Journal: Frontiers in Genetics

Article Title: Identification of a Ferroptosis Gene Set That Mediates the Prognosis of Squamous Cell Carcinoma of the Head and Neck

doi: 10.3389/fgene.2021.698040

Figure Lengend Snippet: GEPIA verification of ferroptosis-related gene (FRG) expression levels and their relationship with prognosis. (A–E) Expression levels of SLC7A11 , AKR1C3 , AURKA , CAV1 , and TRIB3 . (F–J) Kaplan-Meier curves for OS or disease-free survival based on the five FRGs. TRIB3 and CAV1 are significantly related to OS, AURKA is closely related to patient disease-free survival ( P < 0.05).

Article Snippet: IHC was used to detect protein expression using AKR1C3 (1:100, A13568, Abclonal), CAV1 (1:100, A1555, Abclonal), AURKA (1:200, bs2749R, bioss), and TRIB3 (1:200, bs7538R, bioss) antibodies.

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Identification of a Ferroptosis Gene Set That Mediates the Prognosis of Squamous Cell Carcinoma of the Head and Neck

doi: 10.3389/fgene.2021.698040

Figure Lengend Snippet: Relative expression levels of the five genes in normal and squamous cell carcinoma of the head and neck tissues (HNSCC). SLC7A11 (A) , AKR1C3 (B) , AURKA (C) , CAV1 (D) , and TRIB3 (E) are significantly up-regulated in HNSCC tissues. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: IHC was used to detect protein expression using AKR1C3 (1:100, A13568, Abclonal), CAV1 (1:100, A1555, Abclonal), AURKA (1:200, bs2749R, bioss), and TRIB3 (1:200, bs7538R, bioss) antibodies.

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Identification of a Ferroptosis Gene Set That Mediates the Prognosis of Squamous Cell Carcinoma of the Head and Neck

doi: 10.3389/fgene.2021.698040

Figure Lengend Snippet: Representative immunohistochemistry results. (A,C,E,G) represent normal tissue. (B,D,F,H) represents tumor tissue (the scale bar represents 100 μm). (A,B) AKR1C3 protein. (C,D) AURKA protein. (E,F) CAV1protein. (G,H) TRIB3 protein.

Article Snippet: IHC was used to detect protein expression using AKR1C3 (1:100, A13568, Abclonal), CAV1 (1:100, A1555, Abclonal), AURKA (1:200, bs2749R, bioss), and TRIB3 (1:200, bs7538R, bioss) antibodies.

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: Spatially distinct inputs modulate the amount of active Mitotic-phase GAP to locally restrict RhoA signaling for successful cell division

doi: 10.1101/2023.08.08.552464

Figure Lengend Snippet: A) Aurora A kinase could facilitate RhoA inactivation by activating the RhoA GAP MP-GAP. B) Schematic of MP-GAP domain organization highlighting the three fragments and putative phosphosites tested in the kinase assays. C) Coomassie gel and autoradiography images of Aurora A kinase assays with GST-tagged GAP domain and F1 and F2 region of MP-GAP. Different lanes of the same gel are shown. D) Sequence alignment of the F1 region of MP-GAP. The conserved putative Aurora A phosphosites are highlighted in red. E, F) Coomassie gel and autoradiography images of Aurora A kinase assays with the myelin basic protein (MBP, positive control), GST (negative control), GST-tagged F1-WT, F1-C1 (S285A, S304A, S345A) and F1-C2 (S403A, S434A, S500A, T508A) (E) or F1-S285A, F1-S304A, and F1-S345A (F) of MP-GAP.

Article Snippet: Proximity ligation assays with Aurora A kinase and MP-GAP were performed in HeLa cells using mouse anti-Aurora A Kinase antibody (1:500, A1231 Merck), rabbit anti-MP-GAP antibody (1:250, self-made), Duolink In Situ PLA Probe Anti-Mouse MINUS (DUO92004, Sigma-Aldrich), Duolink In Situ PLA Probe Anti-Rabbit PLUS (DUO92002, Sigma-Aldrich), and Duolink In Situ Detection Reagents Orange (DUO92007, Sigma-Aldrich).

Techniques: Autoradiography, Sequencing, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Spatially distinct inputs modulate the amount of active Mitotic-phase GAP to locally restrict RhoA signaling for successful cell division

doi: 10.1101/2023.08.08.552464

Figure Lengend Snippet: A) Merged immunofluorescent images showing PLA foci (white) and DNA (red) for metaphase and anaphase HeLa cell. Staining was performed either with the anti-Aurora A and anti-MP-GAP together (positive) or only with one antibody (negative). B) Distribution of the PLA foci in % at the cell periphery, middle or center during metaphase and anaphase. C) The number of PLA foci per 100 µm 2 is plotted for the central or polar area of the cell. D) Immunofluorescence images of F-actin (white) and DNA (red) stained HeLa cells treated with DMSO or 2.5 µM Latrunculin A (LatA). E) Immunofluorescence images of MP-GAP (white) and DNA (red) stained HeLa cells treated with DMSO or 2.5 µM Latrunculin A. F) MP-GAP fluorescence intensity is plotted for an equatorial linescan. Examples of an equatorial and polar linescan are shown for illustration. G) Quantification of the mean cortical MP-GAP intensity as depicted in (F) for the entire cell cortex in metaphase and for the poles and equator in anaphase cells for indicated treatments. Values above the dotted line represent cortical MP-GAP enrichment. H) Shown are immunofluorescence images of metaphase and anaphase HeLa cells treated with DMSO or MK-5108. Cells were stained for MP-GAP (white) and DNA (red). I) Shown are immunofluorescence images of metaphase and anaphase HeLa cells expressing GFP-MP-GAP WT or GFP-MP-GAP S3A and treated with MP-GAP siRNA. Cells were stained for MP-GAP (white) and DNA (red). J) Quantification of the mean cortical MP-GAP intensity as depicted in (F) for the entire cell cortex in metaphase and for the poles and equator in anaphase cells for indicated treatments. For all n = number of cells, and ≥2 independent experiments were performed for each condition. Error bars are SEM, and p values were calculated with the Kruskal-Wallis test. Scale bars are 5 µm.

