-181, Novus Biologicals) were used in the Western blots, and β-actin was used as an internal control.

Techniques: Immunofluorescence, Staining, Expressing, Labeling

Journal: Chemmedchem

Article Title: Cytotoxicity of Atropisomeric [1,1′‐Binaphthalene]‐2,2′‐Diamines (BINAM) and Analogs in Human Cancer Cells: Enantioselectivity, Structure–Activity Relationships, and Mechanism

doi: 10.1002/cmdc.202500426

Figure Lengend Snippet: Impact of 1( R ) and 1( S ) on the motor proteins KIF1C, Eg5, and the microtubule destabilizing protein stathmin. Nuclear DNA is displayed in blue in all images. The scale bar (10 µm) is valid for the respective row of images. A) 1( R ) has no effects on the distribution of KIF1C (green). B) Eg5 (green) is diffusely distributed in mitotic cells treated with 1( R ) . C) 1( R ) treated cells do not show an induction of stathmin (green).

Article Snippet: Antibodies used were as follows: Alexa Fluor 488 conjugate (#4412, CST, 1:500), Alexa Fluor 594 conjugate (#8890, CST, 1:500), alpha‐tubulin (#11224‐1‐AP, Proteintech, 1:100), acetyl alpha‐tubulin (#5335, CST, 1:100), centrin (#ZMS1054, Sigma–Aldrich, 1:100), detyrosinated alpha‐tubulin (#abx375026, Abbexa, 1:100), EB1 (#17717‐1‐AP, Proteintech, 1:200), Eg5 (#23333‐1‐AP, Proteintech, 1:200), gamma‐tubulin (#CL594‐66320, Proteintech, 1:100), KIF1C (#12760‐1‐AP, Proteintech, 1:100), stathmin 1 (#11157‐1‐AP, Proteintech, 1:100).

Techniques: