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Addgene inc h2b mcherry
Sp1 regulates mitotic progression. (A,B) Live cell imaging of mAID-Sp1; <t>H2B-mCherry</t> cells following the indicated treatments. While images were taken every three minutes, the above image sequence represents images taken every nine minutes to best highlight the differences between the treatments. Time = h:min. Scale bar = 5 μm. (C) Time (m) from nuclear envelope breakdown to G 1 . 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.016, unpaired t -test‥ (D) Time (m) from nuclear envelope breakdown to anaphase. 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.042, unpaired t -test‥ (E) Schematic outlining the experimental strategy for (F). (F) Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 μm. (F) Quantification of (E). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.0037, unpaired t -test. All images are representative.
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Images

1) Product Images from "Transcription factor Sp1 regulates mitotic fidelity through Aurora B kinase-mediated condensin I localization). The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data:"

Article Title: Transcription factor Sp1 regulates mitotic fidelity through Aurora B kinase-mediated condensin I localization). The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data:

Journal: bioRxiv

doi: 10.1101/2020.06.19.158030

Sp1 regulates mitotic progression. (A,B) Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every three minutes, the above image sequence represents images taken every nine minutes to best highlight the differences between the treatments. Time = h:min. Scale bar = 5 μm. (C) Time (m) from nuclear envelope breakdown to G 1 . 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.016, unpaired t -test‥ (D) Time (m) from nuclear envelope breakdown to anaphase. 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.042, unpaired t -test‥ (E) Schematic outlining the experimental strategy for (F). (F) Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 μm. (F) Quantification of (E). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.0037, unpaired t -test. All images are representative.
Figure Legend Snippet: Sp1 regulates mitotic progression. (A,B) Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every three minutes, the above image sequence represents images taken every nine minutes to best highlight the differences between the treatments. Time = h:min. Scale bar = 5 μm. (C) Time (m) from nuclear envelope breakdown to G 1 . 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.016, unpaired t -test‥ (D) Time (m) from nuclear envelope breakdown to anaphase. 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.042, unpaired t -test‥ (E) Schematic outlining the experimental strategy for (F). (F) Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 μm. (F) Quantification of (E). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.0037, unpaired t -test. All images are representative.

Techniques Used: Live Cell Imaging, Sequencing, Staining

2) Product Images from "APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability"

Article Title: APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability

Journal: Cancer discovery

doi: 10.1158/2159-8290.CD-16-0645

APC/C dysfunction results in CIN buffering and adaptation to extreme CIN. A and B, RPE1/H2B-mCherry cells were arrested in mitosis using 40 nmol/L STLC, collected by shake-off, and released into media containing 3 μmol/L proTAME or DMSO (t = 0h). Stacks were acquired every 3 minutes to determine anaphase onset ( A ) and the presence of lagging chromosomes ( B ; error bars, mean ± 95% CI). Sample images from movies are shown, and the arrow indicates a lagging chromosome in a cell without proTAME. Representative images are shown (scale bar, 10 μm). C, Degradation kinetics of cyclin A2-Venus fluorescence quantified from unsynchronized single cells as they progress through mitosis. 1.5 μmol/L proTAME or DMSO was added 2 hours before imaging. Total cell fluorescence was quantified and normalized to the level at NEBD. Curves end at anaphase onset ( n = 24 cells per condition; error bars indicate SD; ***, P
Figure Legend Snippet: APC/C dysfunction results in CIN buffering and adaptation to extreme CIN. A and B, RPE1/H2B-mCherry cells were arrested in mitosis using 40 nmol/L STLC, collected by shake-off, and released into media containing 3 μmol/L proTAME or DMSO (t = 0h). Stacks were acquired every 3 minutes to determine anaphase onset ( A ) and the presence of lagging chromosomes ( B ; error bars, mean ± 95% CI). Sample images from movies are shown, and the arrow indicates a lagging chromosome in a cell without proTAME. Representative images are shown (scale bar, 10 μm). C, Degradation kinetics of cyclin A2-Venus fluorescence quantified from unsynchronized single cells as they progress through mitosis. 1.5 μmol/L proTAME or DMSO was added 2 hours before imaging. Total cell fluorescence was quantified and normalized to the level at NEBD. Curves end at anaphase onset ( n = 24 cells per condition; error bars indicate SD; ***, P

Techniques Used: Fluorescence, Imaging

APC/C subunit mutational status affects CIN in cancer cells. A and B, H2B-mCherry was introduced in H2030, U251, and SW480 cell lines and NEBD–anaphase duration ( A ) as well as the frequency of anaphase segregation errors ( B ) with and without proTAME was determined by time-lapse fluorescence microscopy. Stacks were acquired every 3 minutes, and an example of segregation error is shown for each cell line (scale bar, 10 μm; in A, bars, mean ± 95% CI of a representative experiment; in B, P values from Fisher exact test). C, Experimental procedure used to generate CRISPR/Cas9 edited H2030 and HT29 cells used for plots D to I. In each case, a single clone was infected (lentiCRISPR/CDC27 for H2030) or transfected (espCas9(1.1)/CDC23 + ssDNA donor) in a well of a 96-well plate before the colony reached confluency. Following transfection or transduction, the colony was dispersed by limiting dilution into 96-well plates. Clones were then screened for heterozygous disruption of CDC27 in H2030 cells or correction of the heterozygous E245 nonsense mutation in HT29 cells. Nonedited clones identified during screening were used as controls. Phenotypic analysis of all newly derived cell lines was performed following minimal clonal expansion to limit phenotypic diversity that may be acquired due to ongoing CIN. D, Lollipop plot of CDC27 showing only truncating mutations reported in TCGA and the location of the guide RNA used to disrupt CDC27 in H2030 cells. The clone isolated contained a heterozygous 35-bp deletion creating the truncation I442Sfs*15. E, NEBD–anaphase duration was determined for the H2030 clones using phase–contrast time-lapse microscopy (3 minutes/frame; bars, average ± 95% CI). F, The frequency of anaphase lagging chromosomes was determined on fixed cells by indirect IF microscopy (*, P
Figure Legend Snippet: APC/C subunit mutational status affects CIN in cancer cells. A and B, H2B-mCherry was introduced in H2030, U251, and SW480 cell lines and NEBD–anaphase duration ( A ) as well as the frequency of anaphase segregation errors ( B ) with and without proTAME was determined by time-lapse fluorescence microscopy. Stacks were acquired every 3 minutes, and an example of segregation error is shown for each cell line (scale bar, 10 μm; in A, bars, mean ± 95% CI of a representative experiment; in B, P values from Fisher exact test). C, Experimental procedure used to generate CRISPR/Cas9 edited H2030 and HT29 cells used for plots D to I. In each case, a single clone was infected (lentiCRISPR/CDC27 for H2030) or transfected (espCas9(1.1)/CDC23 + ssDNA donor) in a well of a 96-well plate before the colony reached confluency. Following transfection or transduction, the colony was dispersed by limiting dilution into 96-well plates. Clones were then screened for heterozygous disruption of CDC27 in H2030 cells or correction of the heterozygous E245 nonsense mutation in HT29 cells. Nonedited clones identified during screening were used as controls. Phenotypic analysis of all newly derived cell lines was performed following minimal clonal expansion to limit phenotypic diversity that may be acquired due to ongoing CIN. D, Lollipop plot of CDC27 showing only truncating mutations reported in TCGA and the location of the guide RNA used to disrupt CDC27 in H2030 cells. The clone isolated contained a heterozygous 35-bp deletion creating the truncation I442Sfs*15. E, NEBD–anaphase duration was determined for the H2030 clones using phase–contrast time-lapse microscopy (3 minutes/frame; bars, average ± 95% CI). F, The frequency of anaphase lagging chromosomes was determined on fixed cells by indirect IF microscopy (*, P

Techniques Used: Fluorescence, Microscopy, CRISPR, Infection, Transfection, Transduction, Clone Assay, Mutagenesis, Derivative Assay, Isolation, Time-lapse Microscopy

