Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: ETV2 binds with KDM4A (A and B) Rosa26-CreERT2;Kdm4a/4c f/f mESCs ±4′-OH-tamoxifen were differentiated and analyzed on day 4 for RT-qPCR (A, n = 3) and flow cytometry (B, B′, n = 3). (B) Representative data from three independent experiments. (B′) Quantification data of flow cytometry. Con, wild-type control; DKO, double knockout; FL1, empty channel of flow cytometry. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C) Overexpression of ETV2 increases genes downregulated in Kdm4a/4c DKO mESCs. Kdm4a/4c DKO mESCs overexpressing Etv2 were differentiated and subjected to RT-qPCR. n = 3. ∗∗∗ p < 0.001. (D) Enhanced H3K9me3 in endothelial and hematopoietic genes in the absence of ETV2. Etv2 −/− mESCs were differentiated, harvested at day 4 and cross-linked. Subsequently, the nuclear genomic DNAs were sonicated, and the fragmented genomic DNAs were immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter or enhancer regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05. (E–G) (E) Interaction of ETV2 and KDM4A. HEK/293T cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and western blot analysis. Anti-TUBULIN antibody was used for the internal loading control. (F) In vitro translated FLAG-ETV2 and HA-KDM4A proteins were subjected to immunoprecipitation with an anti-FLAG antibody and western blot analysis with the indicated antibodies. (G) The differentiated wild type mESCs were subjected to immunoprecipitation with an anti-KDM4A antibody, followed by a WB blot analysis with an anti-ETV2 antibody. (H) The cooperative function of ETV2-KDM4A interaction in inducing Flk1 promoter/enhancer ( p/e ) activity. Each construct expressing ETV2 or KDM4A (wt or H188A, a defective mutant of demethylation activity of KDM4A) was introduced into HEK/293T cells together with the Firefly luciferase reporter construct, pGL3-Flk1(p/e) . The Firefly luciferase activity was normalized with Renilla luciferase activity. Data are mean ± SEM of three independent experiments with triplicate assays/experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Goat polyclonal anti-α-tubulin antibody , Origene , Cat# AB0046-200; RRID: N/A.

Techniques: Quantitative RT-PCR, Flow Cytometry, Control, Double Knockout, Over Expression, Sonication, Immunoprecipitation, Amplification, Transfection, Construct, Western Blot, In Vitro, Activity Assay, Expressing, Mutagenesis, Luciferase

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet:

Article Snippet: Goat polyclonal anti-α-tubulin antibody , Origene , Cat# AB0046-200; RRID: N/A.

Techniques: Plasmid Preparation, Recombinant, Virus, Control, Transfection, Staining, Knock-Out, Protease Inhibitor, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Cell Culture, DNA Purification, DNA Methylation Assay, Reporter Assay, Software

Journal: PLoS ONE

Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

doi: 10.1371/journal.pone.0114208

Figure Lengend Snippet: A. VK2/E6E7 cells were synchronized and bacteria were allowed to adhere and invade cells for 24 h. Cells were then fixed and stained with DAPI. The nuclear area of 300 infected and 300 uninfected cells were measured. Data were analyzed using a paired 2-tailed Student's t -test. The average nuclear areas observed in 3 independent experiments are shown (* p <0.05). Error bars represent S.E.M. B. Cells were infected with N. gonorrhoeae and observed for 24 h by live-cell microscopy. DIC images were captured every 15 min at randomly selected positions, and times spent transitioning from prophase to cytokinesis were measured for 150 mitotic cells. The graph shows mean ± standard deviation values for the time required for progression from prophase to cytokinesis, measured in 3 independent experiments. C. VK2/E6E7 cells were infected with N. gonorrhoeae for 24 h. RNA from control cells and infected cells was isolated and qPCR was performed. Average mRNA levels of MAD1L1 and MAD2L1 from 3 independent experiments are shown (* p <0.05). Error bars represent S.E.M. Values were normalized against TUBA1A expression and analyzed using a paired 2-tailed Student's t -test. D. Protein expression in infected cells and control cells was analyzed in western blots using antibodies against of MAD1 and MAD2. GAPDH or α-tubulin was used as loading controls. E. Western blot band intensities were quantified and analyzed with a paired 2-tailed Student's t -test, using GAPDH or α-tubulin expression as controls. Mean expression levels of each protein observed in 3 independent western blots are shown (* p <0.05). Error bars represent S.E.M.

