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: PLOS One

Article Title: KIF11 promotes rheumatoid arthritis pathogenesis by activating M1 macrophage polarization and promoting inflammatory cytokine secretion

doi: 10.1371/journal.pone.0347313

Figure Lengend Snippet: (A) A petal plot demonstrated 672 overlapping DEGs identified from five RA synovial tissue datasets ( GSE55457 , GSE55235 , GSE2053 , GSE12021 , and GSE1919 ). DEGs were selected with thresholds of |log2FC| ≥ 0.5 and adjusted p-value < 0.05. (B) The mRNA expression levels of KIF11 in rheumatoid arthritis (RA) and normal control (NC) synovial tissues were analyzed in six independent GEO datasets: GSE1919 , GSE12021 , GSE55235 , GSE55457 , GSE77298 , and GSE2053 . Data are presented as mean ± SD. ***P < 0.001, compared with NC controls. (C) MH7A cells were treated with TNF-α (10 ng/mL) and IL-1β (10 ng/mL) for 24 h, and the expression of KIF11 was determined via western blotting. (D) The relative expression of KIF11 was shown in histogram. ***P < 0.01, compared with untreated group.

Article Snippet: KIF11 human shRNA lentiviral particles (Locus ID 3832, Catalog Number: TL311921V) and their corresponding control lentiviral particles (Catalog Number: TR30021V) were obtained from Origene Inc.

Techniques: Expressing, Control, Western Blot

Journal: PLOS One

Article Title: KIF11 promotes rheumatoid arthritis pathogenesis by activating M1 macrophage polarization and promoting inflammatory cytokine secretion

doi: 10.1371/journal.pone.0347313

Figure Lengend Snippet: (A) The expression of KIF11 was detected by western blotting. (C) Cell viability was measured by MTT assay at 24, 48, and 72 h post-infection with control shRNA (shCtrl) or KIF11 shRNA (shKIF11) lentivirus. n = 3. ***P < 0.001 vs. Ctrl. shRNA group. (D) Edu incorporation assay. Fluorescence imaging results at 24 and 48 hours. E. The proportion of positively stained cells is displayed in a bar graph, ***P < 0.001. F. Colony formation assay. Representative images (E) and quantification (F) of colony formation assay in Ctrl. shRNA- and KIF11 shRNA infected MH7A cells after two-week culture. ***P < 0.001 vs. Ctrl.shRNA group, ###p < 0.001 vs.untreated cells .

Article Snippet: KIF11 human shRNA lentiviral particles (Locus ID 3832, Catalog Number: TL311921V) and their corresponding control lentiviral particles (Catalog Number: TR30021V) were obtained from Origene Inc.

Techniques: Expressing, Western Blot, MTT Assay, Infection, Control, shRNA, Fluorescence, Imaging, Staining, Colony Assay

Journal: PLOS One

Article Title: KIF11 promotes rheumatoid arthritis pathogenesis by activating M1 macrophage polarization and promoting inflammatory cytokine secretion

doi: 10.1371/journal.pone.0347313

Figure Lengend Snippet: (A) Transwell migration assay of shCtrl- and shKIF11-infected MH7A cells at 24 h and 48 h. Representative images (40 × magnification) and migrated cell quantification (right). Data are mean ± SD ( n = 3). *P < 0.05, **P < 0.01 vs. CtrlshRNA group. (B) The expression of IL-1β, IL-6, and IL-8 was detected by ELISA assay in cell supernatant. ***P < 0.001. (C) Rescue of KIF11 expression in knockdown cells. MH7A cells with stable KIF11 knockdown (shKIF11) were transfected with either a human KIF11 cDNA overexpression plasmid (KIF11-OE) or the empty control vector (Pcmv3 vector). After 24 hours, KIF11 protein expression was analyzed by Western blotting. β-actin served as a loading control. (D) The relative KIF11 protein levels were shown in histogram. Data are presented as mean ± SD (n = 3). ***P < 0.001 vs. the vector-transfected shKIF11 group. (E) Rescue of pro-inflammatory cytokine secretion. The cell culture supernatants from the experiment described in (D) were collected, and the concentrations of TNF-α, IL-1β, IL-6, and IL-8 were measured by ELISA. Data are presented as mean ± SD (n = 3). ***p < 0.001 vs. the control vector-transfected shKIF11 group.

Article Snippet: KIF11 human shRNA lentiviral particles (Locus ID 3832, Catalog Number: TL311921V) and their corresponding control lentiviral particles (Catalog Number: TR30021V) were obtained from Origene Inc.

Techniques: Transwell Migration Assay, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Transfection, Over Expression, Plasmid Preparation, Control, Western Blot, Cell Culture

Journal: PLOS One

Article Title: KIF11 promotes rheumatoid arthritis pathogenesis by activating M1 macrophage polarization and promoting inflammatory cytokine secretion

doi: 10.1371/journal.pone.0347313

Figure Lengend Snippet: (A) KIF11 knockdown inhibited NF-κB p65 phosphorylation. MH7A cells were infected with control shRNA (shCtrl) or KIF11 shRNA (shKIF11) lentivirus for 24 hours. Whole-cell lysates were subjected to Western blotting using antibodies against phospho-p65 (p-p65), total p65, and KIF11. β-actin served as a loading control. (B) The histogram shows the relative ratio of p-p65 to total p65. Data are presented as mean ± SD (n = 3). ***P < 0.001 vs. shCtrl group. (C) KIF11 knockdown impaired nuclear translocation of NF-κB p65. Cytoplasmic and nuclear fractions were extracted from shCtrl- and shKIF11-infected MH7A cells. The distribution of p65 in the cytoplasm (Cyto) and nucleus (Nuc) was analyzed by Western blotting. Lamin B1 and α-tubulin were used as markers for nuclear and cytoplasmic fractions, respectively. (D) The relative abundance of p65 in the nuclear and cytoplasmic fractions were shown in histograms, normalized to their respective loading controls. Data are presented as mean ± SD (n = 3). ***P < 0.001 vs. shCtrl group.

Article Snippet: KIF11 human shRNA lentiviral particles (Locus ID 3832, Catalog Number: TL311921V) and their corresponding control lentiviral particles (Catalog Number: TR30021V) were obtained from Origene Inc.

Techniques: Knockdown, Phospho-proteomics, Infection, Control, shRNA, Western Blot, Translocation Assay

Journal: PLOS One

Article Title: KIF11 promotes rheumatoid arthritis pathogenesis by activating M1 macrophage polarization and promoting inflammatory cytokine secretion

doi: 10.1371/journal.pone.0347313

Figure Lengend Snippet: A. KIF11 was knock down in M1 macrophages for 24 h. B. The histogram of gray value was shown IN Ctrl. shRNA or KIF11 shRNA lentiviruses infected M1 macrophages. **P < 0.01. Interference with KIF11 in M1 macrophages decreased the expression of CD86. The intensity of surface molecules CD14 and CD86 (C) and CD86(D) were determined by flow cytometry in THP-1-derived M0 and M1 cells, shKIF11 lentivirus or ctrl. shRNA lentivirus infected M1 macrophages. E. The histogram of CD86 and CD80 expression was shown. ***P < 0.001.

Article Snippet: KIF11 human shRNA lentiviral particles (Locus ID 3832, Catalog Number: TL311921V) and their corresponding control lentiviral particles (Catalog Number: TR30021V) were obtained from Origene Inc.

Techniques: Knockdown, shRNA, Infection, Expressing, Flow Cytometry, Derivative Assay