Identification of APC/C subunits in a genome-wide siRNA screen for CIN survival. A, Chromosome segregation error rates determined by FISH in postmitotic daughter cells. RPE1 cells were treated with reversine for 2 hours, then mitotic cells were collected by shake-off and allowed to reattach in the presence of reversine, before fixation. Graph represents the average rate measured for chromosomes 6, 7, 8, and 10 (bars, average ± 95% CI). B, HCT116 wild-type and p53 −/− isogenic lines were imaged for 72 hours by live-cell imaging. Cell density for each drug concentration was normalized to DMSO for wild-type and p53 −/− cell separately. Fold difference in cell density of HCT116 p53 −/− relative to wild-type is displayed for each drug concentration. C, Clonogenic assay using isogenic HCT116 cells grown for 10 days in the presence or absence or reversine, as indicated. D, Genome-wide RNAi screen for synthetic viability with MPS1 inhibition. RPE1 cells were synchronized in G 0 –G 1 by contact inhibition, trypsinized, and reverse-transfected at low density in triplicate to allow uniform passage through mitosis. Cells were exposed to 250 nmol/L reversine for 96 hours and fixed. Automated image acquisition and analysis were performed for various parameters, and Z-scores were derived based on median plate normalization for each siRNA pool. E, DAPI staining of fixed cells 48 hours following the indicated siRNA treatments with 250 nmol/L reversine where indicated. F, Chromosome segregation error rates measured by FISH as in A. Cells were transfected with a nontargeting or a CDC16 siRNA pool, and 48 hours later, cells were synchronized with a single thymidine block. Reversine was added 10 hours after thymidine release, prior to mitotic entry. Mitotic cells were collected at 12 hours by shake-off, allowed to reattach on coverslips still in the presence of reversine, and fixed (bars, average ± 95% CI). G, Time-lapse fluorescence microscopy of RPE1 cells expressing H2B-mCherry imaged every 3 minutes, 1 hour following the addition of 350 nmol/L reversine +/− proTAME (pT), as indicated. The duration from NEBD to metaphase and metaphase to anaphase is shown for cells in which all chromosomes congressed to form a metaphase plate. Each row corresponds to a single cell ( n > 60 cells each; bars, mean ± 95% CI). H, Sample images of segregation errors scored in G. Maximum intensity projections are shown for timeframes immediately preceding and following anaphase onset. Shown are examples of a correct division (no error), an example of a lagging chromosome following proper congression at metaphase (middle), and an example where anaphase occurred before all chromosomes congressed to the metaphase plate (congression defect, hence NEBD–metaphase could not be determined). Scale bar, 10 μm.
Figure Legend Snippet: Identification of APC/C subunits in a genome-wide siRNA screen for CIN survival. A, Chromosome segregation error rates determined by FISH in postmitotic daughter cells. RPE1 cells were treated with reversine for 2 hours, then mitotic cells were collected by shake-off and allowed to reattach in the presence of reversine, before fixation. Graph represents the average rate measured for chromosomes 6, 7, 8, and 10 (bars, average ± 95% CI). B, HCT116 wild-type and p53 −/− isogenic lines were imaged for 72 hours by live-cell imaging. Cell density for each drug concentration was normalized to DMSO for wild-type and p53 −/− cell separately. Fold difference in cell density of HCT116 p53 −/− relative to wild-type is displayed for each drug concentration. C, Clonogenic assay using isogenic HCT116 cells grown for 10 days in the presence or absence or reversine, as indicated. D, Genome-wide RNAi screen for synthetic viability with MPS1 inhibition. RPE1 cells were synchronized in G 0 –G 1 by contact inhibition, trypsinized, and reverse-transfected at low density in triplicate to allow uniform passage through mitosis. Cells were exposed to 250 nmol/L reversine for 96 hours and fixed. Automated image acquisition and analysis were performed for various parameters, and Z-scores were derived based on median plate normalization for each siRNA pool. E, DAPI staining of fixed cells 48 hours following the indicated siRNA treatments with 250 nmol/L reversine where indicated. F, Chromosome segregation error rates measured by FISH as in A. Cells were transfected with a nontargeting or a CDC16 siRNA pool, and 48 hours later, cells were synchronized with a single thymidine block. Reversine was added 10 hours after thymidine release, prior to mitotic entry. Mitotic cells were collected at 12 hours by shake-off, allowed to reattach on coverslips still in the presence of reversine, and fixed (bars, average ± 95% CI). G, Time-lapse fluorescence microscopy of RPE1 cells expressing H2B-mCherry imaged every 3 minutes, 1 hour following the addition of 350 nmol/L reversine +/− proTAME (pT), as indicated. The duration from NEBD to metaphase and metaphase to anaphase is shown for cells in which all chromosomes congressed to form a metaphase plate. Each row corresponds to a single cell ( n > 60 cells each; bars, mean ± 95% CI). H, Sample images of segregation errors scored in G. Maximum intensity projections are shown for timeframes immediately preceding and following anaphase onset. Shown are examples of a correct division (no error), an example of a lagging chromosome following proper congression at metaphase (middle), and an example where anaphase occurred before all chromosomes congressed to the metaphase plate (congression defect, hence NEBD–metaphase could not be determined). Scale bar, 10 μm.

Techniques Used: Genome Wide, Fluorescence In Situ Hybridization, Live Cell Imaging, Concentration Assay, Clonogenic Assay, Inhibition, Transfection, Derivative Assay, Staining, Blocking Assay, Fluorescence, Microscopy, Expressing

3) Product Images from "GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence"

Article Title: GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence

Journal: bioRxiv

doi: 10.1101/2021.08.19.456978

Laminin staining can be used to distinguish tumour core from invading cells. Overexposure of laminin reveals background staining that colocalizes with the highest density of H2B-mCherry nuclei at the site of injection. This laminin deposit is used to distinguish cells in the tumour core versus cells that invaded the tissue, indicated with the orange dotted line. Scale bar = 100 μm.
Figure Legend Snippet: Laminin staining can be used to distinguish tumour core from invading cells. Overexposure of laminin reveals background staining that colocalizes with the highest density of H2B-mCherry nuclei at the site of injection. This laminin deposit is used to distinguish cells in the tumour core versus cells that invaded the tissue, indicated with the orange dotted line. Scale bar = 100 μm.

Techniques Used: Staining, Injection

Representative images of organotypic slice cultures injected with the different clonal lines. Representative images of injected I-CTL-H2B-mCherry cells together with H2B-mNeonGreen expressing CTL clones (a), GFAPδ-KO clones (b) or GFAPα-KO clones (c). Scale bar = 100 μm. NG= mNeonGreen, Lam = laminin.
Figure Legend Snippet: Representative images of organotypic slice cultures injected with the different clonal lines. Representative images of injected I-CTL-H2B-mCherry cells together with H2B-mNeonGreen expressing CTL clones (a), GFAPδ-KO clones (b) or GFAPα-KO clones (c). Scale bar = 100 μm. NG= mNeonGreen, Lam = laminin.

Techniques Used: Injection, Expressing, Laser Capture Microdissection

Modification of GFAP isoform expression affects macroscopic growth patterns in organotypic brain slice cultures. (a) Schematic of experimental set-up: H2B-NeonGr expressing control (CTL), GFAPδ-KO and GFAPα-KO clonal lines are injected in organotypic slices together with an H2B-mCherry expressing internal control (I-CTL) and co-cultured for one week. After fixation, whole-mount immunofluorescent staining, clearing and confocal imaging are used to create a 3D reconstruction of the invasion patterns. (b) Representative image of nuclei of an I-CTL (magenta) and a CTL (green) lines within the organotypic slice model. Invading cells are mainly found around the mouse brain vasculature (laminin, cyan). Laminin deposits in the tumour core can be used to distinguish stationary cells from cells invading the tissue, indicated with the orange dotted line. (c) Distribution of nuclei of GFAPδ-KO and I-CTL cell lines in the organotypic slices (n=18 independent experiments, 4 different clones). (d) Representative images of invasion pattern of GFAPδ-KO line 1 and I-CTL 1. (e) Distribution of nuclei of GFAPα-KO cells and I-CTL cells in the organotypic brain slices (n= 20 independent experiments, 4 different clones). (h) Representative image of the invasion pattern of GFAPα-KO line 2 and I-CTL 1. (g) Distribution of I-CTL and CTL nuclei in the organotypic brain slices (n=16 independent experiments, 4 different clones). (h) Schematic depicting the method used to quantify the distribution of nuclei in the organotypic slices. (i) Quantification of the percentage of invaded cells per condition, n= 16 (CTLs), n=18 (GFAPδ-KO), and n= 20 (GFAPα-KO) injected organotypic brain slices derived from 4 different clones per condition. Significance was determined using a two-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 100 µm. The data is shown as mean ± S.E.M, *p
Figure Legend Snippet: Modification of GFAP isoform expression affects macroscopic growth patterns in organotypic brain slice cultures. (a) Schematic of experimental set-up: H2B-NeonGr expressing control (CTL), GFAPδ-KO and GFAPα-KO clonal lines are injected in organotypic slices together with an H2B-mCherry expressing internal control (I-CTL) and co-cultured for one week. After fixation, whole-mount immunofluorescent staining, clearing and confocal imaging are used to create a 3D reconstruction of the invasion patterns. (b) Representative image of nuclei of an I-CTL (magenta) and a CTL (green) lines within the organotypic slice model. Invading cells are mainly found around the mouse brain vasculature (laminin, cyan). Laminin deposits in the tumour core can be used to distinguish stationary cells from cells invading the tissue, indicated with the orange dotted line. (c) Distribution of nuclei of GFAPδ-KO and I-CTL cell lines in the organotypic slices (n=18 independent experiments, 4 different clones). (d) Representative images of invasion pattern of GFAPδ-KO line 1 and I-CTL 1. (e) Distribution of nuclei of GFAPα-KO cells and I-CTL cells in the organotypic brain slices (n= 20 independent experiments, 4 different clones). (h) Representative image of the invasion pattern of GFAPα-KO line 2 and I-CTL 1. (g) Distribution of I-CTL and CTL nuclei in the organotypic brain slices (n=16 independent experiments, 4 different clones). (h) Schematic depicting the method used to quantify the distribution of nuclei in the organotypic slices. (i) Quantification of the percentage of invaded cells per condition, n= 16 (CTLs), n=18 (GFAPδ-KO), and n= 20 (GFAPα-KO) injected organotypic brain slices derived from 4 different clones per condition. Significance was determined using a two-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 100 µm. The data is shown as mean ± S.E.M, *p

Techniques Used: Modification, Expressing, Slice Preparation, Injection, Cell Culture, Staining, Imaging, Derivative Assay, Clone Assay