Article Snippet: Polyclonal antibodies against α-tubulin (MBS316320, MyBioSource, 1∶1,000) or GAPDH (G-9545, Sigma-Aldrich, 1∶5,000) were used for normalizing total protein loaded in each well.

Techniques: Bacteria, Staining, Infection, Microscopy, Standard Deviation, Control, Isolation, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway

doi: 10.1038/s41598-018-26694-w

Figure Lengend Snippet: Tubulin glutamylation in C. elegans sensory cilia is modulated by the environment. ( a ) Schematic diagram of a worm head that illustrates the positions of the ciliated cell bodies (right) and their ending (left). ( b – g ) Results of immunohistochemical analysis of the C. elegans head (around tip of nose) using GT335, which recognises glutamylated tubulin. Signals can be observed in the cilia of amphid, inner labial, and outer labial neurons. Signal intensities were compared between control ( b ) and worms exposed to environmental stimuli ( c – g ). ( h ) Quantified result of tubulin glutamylation described in ( b – g ). All stimuli except physical vibration induced a significant increase in tubulin glutamylation. Asterisks (* p < 0.01, Student’s t test) and double asterisks (** p < 0.001, Student’s t test) indicate significant differences as compared with the wild-type. Environmental stimuli of “heat”, “cold”, “osmotic”, and “starvation” but not of “shake” induced a significant increase in glutamylation. Numbers of animals scored are indicated (h, bottom). Bars indicate mean ± S.E. Scale bars = 5 μm.

Article Snippet: The anti-β-tubulin polyclonal antibody (MBL, Nagoya, Japan) was diluted 1:200.

Techniques: Immunohistochemical staining, Control

Journal: Scientific Reports

Article Title: Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway

doi: 10.1038/s41598-018-26694-w

Figure Lengend Snippet: Tubulin glutamylation in mouse retina is modulated by the light exposure. ( a ) Schematic diagram of a mammalian photoreceptor cell (rod). The region was divided into three areas: outer segment, transition zone, and inner segment. ( b ) Result of immunohistochemical analysis of mouse retina using anti-polyglutamate polyclonal antibody (top, red), α-tubulin (second row, green), merged (third row), and DIC (bottom). Nuclei are shown with DAPI staining (blue). Left column: dark adaptation. Right column: light adaptation. Polyglutamate signals can be observed in connective cilia (red arrow). Tubulin signals were observed in the transition zone and inner segment. Tubulin glutamylation co-localised with tubulin in the transition zone. OS: outer segment, TZ: transition zone, IS: inner segment, ONL: outer nuclear layer. ( c ) Quantitative analysis of five independent mice. All mice have a significant reduction of tubulin glutamylation ( p < 0.05). Scale bars = 20 μm.

Article Snippet: The anti-β-tubulin polyclonal antibody (MBL, Nagoya, Japan) was diluted 1:200.

Techniques: Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway

doi: 10.1038/s41598-018-26694-w

Figure Lengend Snippet: Modification of tubulin glutamylation is mediated by p38 MAPK signalling. ( a – c ) Results of the immunohistochemical analysis of activated p38 MAPK-specific antibody in wild-type ( a ) and a pmk-1 mutant ( b ). Quantified result of activated p38 MAPK in wild-type and the mutants of members of the p38 MAPK signalling pathway ( c ). p38 MAPK is activated in sensory cilia in a manner dependent on the upstream kinases in the pathway. ( d – l ) Results of the immunohistochemical analysis of tubulin glutamylation in wild-type ( d – f ), pmk-1 ( g – i ), and nsy-1 ( j – l ) mutants under control ( d , e and j ), cold ( e , h and k ), and high osmotic ( f , i and l ) conditions. ( m ) Quantified result of tubulin glutamylation. The glutamylation levels after environmental stimuli were relative values for the control (no environmental stimuli). The enhancement of tubulin glutamylation is dependent on p38 MAPK. Y-axis is the relative glutamylation level to control (no environmental stimuli). Thus, the differences of glutamylation level seen in each mutant background were removed. ( n ) Time course analysis of tubulin glutamylation after starvation. Bars indicate mean ± S.E. All scale bars = 5 μm. An asterisk (* p < 0.05, Student’s t test) indicates significant difference as compared with the signal intensity before starvation in wild-type ( n ). Double asterisks (** p < 0.001, Student’s t test) indicate significant difference as compared with the wild-type ( c and m ). Numbers of animals scored are indicated ( c and m , bottom).