Article Snippet: Proximity ligation assays with Aurora A kinase and MP-GAP were performed in HeLa cells using mouse anti-Aurora A Kinase antibody (1:500, A1231 Merck), rabbit anti-MP-GAP antibody (1:250, self-made), Duolink In Situ PLA Probe Anti-Mouse MINUS (DUO92004, Sigma-Aldrich), Duolink In Situ PLA Probe Anti-Rabbit PLUS (DUO92002, Sigma-Aldrich), and Duolink In Situ Detection Reagents Orange (DUO92007, Sigma-Aldrich).

Techniques: Staining, Immunofluorescence, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Spatially distinct inputs modulate the amount of active Mitotic-phase GAP to locally restrict RhoA signaling for successful cell division

doi: 10.1101/2023.08.08.552464

Figure Lengend Snippet: A) Scheme of the possible autoinhibitory conformation of MP-GAP (left). Coomassie gel and anti-His probed immunoblot of pull down assays incubating immobilized GST-tagged MP-GAP fragments (F1, F2 and GAP) with soluble 6xHis-tagged GAP domain (right). B) Scheme of the putative position of the Aurora A phosphorylation sites in either the hinge region (left) or on the binding surface (right) of F1. Coomassie gel and anti-His probed immunoblot of pull down assays incubating immobilized GST-tagged F1 wild type or F1 S3D with soluble 6xHis-tagged GAP domain (right).

Article Snippet: Proximity ligation assays with Aurora A kinase and MP-GAP were performed in HeLa cells using mouse anti-Aurora A Kinase antibody (1:500, A1231 Merck), rabbit anti-MP-GAP antibody (1:250, self-made), Duolink In Situ PLA Probe Anti-Mouse MINUS (DUO92004, Sigma-Aldrich), Duolink In Situ PLA Probe Anti-Rabbit PLUS (DUO92002, Sigma-Aldrich), and Duolink In Situ Detection Reagents Orange (DUO92007, Sigma-Aldrich).

Techniques: Western Blot, Binding Assay

Journal: bioRxiv

Article Title: Spatially distinct inputs modulate the amount of active Mitotic-phase GAP to locally restrict RhoA signaling for successful cell division

doi: 10.1101/2023.08.08.552464

Figure Lengend Snippet: A) In normal cells Aurora A signal is high at the poles and Ect2 autoinhibition is locally released at the cell equator (top). Abolishing Ect2 autoinhibition by the W307A mutation, results in active Ect2 throughout the cell (middle). Without Aurora A signaling, Ect2 autoinhibition is still locally released at the equator (bottom). In all conditions cytokinesis succeeds. B) Merged time-lapse transmission and DNA (SiR-DNA, red) images of HeLa cells expressing Ect2 WT or Ect2 W307A . Cells were treated during live-cell imaging around anaphase onset with DMSO or MK-5108 as indicated. C, D) Percentage of cytokinesis failure (C) and equatorial cell width (D) of the conditions filmed in (B). E) Two spatially distinct regulatory inputs control MP-GAP function at the cell equator and the cell poles during anaphase. At the cell poles F-actin levels are low, but Aurora A kinase activity is high. Aurora A phosphorylates the hinge region between the GAP domain and the F1 region resulting in opening and activation of MP-GAP and RhoA inactivation. At the cell equator Ect2 autoinhibition is released, RhoA is activated and promotes F-actin polymerization. Due to high F-actin levels MP-GAP enriches at the equator and increases the GTP hydrolysis rate of RhoA resulting in high RhoA flux and narrowing of the active RhoA zone. For all n = number of cells, and ≥2 independent experiments were performed for each condition. Scale bars are 5 µm.

Article Snippet: Proximity ligation assays with Aurora A kinase and MP-GAP were performed in HeLa cells using mouse anti-Aurora A Kinase antibody (1:500, A1231 Merck), rabbit anti-MP-GAP antibody (1:250, self-made), Duolink In Situ PLA Probe Anti-Mouse MINUS (DUO92004, Sigma-Aldrich), Duolink In Situ PLA Probe Anti-Rabbit PLUS (DUO92002, Sigma-Aldrich), and Duolink In Situ Detection Reagents Orange (DUO92007, Sigma-Aldrich).

Techniques: Mutagenesis, Transmission Assay, Expressing, Live Cell Imaging, Activity Assay, Activation Assay