In vivo tumour growth dynamics in GFAP modulated tumours. (a) Schematic overview of the experimental setup. U-251-MG GFAP-modulated lines expressing H2B-NeonGreen were implanted in the brain of NSG mice under a CIW. Time-lapse intravital imaging was performed through a CIW to study the tumour growth dynamics of each tumour type. (b) Representative 3D reconstructed tile-scans showing distinct tumours generated by different GFAP-modulated lines. Two clones engineered with different CRISPR-Cas9 guides are presented. Scale bar = 500µm (c) Quantification of tumour density for each indicated tumour type. n=6 (CTLs), n=5 (GFAPδ-KO), and n= 6 (GFAPα-KO) mice. Black dots represent sgRNA1 and white dots represent sgRNA2.The data is shown as mean ± S.E.M, *p
Figure Legend Snippet: In vivo tumour growth dynamics in GFAP modulated tumours. (a) Schematic overview of the experimental setup. U-251-MG GFAP-modulated lines expressing H2B-NeonGreen were implanted in the brain of NSG mice under a CIW. Time-lapse intravital imaging was performed through a CIW to study the tumour growth dynamics of each tumour type. (b) Representative 3D reconstructed tile-scans showing distinct tumours generated by different GFAP-modulated lines. Two clones engineered with different CRISPR-Cas9 guides are presented. Scale bar = 500µm (c) Quantification of tumour density for each indicated tumour type. n=6 (CTLs), n=5 (GFAPδ-KO), and n= 6 (GFAPα-KO) mice. Black dots represent sgRNA1 and white dots represent sgRNA2.The data is shown as mean ± S.E.M, *p

Techniques Used: In Vivo, Expressing, Mouse Assay, Imaging, Generated, Clone Assay, CRISPR

4) Product Images from "Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo"

Article Title: Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo

Journal: Scientific Reports

doi: 10.1038/s41598-019-40959-y

TNF-α stimulated nuclear translocation of NF-κB in HUVECs. HUVECs transfected with GFP-RelA and H2B-mCherry were stimulated with 10 ng/mL TNF-α (a – c ). A time series of GFP-RelA shows strong nuclear concentration at 30 minutes and empty nuclei at 0 and 360 minutes (a) , the population mean of approximate 600 single cell measurements (b) , projected view of the entire cell population (c) . Immunohistochemistry of non-transfected HUVECs treated with 10 ng/mL TNF-α fixed and stained (p65-AF488) at different time points point normalized to unstimulated cells (*p
Figure Legend Snippet: TNF-α stimulated nuclear translocation of NF-κB in HUVECs. HUVECs transfected with GFP-RelA and H2B-mCherry were stimulated with 10 ng/mL TNF-α (a – c ). A time series of GFP-RelA shows strong nuclear concentration at 30 minutes and empty nuclei at 0 and 360 minutes (a) , the population mean of approximate 600 single cell measurements (b) , projected view of the entire cell population (c) . Immunohistochemistry of non-transfected HUVECs treated with 10 ng/mL TNF-α fixed and stained (p65-AF488) at different time points point normalized to unstimulated cells (*p

Techniques Used: Translocation Assay, Transfection, Concentration Assay, Immunohistochemistry, Staining

Schematic diagram of the experimental pipeline: Step 1: Perfusion system including flow chamber, two windkessels, medium reservoir and a peristaltic pump. Cells within the flow chamber were recorded with the fluorescence microscope using LED lamps. Step 2: The raw image of H2B-mCherry and GFP-RelA were processed by enhancing the contrast, correcting non-uniform illumination and removing noise with a median filter. Step 3: H2B-mCherry image was made binary and the nuclei were numbered. Step 4: The numbered nuclei were tracked throughout all time frames. Step 5: The coordinates from the tracked nuclei were used to calculate the nuclear GFP-RelA intensity in the corrected GFP-RelA image. Step 6: nuclear GFP-RelA intensity in each cell was normalized by the time average GFP-RelA intensity. The population mean of the normalized nuclear GFP-RelA intensity including standard deviation was plotted as the result.
Figure Legend Snippet: Schematic diagram of the experimental pipeline: Step 1: Perfusion system including flow chamber, two windkessels, medium reservoir and a peristaltic pump. Cells within the flow chamber were recorded with the fluorescence microscope using LED lamps. Step 2: The raw image of H2B-mCherry and GFP-RelA were processed by enhancing the contrast, correcting non-uniform illumination and removing noise with a median filter. Step 3: H2B-mCherry image was made binary and the nuclei were numbered. Step 4: The numbered nuclei were tracked throughout all time frames. Step 5: The coordinates from the tracked nuclei were used to calculate the nuclear GFP-RelA intensity in the corrected GFP-RelA image. Step 6: nuclear GFP-RelA intensity in each cell was normalized by the time average GFP-RelA intensity. The population mean of the normalized nuclear GFP-RelA intensity including standard deviation was plotted as the result.

Techniques Used: Fluorescence, Microscopy, Standard Deviation

5) Product Images from "Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo"

Article Title: Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo

Journal: Scientific Reports

doi: 10.1038/s41598-019-40959-y

TNF-α stimulated nuclear translocation of NF-κB in HUVECs. HUVECs transfected with GFP-RelA and H2B-mCherry were stimulated with 10 ng/mL TNF-α (a – c ). A time series of GFP-RelA shows strong nuclear concentration at 30 minutes and empty nuclei at 0 and 360 minutes (a) , the population mean of approximate 600 single cell measurements (b) , projected view of the entire cell population (c) . Immunohistochemistry of non-transfected HUVECs treated with 10 ng/mL TNF-α fixed and stained (p65-AF488) at different time points point normalized to unstimulated cells (*p
Figure Legend Snippet: TNF-α stimulated nuclear translocation of NF-κB in HUVECs. HUVECs transfected with GFP-RelA and H2B-mCherry were stimulated with 10 ng/mL TNF-α (a – c ). A time series of GFP-RelA shows strong nuclear concentration at 30 minutes and empty nuclei at 0 and 360 minutes (a) , the population mean of approximate 600 single cell measurements (b) , projected view of the entire cell population (c) . Immunohistochemistry of non-transfected HUVECs treated with 10 ng/mL TNF-α fixed and stained (p65-AF488) at different time points point normalized to unstimulated cells (*p

Techniques Used: Translocation Assay, Transfection, Concentration Assay, Immunohistochemistry, Staining

Schematic diagram of the experimental pipeline: Step 1: Perfusion system including flow chamber, two windkessels, medium reservoir and a peristaltic pump. Cells within the flow chamber were recorded with the fluorescence microscope using LED lamps. Step 2: The raw image of H2B-mCherry and GFP-RelA were processed by enhancing the contrast, correcting non-uniform illumination and removing noise with a median filter. Step 3: H2B-mCherry image was made binary and the nuclei were numbered. Step 4: The numbered nuclei were tracked throughout all time frames. Step 5: The coordinates from the tracked nuclei were used to calculate the nuclear GFP-RelA intensity in the corrected GFP-RelA image. Step 6: nuclear GFP-RelA intensity in each cell was normalized by the time average GFP-RelA intensity. The population mean of the normalized nuclear GFP-RelA intensity including standard deviation was plotted as the result.
Figure Legend Snippet: Schematic diagram of the experimental pipeline: Step 1: Perfusion system including flow chamber, two windkessels, medium reservoir and a peristaltic pump. Cells within the flow chamber were recorded with the fluorescence microscope using LED lamps. Step 2: The raw image of H2B-mCherry and GFP-RelA were processed by enhancing the contrast, correcting non-uniform illumination and removing noise with a median filter. Step 3: H2B-mCherry image was made binary and the nuclei were numbered. Step 4: The numbered nuclei were tracked throughout all time frames. Step 5: The coordinates from the tracked nuclei were used to calculate the nuclear GFP-RelA intensity in the corrected GFP-RelA image. Step 6: nuclear GFP-RelA intensity in each cell was normalized by the time average GFP-RelA intensity. The population mean of the normalized nuclear GFP-RelA intensity including standard deviation was plotted as the result.

Techniques Used: Flow Cytometry, Fluorescence, Microscopy, Standard Deviation

6) Product Images from "RhoA- and Ran-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes"

Article Title: RhoA- and Ran-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes

Journal: bioRxiv

doi: 10.1101/2020.10.20.348045

Cortical actomyosin polarization reorganizes during anaphase II (live experiments). (A) Live fluorescence imaging of an activated metaphase II oocyte undergoing spindle rotation. The oocyte has been injected with a combination of the H2b-mCherry DNA marker and the eGFP-UtrCH F-actin marker (S8 Movie). The images represent maximum z-projections of multiple confocal planes and the symbols highlight remarkable events occurring during the second meiotic division. (B) 2D maps of the polarized domain showing variation over time of the cortex distance to DNA clusters (color-coded top panel) and the cortical F-actin levels (bottom panel). The maps are extracted from the images shown on the right. The isodistance lines are used as landmarks to delimit region of the cortex equidistant from the DNA clusters. The dashed lines separate the best focus plane for each DNA cluster. (C) 1D representation of the data shown in panel B (see Methods). The graphs show variation over time of the DNA clusters distance to the cortex (top panel, d In and d Out ) and the cortical F-actin levels in each polarized domain (see bottom panel, F In and F Out ). The variation of spindle rotation angle α is also represented on both graphs. The diagrams on the left illustrate the measurements described above. All scale bars represent a length of 10 μm.
Figure Legend Snippet: Cortical actomyosin polarization reorganizes during anaphase II (live experiments). (A) Live fluorescence imaging of an activated metaphase II oocyte undergoing spindle rotation. The oocyte has been injected with a combination of the H2b-mCherry DNA marker and the eGFP-UtrCH F-actin marker (S8 Movie). The images represent maximum z-projections of multiple confocal planes and the symbols highlight remarkable events occurring during the second meiotic division. (B) 2D maps of the polarized domain showing variation over time of the cortex distance to DNA clusters (color-coded top panel) and the cortical F-actin levels (bottom panel). The maps are extracted from the images shown on the right. The isodistance lines are used as landmarks to delimit region of the cortex equidistant from the DNA clusters. The dashed lines separate the best focus plane for each DNA cluster. (C) 1D representation of the data shown in panel B (see Methods). The graphs show variation over time of the DNA clusters distance to the cortex (top panel, d In and d Out ) and the cortical F-actin levels in each polarized domain (see bottom panel, F In and F Out ). The variation of spindle rotation angle α is also represented on both graphs. The diagrams on the left illustrate the measurements described above. All scale bars represent a length of 10 μm.