Article Snippet: The anti-β-tubulin polyclonal antibody (MBL, Nagoya, Japan) was diluted 1:200.

Techniques: Modification, Immunohistochemical staining, Mutagenesis, Control

Journal: Scientific Reports

Article Title: Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway

doi: 10.1038/s41598-018-26694-w

Figure Lengend Snippet: Tubulin glutamylation is regulated by the phosphorylation of tubulin glutamate ligase TTLL-4. ( a ) Schematic structure of TTLL-4. Blue area marks the TTLL domain, which is essential for the enzymatic activity. Ser or Thr residues that can be putatively phosphorylated by MAPK are indicated. ( b – h ) Tubulin glutamylation induced by exogenous expression of wild-type and mutant forms of TTLL-4. ( i ) Time course analysis of tubulin glutamylation after starvation. Wild-type: ttll-4 mutant exogenously expressing wild-type TTLL-4; T446A: ttll-4 mutant exogenously expressing phosphor-null TTLL-4; T446E: ttll-4 mutant exogenously expressing phosphomimetic TTLL-4. An asterisk (* p < 0.05, Student’s t test) indicates significant difference as compared with the signal intensity before starvation in wild-type ( i ). Numbers of animals measured are indicated (bottom). Bars indicate mean ± S.E. Scale bar = 5 μm.

Article Snippet: The anti-β-tubulin polyclonal antibody (MBL, Nagoya, Japan) was diluted 1:200.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Mutagenesis

Journal: Scientific Reports

Article Title: Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway

doi: 10.1038/s41598-018-26694-w

Figure Lengend Snippet: Starvation-induced attenuation of osmotic avoidance behaviour is dependent on tubulin glutamylation. ( a ) Schematic diagram of the sensitised osmotic avoidance assay. ( b ) TTLL-4-dependent suppression of avoidance behaviour is induced by starvation. Numbers of assay plates measured are indicated. Numbers in brackets represent the number of independent experiments. Double asterisk ( ** p < 0.01, Student’s t test) indicates significant difference as compared with the avoidance index after starvation in wild-type. Bars indicate mean ± S.E.

Article Snippet: The anti-β-tubulin polyclonal antibody (MBL, Nagoya, Japan) was diluted 1:200.

Techniques:

Journal:

Article Title: The EF-Hand Ca 2+ -binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca 2+ -binding Requirements

doi: 10.1091/mbc.E03-07-0500

Figure Lengend Snippet: Characterization of the intracellular distribution of p22. BHK (A-F) or BHK-ER (G-O) cells were fixed in 4% paraformaldehyde and processed for immunofluorescence by using antibodies against p22 (B, E, H, K, and N), tubulin (A), and GM130 (D). BHK-ER cells were used for ER staining (G, J, and M). J-L represent magnifications of the regions of interest (square) shown in G-I. In M-O, BHK-ER cells were treated with 5 μg/ml nocodazole for 1 h before cell fixation. Arrows in D-F indicate colocalization between p22 and GM130 structures. Arrowheads in J-O indicate colocalization between p22 and ER structures. Bars, 10 μm.

Article Snippet: Polyclonal anti-tubulin antibodies were purchased from Biogenesis Inc. (Kingston, NH) to couple taxol-stabilized microtubules to magnetic beads.

Techniques: Immunofluorescence, Staining

Journal:

Article Title: The EF-Hand Ca 2+ -binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca 2+ -binding Requirements

doi: 10.1091/mbc.E03-07-0500

Figure Lengend Snippet: Role of p22 in the interactions between ER membranes and microtubules. (A) Schematic representation of the two-step microtubule-membrane binding assay. (B) Top, DYNABEADS M-280 tosylactivated were covered with taxol-polymerized microtubules (lanes 1 and 2 and 4-6) or not (lane 3) and incubated with or without rat liver cytosol (lanes 1, 5, and 6) and/or myr-p22 (lanes 1, 3, 4, 6) in the first step. Then, beads were incubated with (lanes 2-6) or without (lane 1) microsomal membranes in the second step and immunoblotted with calnexin antibodies. A value of 100% was assigned to the relative binding of microsomal membranes to microtubule-covered beads after incubation with cytosol in the absence of myr-p22 (lane 5). Data represents mean ± SD of three experiments. (B) Bottom, equal amounts of reaction mixtures were analyzed by SDS-PAGE and immunoblotting by using antibodies against tubulin (tubulin) and p22 (myr-p22). (C) Top, microtubule-membrane binding bead assays were performed in the presence of cytosol with (lanes 3 and 4) or without (lanes 1 and 2) myr-p22 in the first step and in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 40 μM free Ca2+ during the second step. A value of 100% was assigned to the relative amount of microsomal membrane binding shown in lane 1, as described above. Data represents mean ± SD of three experiments. (C) Bottom, equal amounts of reaction mixtures were analyzed by SDS-PAGE and immunoblotting by using antibodies against tubulin (tubulin) and p22 (myr-p22).