Techniques Used: Fluorescence, Imaging, Injection, Marker

The polarized cortex exerts attraction forces on chromatid clusters. (A) Simplified diagram of the RanGTP gradient, emanating from the chromosomes, and leading to the cortical accumulation of F-actin through the activation of the Cdc42, N-WASP and Arp2/3 pathway. The proximity of the chromosomes promotes cortical F-actin polarity, which in turn attracts the chromosomes to the cortex. (B) Fixed F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) imaging of metaphase II arrested oocytes expressing dominant negative forms of the Ran and Cdc42 GTPase (respectively RanT24N and Cdc42T17N). Control oocytes have been injected with water. (C) Line graphs: averaged ± s.d. cortical F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) fluorescence intensities as a function of the cortex distance to DNA clusters. The fluorescence intensities are normalized to the mean of each profile. Quantifications have been performed on 10 controls, 11 RanT24N and 15 Cdc42T17N injected metaphase II oocytes, gathered from 2 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes injected with the H2b-mCherry DNA marker alone (control) or a combination of H2b-mCherry and a Cdc42 dominant negative form (Cdc42T17N, group #1 and #2) (S9 Movie). The Cdc42T17N injected oocytes present two characteristic phenotypes, with a first group of gametes extruding a small PBII (as compared to controls) and a second group whose spindle relocates towards the center of the cell. (E) The graph shows variation over time of the distance between the oocyte centroid and the DNA clusters mid-point. The grey profiles represent controls (H2b-mCherry alone) while the light and dark blue profiles represent Cdc42T17N injected oocytes (group #1 and #2, respectively). The red line shows a linear fit, from 0 to 60 min, of the Cdc42T17N group #2. (F) Distribution of the distance between the oocyte centroid and the DNA clusters mid-point after 120 min of recording. Quantifications have been performed on 34 control and 23 Cdc42T17N injected oocytes, gathered respectively from 9 and 3 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Cdc42T17N injected oocyte (S10 Movie). The presented images are extracted from timepoint t = 15 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.3 μm.min −1 vector. All scale bars represent a length of 10 μm.
Figure Legend Snippet: The polarized cortex exerts attraction forces on chromatid clusters. (A) Simplified diagram of the RanGTP gradient, emanating from the chromosomes, and leading to the cortical accumulation of F-actin through the activation of the Cdc42, N-WASP and Arp2/3 pathway. The proximity of the chromosomes promotes cortical F-actin polarity, which in turn attracts the chromosomes to the cortex. (B) Fixed F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) imaging of metaphase II arrested oocytes expressing dominant negative forms of the Ran and Cdc42 GTPase (respectively RanT24N and Cdc42T17N). Control oocytes have been injected with water. (C) Line graphs: averaged ± s.d. cortical F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) fluorescence intensities as a function of the cortex distance to DNA clusters. The fluorescence intensities are normalized to the mean of each profile. Quantifications have been performed on 10 controls, 11 RanT24N and 15 Cdc42T17N injected metaphase II oocytes, gathered from 2 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes injected with the H2b-mCherry DNA marker alone (control) or a combination of H2b-mCherry and a Cdc42 dominant negative form (Cdc42T17N, group #1 and #2) (S9 Movie). The Cdc42T17N injected oocytes present two characteristic phenotypes, with a first group of gametes extruding a small PBII (as compared to controls) and a second group whose spindle relocates towards the center of the cell. (E) The graph shows variation over time of the distance between the oocyte centroid and the DNA clusters mid-point. The grey profiles represent controls (H2b-mCherry alone) while the light and dark blue profiles represent Cdc42T17N injected oocytes (group #1 and #2, respectively). The red line shows a linear fit, from 0 to 60 min, of the Cdc42T17N group #2. (F) Distribution of the distance between the oocyte centroid and the DNA clusters mid-point after 120 min of recording. Quantifications have been performed on 34 control and 23 Cdc42T17N injected oocytes, gathered respectively from 9 and 3 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Cdc42T17N injected oocyte (S10 Movie). The presented images are extracted from timepoint t = 15 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.3 μm.min −1 vector. All scale bars represent a length of 10 μm.

Techniques Used: Activation Assay, Staining, Immunostaining, Imaging, Expressing, Dominant Negative Mutation, Injection, Fluorescence, Marker, Plasmid Preparation

The central spindle/RhoA pathway is required for spindle rotation. (A) Simplified diagram of the central spindle pathway leading to the activation of the RhoA GTPase and the assembly of the cytokinesis ring. (B) Fixed RhoA (immuno-staining) imaging of differentially staged oocytes undergoing spindle rotation. The top panel represents single confocal planes while the bottom panels represent maximum z-projections of multiple confocal planes. The inserted diagrams in the top panel illustrate the progressive closure of the cytokinesis ring (C) Inhibiting the central spindle pathway, using the Bi-2536 (PLK1 inhibitor), prevents the cortical recruitment of RhoA (see white arrow) and the emission of the PBII. The left images show the RhoA localization in fixed oocytes treated with or without the Bi-2536. The right histograms show the PBII emission rate from 81 control and 105 Bi-2536 treated oocytes, both gathered from 4 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes treated with or without the Bi-2536 (S6 Movie). The oocytes have been injected with the H2b-mCherry DNA marker. (E) Averaged variation over time ± s.d. of the spindle rotation angle α in oocytes treated with or without Bi-2536. (F) Box plots: distribution of early (0-20 min) and late (100-120 min) spindle rotation angle α in 70 control and 35 Bi-2536 treated oocytes, gathered respectively from 9 and 6 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Bi-2536 treated oocyte (S7 Movie). The presented images are extracted from timepoint t = 48 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.25 μm.min −1 vector. Box plots in F extend from the first (Q1) to the third (Q3) quartile (where Q3–Q1 is the interquartile range (IQR)); whiskers are Q1 or Q3 ± 1.5 × IQR; horizontal lines represent the median; and black squares represent the mean. Statistics in F were obtained using a two-sided Mann–Whitney test. The exact p values are shown directly above the graphs. All scale bars represent a length of 10 μm.
Figure Legend Snippet: The central spindle/RhoA pathway is required for spindle rotation. (A) Simplified diagram of the central spindle pathway leading to the activation of the RhoA GTPase and the assembly of the cytokinesis ring. (B) Fixed RhoA (immuno-staining) imaging of differentially staged oocytes undergoing spindle rotation. The top panel represents single confocal planes while the bottom panels represent maximum z-projections of multiple confocal planes. The inserted diagrams in the top panel illustrate the progressive closure of the cytokinesis ring (C) Inhibiting the central spindle pathway, using the Bi-2536 (PLK1 inhibitor), prevents the cortical recruitment of RhoA (see white arrow) and the emission of the PBII. The left images show the RhoA localization in fixed oocytes treated with or without the Bi-2536. The right histograms show the PBII emission rate from 81 control and 105 Bi-2536 treated oocytes, both gathered from 4 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes treated with or without the Bi-2536 (S6 Movie). The oocytes have been injected with the H2b-mCherry DNA marker. (E) Averaged variation over time ± s.d. of the spindle rotation angle α in oocytes treated with or without Bi-2536. (F) Box plots: distribution of early (0-20 min) and late (100-120 min) spindle rotation angle α in 70 control and 35 Bi-2536 treated oocytes, gathered respectively from 9 and 6 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Bi-2536 treated oocyte (S7 Movie). The presented images are extracted from timepoint t = 48 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.25 μm.min −1 vector. Box plots in F extend from the first (Q1) to the third (Q3) quartile (where Q3–Q1 is the interquartile range (IQR)); whiskers are Q1 or Q3 ± 1.5 × IQR; horizontal lines represent the median; and black squares represent the mean. Statistics in F were obtained using a two-sided Mann–Whitney test. The exact p values are shown directly above the graphs. All scale bars represent a length of 10 μm.

Techniques Used: Activation Assay, Immunostaining, Imaging, Fluorescence, Injection, Marker, Plasmid Preparation, MANN-WHITNEY