Article Snippet: Polyclonal anti-tubulin antibodies were purchased from Biogenesis Inc. (Kingston, NH) to couple taxol-stabilized microtubules to magnetic beads.

Techniques: Binding Assay, Incubation, SDS Page, Western Blot

Journal:

Article Title: The EF-Hand Ca 2+ -binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca 2+ -binding Requirements

doi: 10.1091/mbc.E03-07-0500

Figure Lengend Snippet: Effect of bulk microinjection of anti-p22 APpep2 antibodies on the organization of the microtubule cytoskeleton. BHK cells were bulk microinjected with APpep2 antibodies (APpep2: A-F and M-O) or APpep2 previously preincubated with pep2 peptide (pep2-competition: G-L) and then allowed to recover at 37°C for 2 h (A-L, 2 h postdig) or 4 h (M-O, 4 h postdig) before fixation. Cells were processed for immunofluorescence by using tubulin antibodies (B, E, H, K, and N). A, D, G, J, and M show APpep2 staining, indicating bulk-microinjected cells. C, F, I, L, and O represent merged images. D-F and J-L are magnifications of the regions of interest shown in A-C and G-I, respectively. Bars, 10 μm.

Article Snippet: Polyclonal anti-tubulin antibodies were purchased from Biogenesis Inc. (Kingston, NH) to couple taxol-stabilized microtubules to magnetic beads.

Techniques: Immunofluorescence, Staining

Journal:

Article Title: The EF-Hand Ca 2+ -binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca 2+ -binding Requirements

doi: 10.1091/mbc.E03-07-0500

Figure Lengend Snippet: Effect of bulk microinjection of myr-p22 or myr-p22-E134A on the organization of the microtubule cytoskeleton. BHK cells were mock (A-C), myr-p22 (D-F and J-L) or myr-p22-E134A (G-I) bulk microinjected. After 2 h at 37°C, cells were fixed and processed for immunofluorescence by using APpep2 (A, D, and G) and tubulin (B, E, and H) antibodies. C, F, and I represent merged images. Bars, 10 μm. Arrowheads in D-F indicate p22-positive microtubule bundles.

Article Snippet: Polyclonal anti-tubulin antibodies were purchased from Biogenesis Inc. (Kingston, NH) to couple taxol-stabilized microtubules to magnetic beads.

Techniques: Immunofluorescence

Journal:

Article Title: The EF-Hand Ca 2+ -binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca 2+ -binding Requirements

doi: 10.1091/mbc.E03-07-0500

Figure Lengend Snippet: ER structures accumulate at myr-p22-induced microtubule bundles. (A) BHK-ER cells were mock (a and b), myr-p22 (c and d), or myr-p22-E134A (e and f) bulk microinjected, allowed to recover for 2 h and then processed for immunofluorescence by using tubulin antibodies. a, c, and e show the ECFP-ER fluorescence pattern, and b, d, and f show the microtubule staining. Lines were drawn from the periphery to the perinuclear region in mock (lines 1 and 2) and myr-p22 (lines 6 and 7) or myr-p22-E134A (lines 11 and 12) bulk-microinjected cells, and the corresponding plot profiles are shown in B. These lines were made to cross several myr-p22 (lines 6 and 7) or myr-p22-E134A (lines 11 and 12) induced microtubule bundles. Bars, 10 μm. (B) ImageJ software was used to produce plot profiles of the microtubule (MT: dashed lines) and ER (solid lines) networks along the lines drawn in the cells shown in A and in other not-shown cells. ER1/MT1-ER5/MT5: profiles for mock bulk-microinjected cells. ER6/MT6-ER10/MT10: profiles for myr-p22 bulk-microinjected cells. ER11/MT11-ER15/MT15: profiles for myr-E134A-p22 bulk-microinjected cells.