Stochastic symmetry breaking underlies spindle rotation. (A) Live fluorescence and DIC imaging of activated metaphase II oocytes undergoing spindle rotation. The oocytes have been injected either with the H2b-mCherry DNA marker alone (main DIC time series, S1 Movie) or with a combination of H2b-mCherry and the eGFP-MAP4 microtubule marker (inserted spindle time series, S2 Movie). The white arrows show membrane invaginations leading to PBII closure, except in inserted spindle image 20 min where it indicates the central spindle position. (B) High temporal resolution montage of the main DIC time series presented in panel A. The symbols highlight remarkable events occurring during the second meiotic division. (C) Automated 3D segmentation and tracking procedure to monitor the position of the DNA clusters within the shell of the oocyte (see Methods and S4 Movie). (D) Diagram showing the method used to quantify DNA clusters distance d and the spindle rotation angle α . (E) Variation over time of DNA clusters distance d in a selected oocyte (main graph). The red line shows the linear fit used to register in time oocyte population (inserted graphs). (F) Variation over time of spindle rotation angle α in a selected oocyte. The red curve shows the logistic fit used to extract rotation parameters. The initial time of rotation (t i rotation) is defined as the time when the rotation reaches 5% of the total fitted rotation. (G) Two examples of H2b-mCherry injected oocytes illustrating the stochastic triggering of spindle rotation (see S5 Movie). The white arrows indicate the approximate start of the rotation. (H) Distribution of the t i rotation for a control population of 70 oocytes gathered from 9 independent experiments (main graph). Probability density function (pdf) of the t i rotation distribution used to determine three equiprobable categories of increasing t i rotation (inserted graph). (I) Averaged variation over time ± s.d. of the DNA clusters distance d (top graph) and spindle rotation angle α (bottom graph) averaged per category as defined in panel H. All scale bars represent a length of 10 μm.
Figure Legend Snippet: Stochastic symmetry breaking underlies spindle rotation. (A) Live fluorescence and DIC imaging of activated metaphase II oocytes undergoing spindle rotation. The oocytes have been injected either with the H2b-mCherry DNA marker alone (main DIC time series, S1 Movie) or with a combination of H2b-mCherry and the eGFP-MAP4 microtubule marker (inserted spindle time series, S2 Movie). The white arrows show membrane invaginations leading to PBII closure, except in inserted spindle image 20 min where it indicates the central spindle position. (B) High temporal resolution montage of the main DIC time series presented in panel A. The symbols highlight remarkable events occurring during the second meiotic division. (C) Automated 3D segmentation and tracking procedure to monitor the position of the DNA clusters within the shell of the oocyte (see Methods and S4 Movie). (D) Diagram showing the method used to quantify DNA clusters distance d and the spindle rotation angle α . (E) Variation over time of DNA clusters distance d in a selected oocyte (main graph). The red line shows the linear fit used to register in time oocyte population (inserted graphs). (F) Variation over time of spindle rotation angle α in a selected oocyte. The red curve shows the logistic fit used to extract rotation parameters. The initial time of rotation (t i rotation) is defined as the time when the rotation reaches 5% of the total fitted rotation. (G) Two examples of H2b-mCherry injected oocytes illustrating the stochastic triggering of spindle rotation (see S5 Movie). The white arrows indicate the approximate start of the rotation. (H) Distribution of the t i rotation for a control population of 70 oocytes gathered from 9 independent experiments (main graph). Probability density function (pdf) of the t i rotation distribution used to determine three equiprobable categories of increasing t i rotation (inserted graph). (I) Averaged variation over time ± s.d. of the DNA clusters distance d (top graph) and spindle rotation angle α (bottom graph) averaged per category as defined in panel H. All scale bars represent a length of 10 μm.

Techniques Used: Fluorescence, Imaging, Injection, Marker

7) Product Images from "An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis"

Article Title: An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis

Journal: Molecular Cell

doi: 10.1016/j.molcel.2019.10.016

CDK1 Phosphorylates Autophagy Regulators at Known Repressive mTORC1-Directed Sites (A) CCNB1-CDK1 kinase assays were performed using GST-tagged protein fragments as substrates and [γ- 32 P] ATP with or without 300 nM RO-3306 or 500 nM NU6102 ( ∗ heavy-chain antibody from immunoprecipitation). (B) Active CCNB1-CDK1 was treated with 300 nM RO-3306 where indicated, and “cold” CDK1 kinase assays were performed using GST-TFEB (76–160) as substrate and probed with the indicated antibodies. (C) HeLa WT-TFEB-GFP cells were treated with 50 nM paclitaxel (16 h) and/or 1 μM AZD8055 (2 h). Input lysates and anti-GFP immunoprecipitates are shown; note that detection of specific p-S122 TFEB signal required immunoprecipitation of the protein ( Vega-Rubin-de-Celis et al., 2017 ). (D) Montage from Video S4 . Asynchronous HeLa TFEB-GFP H2B-mCherry were treated with 1 μM AZD8055 for 1 h before transfer to a live-cell imaging incubator. (E) HAP1 cells were treated with 50 nM paclitaxel (16 h) and/or 1μM AZD8055 (2 h). (F) Quantification from fluorescent Li-Cor western blotting (E) is provided. p values were calculated using a one-way ANOVA (Tukey). ∗ p
Figure Legend Snippet: CDK1 Phosphorylates Autophagy Regulators at Known Repressive mTORC1-Directed Sites (A) CCNB1-CDK1 kinase assays were performed using GST-tagged protein fragments as substrates and [γ- 32 P] ATP with or without 300 nM RO-3306 or 500 nM NU6102 ( ∗ heavy-chain antibody from immunoprecipitation). (B) Active CCNB1-CDK1 was treated with 300 nM RO-3306 where indicated, and “cold” CDK1 kinase assays were performed using GST-TFEB (76–160) as substrate and probed with the indicated antibodies. (C) HeLa WT-TFEB-GFP cells were treated with 50 nM paclitaxel (16 h) and/or 1 μM AZD8055 (2 h). Input lysates and anti-GFP immunoprecipitates are shown; note that detection of specific p-S122 TFEB signal required immunoprecipitation of the protein ( Vega-Rubin-de-Celis et al., 2017 ). (D) Montage from Video S4 . Asynchronous HeLa TFEB-GFP H2B-mCherry were treated with 1 μM AZD8055 for 1 h before transfer to a live-cell imaging incubator. (E) HAP1 cells were treated with 50 nM paclitaxel (16 h) and/or 1μM AZD8055 (2 h). (F) Quantification from fluorescent Li-Cor western blotting (E) is provided. p values were calculated using a one-way ANOVA (Tukey). ∗ p

Techniques Used: Immunoprecipitation, Live Cell Imaging, Western Blot

8) Product Images from "Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition"

Article Title: Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition

Journal: bioRxiv

doi: 10.1101/2022.09.03.506460

AQP1 is localized in the plasma and vacuole membranes of endothelial cells in the ventral side of the dorsal aorta. (A–C) Optical cross-sections of a tg(pLSiΔAeGFP) embryo at E3.5. (B) Enlarged view of the rectangular areas in (A). (C) Enlarged view of rectangular areas in (B). Endothelial cells in the dorsal aortic floor show eGFP-negative areas (asterisks). AQP1 is localized in the plasma membrane (PM) and boundaries of the eGFP-positive and -negative areas (arrowheads). (D) Immunoelectron microscopy of wild-type (WT) quail embryos at E3.5. Endothelial cells in the dorsal aortic floor contain large vacuoles (asterisks). An enlarged view of the square area is shown in the right panel. AQP1 (6-nm gold particles) was localized in the plasma and vacuole membrane (VM). (E) Optical cross-sections of the dorsal aortic floor in a WT quail embryo at E3.5. AQP1 is localized in the plasma and vacuole membranes in Runx1-positive (arrows) and –negative (arrowheads) cells. (F) Expression vectors for eGFP and Lyn-mCherry were co-electroporated into progenitor cells on the primitive streak at E0.75 (HH4). Optical cross-sections of the dorsal aortic floor (upper two panels) in electroporated quail embryos at E3.5. In a hemispherical cell, an eGFP-negative cavity was observed on the basal side (arrows). In another hemispherical cell, eGFP-negative vacuoles (asterisks) delineated by a Lyn-mCherry-positive membrane were completely separated from the PM. A small vacuole was observed in the rounded cell (arrowheads). Lower: Optically reconstructed horizontal sections. Dashed lines indicate optical slice positions. Scale bars: 50 μm in (A), 20 μm in (B), 5 μm in (C, E), 2 μm in (D, left), 100 nm in (D, right), 5 μm in (E), and 10 μm in (F).
Figure Legend Snippet: AQP1 is localized in the plasma and vacuole membranes of endothelial cells in the ventral side of the dorsal aorta. (A–C) Optical cross-sections of a tg(pLSiΔAeGFP) embryo at E3.5. (B) Enlarged view of the rectangular areas in (A). (C) Enlarged view of rectangular areas in (B). Endothelial cells in the dorsal aortic floor show eGFP-negative areas (asterisks). AQP1 is localized in the plasma membrane (PM) and boundaries of the eGFP-positive and -negative areas (arrowheads). (D) Immunoelectron microscopy of wild-type (WT) quail embryos at E3.5. Endothelial cells in the dorsal aortic floor contain large vacuoles (asterisks). An enlarged view of the square area is shown in the right panel. AQP1 (6-nm gold particles) was localized in the plasma and vacuole membrane (VM). (E) Optical cross-sections of the dorsal aortic floor in a WT quail embryo at E3.5. AQP1 is localized in the plasma and vacuole membranes in Runx1-positive (arrows) and –negative (arrowheads) cells. (F) Expression vectors for eGFP and Lyn-mCherry were co-electroporated into progenitor cells on the primitive streak at E0.75 (HH4). Optical cross-sections of the dorsal aortic floor (upper two panels) in electroporated quail embryos at E3.5. In a hemispherical cell, an eGFP-negative cavity was observed on the basal side (arrows). In another hemispherical cell, eGFP-negative vacuoles (asterisks) delineated by a Lyn-mCherry-positive membrane were completely separated from the PM. A small vacuole was observed in the rounded cell (arrowheads). Lower: Optically reconstructed horizontal sections. Dashed lines indicate optical slice positions. Scale bars: 50 μm in (A), 20 μm in (B), 5 μm in (C, E), 2 μm in (D, left), 100 nm in (D, right), 5 μm in (E), and 10 μm in (F).