Article Snippet: Polyclonal anti-tubulin antibodies were purchased from Biogenesis Inc. (Kingston, NH) to couple taxol-stabilized microtubules to magnetic beads.

Techniques: Immunofluorescence, Fluorescence, Staining, Software

Journal: Oncotarget

Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

doi: 10.18632/oncotarget.12482

Figure Lengend Snippet: ( A and B ) ASCs were treated with indicated Plk1 inhibitors (50 nM BI 2536, BI 6727 or 50 μM Poloxin) for 48 h and cell cycle analyses were performed for visceral and subcutaneous ASCs. The results are based on three independent experiments, presented as mean ± SEM and statistically analyzed compared to DMSO treated cells. ** p < 0.01. (C and E) ASCs were treated as in (A and B) and stained for α-tubulin, pericentrin, ACA (anti-centromere antibody) and DNA for immunofluorescence microscopy. Mitotic arrested ASCs were evaluated. Representatives are shown for visceral ASCs ( C ) and subcutaneous ASCs ( E ). White arrows indicate mitotic cells; red arrows indicate fragmented cell nuclei. Scale: 25 μm. ( D and F ) Quantification of mitotic cells ( n = 150 analyzed cells for each condition). The results are from three independent experiments and presented as mean ± SEM. ** p < 0.01, *** p < 0.001. ( G and H ) Cellular extracts from treated ASCs were prepared for Western blot analyses with indicated antibodies. β-actin served as loading and DMSO treated cells as vehicle control.

Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

Techniques: Staining, Immunofluorescence, Microscopy, Western Blot, Control

Journal: Oncotarget

Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

doi: 10.18632/oncotarget.12482

Figure Lengend Snippet: ASCs were subjected to Plk1 compounds (25 nM BI 2536, 25 nM BI 6727 or 25 μM Poloxin) for 4 days and 14 days. Media and compounds were renewed every 3 days. (A and B) To evaluate the induction of DNA damage, treated cells were stained for the DNA damage markers γ-H2AX and 53BP1, α-tubulin and DNA. Representatives are shown. White arrows indicate fragmented cell nuclei and red arrows depict abnormal enlarged cell nuclei. Scale: 25 μm. Insets are a four time magnification of boxed regions in leftist panels. Scale: 6.5 μm. ( C and D ) Quantification of γ-H2AX and 53BP1 double positive cells. The results are based on three independent experiments and presented as mean ± SEM. ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

Techniques: Staining

Journal: Oncotarget

Article Title: Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells

doi: 10.18632/oncotarget.12482

Figure Lengend Snippet: ASCs were treated with Plk1 inhibitors (50 nM BI 2536 or 50 nM BI 6727) for 9 h and released into fresh media. Treated ASCs and breast cancer cells were seeded in separated chambers of an culture insert and cultured for 0 h, 8 h and 15 h. Cells were then stained for microtubule marker acetylated α-tubulin, actin marker phalloidin and DNA for analyzing the number of membrane protrusions, including lamellipodia and filopodia, on the migration front of ASCs toward breast cancer cells. ( A ) Evaluation of protrusions in subcutaneous ASCs toward MDA-MB-231 and MCF-7 cells. Each experiment was performed in triplicate, and the results are from three independent experiments and shown as mean ± SEM. ** p < 0.01. ( B ) Representatives are depicted. Left side: subcutaneous ASCs; right side: breast cancer cells. Scale: 25 μm. ( C ) Evaluation of protrusions in visceral ASCs toward MDA-MB-231 and MCF-7 cells. The results are from three independent experiments and shown as mean ± SEM. ( D ) Representatives are shown. Left side: visceral ASCs; right side: breast cancer cells. Scale: 25 μm.

Article Snippet: The following primary antibodies were used: rat polyclonal antibody against α-tubulin (Biozol, Eching), rabbit polyclonal antibodies against pericentrin, human monoclonal antibody against ACA (anti-centromere antibody) (ImmunoVision, Springdale), mouse monoclonal anti-phospho-histone γ-H2AX (Ser139) (Merck Millipore, Darmstadt) and polyclonal rabbit antibodies against 53BP1 (Novus, Cambridge, UK).

Techniques: Cell Culture, Staining, Marker, Membrane, Migration