Techniques Used: Immuno-Electron Microscopy, Expressing

AQP1 overexpression leads to ectopic vacuole expansion and cell rounding. (A) Optical cross-sections of mCherry-CAAX (control)-, AQP1-mRFP-, and AQP1(R196H)-mRFP-overexpressing quail embryos at E4. eGFP was coexpressed for vacuole measurements. Runx1-positive HECs are indicated by arrowheads. AQP1-mRFP-overexpressing endothelial cells ectopically formed large vacuoles without Runx1 expression in the roof of the dorsal aorta (cells 3 and 4 in the middle panels). Large vacuoles were not observed in AQP1(R196H)-mRFP-overexpressing endothelial cells (cells 5 and 6 in the lower panels). (B) Representative optical cross- (B) and horizontal (B’) sections of mCherry-CAAX-, AQP1-mRFP-, and AQP1 (R196H)-mRFP-overexpressing cells in the roof. Thick dashed lines indicate optical slice positions. A large vacuole was formed ectopically in AQP1-mRFP-overexpressing cells (asterisks in the middle panels). In AQP1(R196H)-mRFP-overexpressing cells, vacuoles were smaller than those in AQP1-mRFP-overexpressing cells (arrows in the lower panels). (C) Sizes of the cross-sectional areas of vacuoles in the roof. AQP1-mRFP-overexpressing cells formed significantly larger vacuoles. (D) Roundness of vacuolated cell cross-sections. AQP1-mRFP-overexpressing cells were rounded compared with mRFP-CAAX- and AQP1(R196H)-mRFP-overexpressing cells. (E) Representative images of co-electroporated eGFP in AQP1-mRFP-overexpressing cells in the roof. Values in brackets are the vacuole size (V) and cell roundness (R). (F) Scatter plot of vacuole size and cell roundness shown in (C and D). AQP1-mRFP-overexpressing cells were characterized by large vacuoles and increased roundness. The correlation coefficients were r = 0.2466 (p = 0.0459) for mRFP-CAAX, r = 0.04878 (p = 0.6229) for AQP1-mRFP, and r = 0.5672 (p
Figure Legend Snippet: AQP1 overexpression leads to ectopic vacuole expansion and cell rounding. (A) Optical cross-sections of mCherry-CAAX (control)-, AQP1-mRFP-, and AQP1(R196H)-mRFP-overexpressing quail embryos at E4. eGFP was coexpressed for vacuole measurements. Runx1-positive HECs are indicated by arrowheads. AQP1-mRFP-overexpressing endothelial cells ectopically formed large vacuoles without Runx1 expression in the roof of the dorsal aorta (cells 3 and 4 in the middle panels). Large vacuoles were not observed in AQP1(R196H)-mRFP-overexpressing endothelial cells (cells 5 and 6 in the lower panels). (B) Representative optical cross- (B) and horizontal (B’) sections of mCherry-CAAX-, AQP1-mRFP-, and AQP1 (R196H)-mRFP-overexpressing cells in the roof. Thick dashed lines indicate optical slice positions. A large vacuole was formed ectopically in AQP1-mRFP-overexpressing cells (asterisks in the middle panels). In AQP1(R196H)-mRFP-overexpressing cells, vacuoles were smaller than those in AQP1-mRFP-overexpressing cells (arrows in the lower panels). (C) Sizes of the cross-sectional areas of vacuoles in the roof. AQP1-mRFP-overexpressing cells formed significantly larger vacuoles. (D) Roundness of vacuolated cell cross-sections. AQP1-mRFP-overexpressing cells were rounded compared with mRFP-CAAX- and AQP1(R196H)-mRFP-overexpressing cells. (E) Representative images of co-electroporated eGFP in AQP1-mRFP-overexpressing cells in the roof. Values in brackets are the vacuole size (V) and cell roundness (R). (F) Scatter plot of vacuole size and cell roundness shown in (C and D). AQP1-mRFP-overexpressing cells were characterized by large vacuoles and increased roundness. The correlation coefficients were r = 0.2466 (p = 0.0459) for mRFP-CAAX, r = 0.04878 (p = 0.6229) for AQP1-mRFP, and r = 0.5672 (p

Techniques Used: Over Expression, Expressing

Redundant AQP function is required for hemogenic endothelial cell rounding and detachment. (A) Oblique views of z-stacked images of the dorsal aortic floor at E4 (upper panels). Electroporated cell bodies and internal vacuoles were identified by mRFP. Endothelial cell membrane is detected by QH1. Arrows in the left panel: mRFP + /Runx1 + cells in control gRNA-electroporated embryos. Arrows in the right panel: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryos. Lower panels: optical cross-sections of each mRFP + /Runx1 + cell. (B) Optical cross-sections of the aortic floor at E4. Upper: mRFP + /Runx1 + cells in control embryos. Lower: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryos. Arrows: mRFP + /Runx1 + flat cells. (C) Percentage of vacuolated mRFP + /Runx1 + cells among the total Runx1 + cells in the control (23 slices, n = 13) and AQP1/5/8/9 gRNA-electroporated embryos (28 slices, n = 14) at E4. (D) Enlarged views of representative electroporated HECs (mRFP + /Runx1 + ). Vacuoles are indicated by arrowheads. (E) Cross-sectional area sizes of the vacuoles in control (46 cells, n = 13) and AQP1/5/8/9 gRNA-electroporated embryos (27 cells, n = 14) at E4. (F) Cell roundness of mRFP + /Runx1 + cells in control (46 cells, n = 13) and AQP1/5/8/9 gRNA-electroporated embryos at E4 (27 vacuolated and 46 non-vacuolated cells, n = 14). (G) Oblique views of z-stacked images of the dorsal aortic floor at E5 (upper panels). Left: mRFP + /Runx1 + cells in control embryos. Arrowheads: Runx1 + cells. Arrows: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryos. Lower: optical cross-sections of each mRFP + /Runx1 + cell. (H) Optical cross-sections at E5. Arrows: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryo. (I) Percentages of mRFP + /Runx1 + cells among eYFP+ endothelial cells in the dorsal aortic floors of control gRNA (26 slices, n = 5 at E4; 30 slices, n = 5 at E5) and AQP1/5/8/9 gRNA (28 slices, n = 6 at E4; 33 slices, n = 9 at E5) electroporated tg(tie1:H2B-eYFP) embryos (refer to Fig. S6). (J) Model depicting HEC rounding. Error bars indicate SD. ns: not significant, *** P
Figure Legend Snippet: Redundant AQP function is required for hemogenic endothelial cell rounding and detachment. (A) Oblique views of z-stacked images of the dorsal aortic floor at E4 (upper panels). Electroporated cell bodies and internal vacuoles were identified by mRFP. Endothelial cell membrane is detected by QH1. Arrows in the left panel: mRFP + /Runx1 + cells in control gRNA-electroporated embryos. Arrows in the right panel: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryos. Lower panels: optical cross-sections of each mRFP + /Runx1 + cell. (B) Optical cross-sections of the aortic floor at E4. Upper: mRFP + /Runx1 + cells in control embryos. Lower: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryos. Arrows: mRFP + /Runx1 + flat cells. (C) Percentage of vacuolated mRFP + /Runx1 + cells among the total Runx1 + cells in the control (23 slices, n = 13) and AQP1/5/8/9 gRNA-electroporated embryos (28 slices, n = 14) at E4. (D) Enlarged views of representative electroporated HECs (mRFP + /Runx1 + ). Vacuoles are indicated by arrowheads. (E) Cross-sectional area sizes of the vacuoles in control (46 cells, n = 13) and AQP1/5/8/9 gRNA-electroporated embryos (27 cells, n = 14) at E4. (F) Cell roundness of mRFP + /Runx1 + cells in control (46 cells, n = 13) and AQP1/5/8/9 gRNA-electroporated embryos at E4 (27 vacuolated and 46 non-vacuolated cells, n = 14). (G) Oblique views of z-stacked images of the dorsal aortic floor at E5 (upper panels). Left: mRFP + /Runx1 + cells in control embryos. Arrowheads: Runx1 + cells. Arrows: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryos. Lower: optical cross-sections of each mRFP + /Runx1 + cell. (H) Optical cross-sections at E5. Arrows: mRFP + /Runx1 + cells in AQP1/5/8/9 gRNA-electroporated embryo. (I) Percentages of mRFP + /Runx1 + cells among eYFP+ endothelial cells in the dorsal aortic floors of control gRNA (26 slices, n = 5 at E4; 30 slices, n = 5 at E5) and AQP1/5/8/9 gRNA (28 slices, n = 6 at E4; 33 slices, n = 9 at E5) electroporated tg(tie1:H2B-eYFP) embryos (refer to Fig. S6). (J) Model depicting HEC rounding. Error bars indicate SD. ns: not significant, *** P

Techniques Used:

9) Product Images from "Transcription factor Sp1 regulates mitotic chromosome assembly and segregation"

Article Title: Transcription factor Sp1 regulates mitotic chromosome assembly and segregation

Journal: Chromosoma

doi: 10.1007/s00412-022-00778-z

Sp1 regulates mitotic progression. a and b Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every 3 min, the above image sequence represents images taken every 9 min to best highlight the differences between the treatments. Time = h:min., Scale bar = 5 µm. c Time (m) from nuclear envelope breakdown to G 1 . Forty cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.016, unpaired t -test. d Time (m) from nuclear envelope breakdown to anaphase. Forty cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.042, unpaired t -test. e Schematic outlining the experimental strategy for ( f ). f Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 µm. f Quantification of ( e ). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.0037, unpaired t -test. All images are representative
Figure Legend Snippet: Sp1 regulates mitotic progression. a and b Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every 3 min, the above image sequence represents images taken every 9 min to best highlight the differences between the treatments. Time = h:min., Scale bar = 5 µm. c Time (m) from nuclear envelope breakdown to G 1 . Forty cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.016, unpaired t -test. d Time (m) from nuclear envelope breakdown to anaphase. Forty cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.042, unpaired t -test. e Schematic outlining the experimental strategy for ( f ). f Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 µm. f Quantification of ( e ). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m., p = 0.0037, unpaired t -test. All images are representative

Techniques Used: Live Cell Imaging, Sequencing, Staining

10) Product Images from "VISAGE Reveals a Targetable Mitotic Spindle Vulnerability in Cancer Cells"

Article Title: VISAGE Reveals a Targetable Mitotic Spindle Vulnerability in Cancer Cells

Journal: Cell systems

doi: 10.1016/j.cels.2019.05.009

Cell death from TH588/BI2536 co-treatment occurs by prolonged mitotic arrest, requires the SAC, and is MTH1-independent. (A) Quantification of phenotypic outcomes of C4–2 CRPC cells expressing mEmerald-tubulin and H2B-mCherry treated with a dose-response matrix of BI2536 and TH588, and imaged at 15 min intervals. Each bar represents a single cell, with color indicating its phenotype/cell cycle state over the time course of the experiment. Thirty cells that initiated and exited mitosis during the time-lapse were analyzed per condition. (B) Violin plots depicting the distribution in duration of mitosis of C4–2 cells from panel (A) above. Position of horizontal lines on the y-axis indicates duration of mitosis of individual cells. (C) Disruption of the SAC by MAD2L1 knockdown ablates sensitivity to TH588 and entirely prevents synergy with BI2536. Cells were treated with the indicated drugs 48 hours after control or MAD2L1 siRNA transfection and assessed for viability at 5 days. Bottom left inset on the right graph shows immunoblots of lysates from cells 48 hours after transfection of the indicated siRNA, confirming loss of MAD2. Mean ± SEM (n = 3) is shown. (D) HCT116 cells, or single-cell clones expressing a control guide RNA ( AAVS1 -1), the MTH1 KO 9–4, clone, and the same clone transduced with constructs containing HA-MTH1 or HAMTH1 E56A were treated with the indicated drugs for 16 hours, fixed, stained with DAPI and antibodies against pHH3, and analyzed by flow cytometry to assess the percentage of mitotically arrested cells. Mean ± SEM (n = 3) is shown. (E) Parental HCT116 cells, as well as the AAVS1 -1 and MTH1 KO 9–4 clones were treated with 5 μM TH588 for 16 hours, fixed and stained for DNA (DAPI; blue), pericentrin (red) and tubulin (green). Representative deconvolved Z-stack projections are shown. Scale bar at bottom right indicates 10 μm. White arrowheads indicate cells undergoing abnormal mitosis.
Figure Legend Snippet: Cell death from TH588/BI2536 co-treatment occurs by prolonged mitotic arrest, requires the SAC, and is MTH1-independent. (A) Quantification of phenotypic outcomes of C4–2 CRPC cells expressing mEmerald-tubulin and H2B-mCherry treated with a dose-response matrix of BI2536 and TH588, and imaged at 15 min intervals. Each bar represents a single cell, with color indicating its phenotype/cell cycle state over the time course of the experiment. Thirty cells that initiated and exited mitosis during the time-lapse were analyzed per condition. (B) Violin plots depicting the distribution in duration of mitosis of C4–2 cells from panel (A) above. Position of horizontal lines on the y-axis indicates duration of mitosis of individual cells. (C) Disruption of the SAC by MAD2L1 knockdown ablates sensitivity to TH588 and entirely prevents synergy with BI2536. Cells were treated with the indicated drugs 48 hours after control or MAD2L1 siRNA transfection and assessed for viability at 5 days. Bottom left inset on the right graph shows immunoblots of lysates from cells 48 hours after transfection of the indicated siRNA, confirming loss of MAD2. Mean ± SEM (n = 3) is shown. (D) HCT116 cells, or single-cell clones expressing a control guide RNA ( AAVS1 -1), the MTH1 KO 9–4, clone, and the same clone transduced with constructs containing HA-MTH1 or HAMTH1 E56A were treated with the indicated drugs for 16 hours, fixed, stained with DAPI and antibodies against pHH3, and analyzed by flow cytometry to assess the percentage of mitotically arrested cells. Mean ± SEM (n = 3) is shown. (E) Parental HCT116 cells, as well as the AAVS1 -1 and MTH1 KO 9–4 clones were treated with 5 μM TH588 for 16 hours, fixed and stained for DNA (DAPI; blue), pericentrin (red) and tubulin (green). Representative deconvolved Z-stack projections are shown. Scale bar at bottom right indicates 10 μm. White arrowheads indicate cells undergoing abnormal mitosis.

Techniques Used: Expressing, Transfection, Western Blot, Clone Assay, Transduction, Construct, Staining, Flow Cytometry

11) Product Images from "KIF24 depletion induces clustering of supernumerary centrosomes in PDAC cells"

Article Title: KIF24 depletion induces clustering of supernumerary centrosomes in PDAC cells

Journal: Life Science Alliance

doi: 10.26508/lsa.202201470

KIF24 depletion restores impaired mitotic events in Panc1 cells. (A) Frames from live cell imaging of Panc1 or Kif24-3 cells stably expressing H2B-mCherry. Scale bar, 10 μm. (B) Time from nuclear envelope breakdown to the anaphase onset in indicated cells was measured. n = 119 (Panc1_H2B-mCherry), 135 (Kif24-3_H2B-mCherry). (C) The percentage of cells that entered and remained in mitosis for > 180 min. The average of three independent experiments is shown; > 40 cells were scored each time. (B, C) All data are shown as mean ± SD. Two-tailed t test. ** P
Figure Legend Snippet: KIF24 depletion restores impaired mitotic events in Panc1 cells. (A) Frames from live cell imaging of Panc1 or Kif24-3 cells stably expressing H2B-mCherry. Scale bar, 10 μm. (B) Time from nuclear envelope breakdown to the anaphase onset in indicated cells was measured. n = 119 (Panc1_H2B-mCherry), 135 (Kif24-3_H2B-mCherry). (C) The percentage of cells that entered and remained in mitosis for > 180 min. The average of three independent experiments is shown; > 40 cells were scored each time. (B, C) All data are shown as mean ± SD. Two-tailed t test. ** P

Techniques Used: Live Cell Imaging, Stable Transfection, Expressing, Two Tailed Test

12) Product Images from "Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle"

Article Title: Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle

Journal: Current Biology

doi: 10.1016/j.cub.2020.06.098

Flat cells with a monopolar spindle as a simplified system to study dynamic spindle positioning (A) Immuno-fluorescence confocal images of HeLa cells in mitosis on FN-coated unpatterned substrates. Wild-type cells are round and have a bipolar spindle (left). Overexpression of Rap1 ∗ results in cells that fail to round up in mitosis (middle), and the combined treatment of Rap1 ∗ and STLC results in flat mitotic cells with a monopolar spindle. The dashed line (magenta) shows the elliptical fit of the cell outline. (B) Time-lapse confocal images of a HeLa cell (Rap1 ∗ + STLC) on a uniformly FN-coated substrate as it enters mitosis: at NEB, the two centrosomes fail to separate, resulting in monopolar spindle formation. The monopolar spindle moves freely and continuously. (C) Plot of the trajectories of monopolar spindles in mitotic HeLa cells on fibronectin-coated adhesive substrates (Rap1 ∗ + STLC; n = 15). The trajectory of the spindle shown in (B) is highlighted in black, and the outline of the cell is shown as a dashed line, although other trajectories are shown in gray. (D) Detailed time lapse of the monopolar spindle shown in (B). The centrosome leads the movement, and the rest of the spindle follows. (E) X-Z section of a confocal time lapse of a representative HeLa cell treated with Rap1 ∗ + STLC on a FN-coated unpatterned substrate. Centrosomes lie close to the basal membrane as the spindle moves. (F) Spindle motion still occurs in STLC-treated HeLa cells on FN-coated surfaces under a FN-coated PDMS roof (upper graphic, top row; n = 10), as well as on non-adherent PEG-coated surfaces, held flat under a PEG-coated roof of PDMS (bottom; n = 10). (G) Boxplots of STLC-treated monopolar spindle velocities in cells flattened by different means. Physical confinement with FN or PEG coating (shown in F) or genetic treatments (Rap1 ∗ overexpression; shown in B and C) results in similar monopolar spindle behavior (PDMS + STLC + FN n = 10; PDMS + STLC + PEG n = 10; Rap1* + STLC + FN n = 15; significance tests: p > 0.05; Mann-Whitney U test). Thick bars and boxes indicate median values and lower/upper quartiles, respectively. Whiskers extend to the smallest/largest value, but no further than 1.5 times the interquartile range. (H) Wide-field time-lapse images of a mitotic HeLa cell (Rap1 ∗ + STLC) on a FN-coated line pattern (10 μm width, magenta box). The spindle follows a 1D path, alternating its direction of movement. (I) Phase portrait of centrosome motion in monopolar cells on line-patterns (as in H). The phase portrait reveals that the spindle motion alternates between fast motion and pausing near cell ends as it changes direction. All cells in time-lapse images are expressing tubulin-GFP and H2B-mCherry. All scale bars indicate 10 μm. See also Figure S1 and Video S1 A.
Figure Legend Snippet: Flat cells with a monopolar spindle as a simplified system to study dynamic spindle positioning (A) Immuno-fluorescence confocal images of HeLa cells in mitosis on FN-coated unpatterned substrates. Wild-type cells are round and have a bipolar spindle (left). Overexpression of Rap1 ∗ results in cells that fail to round up in mitosis (middle), and the combined treatment of Rap1 ∗ and STLC results in flat mitotic cells with a monopolar spindle. The dashed line (magenta) shows the elliptical fit of the cell outline. (B) Time-lapse confocal images of a HeLa cell (Rap1 ∗ + STLC) on a uniformly FN-coated substrate as it enters mitosis: at NEB, the two centrosomes fail to separate, resulting in monopolar spindle formation. The monopolar spindle moves freely and continuously. (C) Plot of the trajectories of monopolar spindles in mitotic HeLa cells on fibronectin-coated adhesive substrates (Rap1 ∗ + STLC; n = 15). The trajectory of the spindle shown in (B) is highlighted in black, and the outline of the cell is shown as a dashed line, although other trajectories are shown in gray. (D) Detailed time lapse of the monopolar spindle shown in (B). The centrosome leads the movement, and the rest of the spindle follows. (E) X-Z section of a confocal time lapse of a representative HeLa cell treated with Rap1 ∗ + STLC on a FN-coated unpatterned substrate. Centrosomes lie close to the basal membrane as the spindle moves. (F) Spindle motion still occurs in STLC-treated HeLa cells on FN-coated surfaces under a FN-coated PDMS roof (upper graphic, top row; n = 10), as well as on non-adherent PEG-coated surfaces, held flat under a PEG-coated roof of PDMS (bottom; n = 10). (G) Boxplots of STLC-treated monopolar spindle velocities in cells flattened by different means. Physical confinement with FN or PEG coating (shown in F) or genetic treatments (Rap1 ∗ overexpression; shown in B and C) results in similar monopolar spindle behavior (PDMS + STLC + FN n = 10; PDMS + STLC + PEG n = 10; Rap1* + STLC + FN n = 15; significance tests: p > 0.05; Mann-Whitney U test). Thick bars and boxes indicate median values and lower/upper quartiles, respectively. Whiskers extend to the smallest/largest value, but no further than 1.5 times the interquartile range. (H) Wide-field time-lapse images of a mitotic HeLa cell (Rap1 ∗ + STLC) on a FN-coated line pattern (10 μm width, magenta box). The spindle follows a 1D path, alternating its direction of movement. (I) Phase portrait of centrosome motion in monopolar cells on line-patterns (as in H). The phase portrait reveals that the spindle motion alternates between fast motion and pausing near cell ends as it changes direction. All cells in time-lapse images are expressing tubulin-GFP and H2B-mCherry. All scale bars indicate 10 μm. See also Figure S1 and Video S1 A.

Techniques Used: Fluorescence, Over Expression, MANN-WHITNEY, Expressing

13) Product Images from "Piezo mechanosensory channels regulate centrosome integrity"

Article Title: Piezo mechanosensory channels regulate centrosome integrity

Journal: bioRxiv

doi: 10.1101/2022.04.12.488050

Centriole disengagement upon Piezo1 activation or Piezo inhibition in live and fixed cells. a-d, Movie snapshots of imaged C2C12 cells stably expressing GFP-Centrin2 (green) and H2B-mCherry (magenta) treated with DMSO (0.1%, vehicle control) (a), Yoda1 (10 μM) at metaphase 30 minutes after RO-3306 release (b, top) or after MG132 synchronization (b, bottom), Dooku1 (10 μM) at metaphase or GsMTx4 (5 μM) at both early mitosis and interphase (d). Distances between centrioles are labeled in μm. The DMSO-treated cell contained centrosomes with two centrioles separated by
Figure Legend Snippet: Centriole disengagement upon Piezo1 activation or Piezo inhibition in live and fixed cells. a-d, Movie snapshots of imaged C2C12 cells stably expressing GFP-Centrin2 (green) and H2B-mCherry (magenta) treated with DMSO (0.1%, vehicle control) (a), Yoda1 (10 μM) at metaphase 30 minutes after RO-3306 release (b, top) or after MG132 synchronization (b, bottom), Dooku1 (10 μM) at metaphase or GsMTx4 (5 μM) at both early mitosis and interphase (d). Distances between centrioles are labeled in μm. The DMSO-treated cell contained centrosomes with two centrioles separated by

Techniques Used: Activation Assay, Inhibition, Stable Transfection, Expressing, Labeling

Rapid centriole disengagement following Yoda1 treatment at different cell cycle stages. a-c, Live time-lapse images from mitotic or interphase C2C12 cells stably expressing GFP-Centrin2 (green) and H2B-mCherry (magenta) treated with Yoda1 (10 μM). Cells were synchronized to G2/M by RO-3306 (a, b) or interphase by double thymidine block (c). Yoda1 was introduced 15 min and 45 min after RO-3306 washout to capture cells at prophase (a) and cytokinesis (b), respectively, and to unsynchronized interphase C2C12 cells at G1 and G2 (c). Phase images are shown for (b) to emphasis the mitotic stage. Distances (μm) between mother and daughter centriole pairs are marked, and lagging chromatin in (a) is labeled by white arrows. Rapid centriole disengagement was observed at prophase (a), cytokinesis (b) and interphase (c). However, cells at metaphase did not exhibit centriole disengagement upon Yoda1 introduction ( Extended Data Fig. 7b ). d, Quantitative analysis of IF images of C2C12 cells for supernumerary centrosomes following treatment with DMSO (0.1%) or Yoda1 (10 μM) at different stages of the cell cycle, captured largely as in (a-c) and in Extended Data Fig. 7b for metaphase cells. All images are maximum intensity Z projections. All scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments counting 100-250 cells each. Statistical significance between an experimental group and a control group was assessed by 2-tailed t-test with *** and * for p
Figure Legend Snippet: Rapid centriole disengagement following Yoda1 treatment at different cell cycle stages. a-c, Live time-lapse images from mitotic or interphase C2C12 cells stably expressing GFP-Centrin2 (green) and H2B-mCherry (magenta) treated with Yoda1 (10 μM). Cells were synchronized to G2/M by RO-3306 (a, b) or interphase by double thymidine block (c). Yoda1 was introduced 15 min and 45 min after RO-3306 washout to capture cells at prophase (a) and cytokinesis (b), respectively, and to unsynchronized interphase C2C12 cells at G1 and G2 (c). Phase images are shown for (b) to emphasis the mitotic stage. Distances (μm) between mother and daughter centriole pairs are marked, and lagging chromatin in (a) is labeled by white arrows. Rapid centriole disengagement was observed at prophase (a), cytokinesis (b) and interphase (c). However, cells at metaphase did not exhibit centriole disengagement upon Yoda1 introduction ( Extended Data Fig. 7b ). d, Quantitative analysis of IF images of C2C12 cells for supernumerary centrosomes following treatment with DMSO (0.1%) or Yoda1 (10 μM) at different stages of the cell cycle, captured largely as in (a-c) and in Extended Data Fig. 7b for metaphase cells. All images are maximum intensity Z projections. All scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments counting 100-250 cells each. Statistical significance between an experimental group and a control group was assessed by 2-tailed t-test with *** and * for p

Techniques Used: Stable Transfection, Expressing, Blocking Assay, Labeling

14) Product Images from "An easily transfectable cell line that produces an infectious reporter virus for routine and robust quantitation of Kaposi’s sarcoma-associated herpesvirus reactivation"

Article Title: An easily transfectable cell line that produces an infectious reporter virus for routine and robust quantitation of Kaposi’s sarcoma-associated herpesvirus reactivation

Journal: Journal of virological methods

doi: 10.1016/j.jviromet.2017.04.019

Vero rKHSV.219 cells are highly transfectable BCBL1 or Vero rKSHV.294 cells were transfected with either 2.5ug pcDNA3 or H2b-mCherry and incubated for 48 h prior to immunofluorescence. (A) Percent transfection efficiency was determined by dividing the number of H2b-mCherry positive cells by the total number of DAPI-positive cells. Representative fields of (B) Vero rKSHV.294 (green) and (C) BCBL-1 cells transfected with pcDNA3 (top panels), or H2b-mCherry (bottom panels).
Figure Legend Snippet: Vero rKHSV.219 cells are highly transfectable BCBL1 or Vero rKSHV.294 cells were transfected with either 2.5ug pcDNA3 or H2b-mCherry and incubated for 48 h prior to immunofluorescence. (A) Percent transfection efficiency was determined by dividing the number of H2b-mCherry positive cells by the total number of DAPI-positive cells. Representative fields of (B) Vero rKSHV.294 (green) and (C) BCBL-1 cells transfected with pcDNA3 (top panels), or H2b-mCherry (bottom panels).

Techniques Used: Transfection, Incubation, Immunofluorescence

15) Product Images from "Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes"

Article Title: Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes

Journal: Scientific Reports

doi: 10.1038/srep39632

CDC42, IRSp53, and VASP negatively regulate intercellular vesicle transfer. ( a ) Raw data (dot plots) from a representative experiment showing the transfer of DiD-labeled vesicles to the acceptor population (H2B-mCherry) upon ectopic expression of GFP-CDC42 V12, GFP-CDC42 T17N, RFP-IRSp53, RFP-IRS FP/AA, or GFP-VASP in the donor population. A population of CAD cells was transiently transfected with the different fluorescently-tagged constructs (donor population) and another population was transiently transfected with either H2B-GFP or H2B-mCherry (acceptor population). Internal vesicles of donor cells were labeled with the membrane dye Vybrant DiD as described in the ‘Material and Methods’ section. Donor and acceptor cells were co-cultured for 16 hrs. Cells were then fixed and analyzed by flow cytometry. ( b ) Quantification by flow cytometry of DiD-positive acceptor cells upon ectopic expression of GFP-CDC42 V12, GFP-CDC42 T17N, RFP-IRSp53, RFP-IRS FP/AA, or GFP-VASP in the donor population. The percentage of DiD-positive acceptor cells in the total cell population was evaluated. Data represent the mean (±SEM), normalized to control cells arbitrarily set at 100%, of at least 4 independent experiments. *P
Figure Legend Snippet: CDC42, IRSp53, and VASP negatively regulate intercellular vesicle transfer. ( a ) Raw data (dot plots) from a representative experiment showing the transfer of DiD-labeled vesicles to the acceptor population (H2B-mCherry) upon ectopic expression of GFP-CDC42 V12, GFP-CDC42 T17N, RFP-IRSp53, RFP-IRS FP/AA, or GFP-VASP in the donor population. A population of CAD cells was transiently transfected with the different fluorescently-tagged constructs (donor population) and another population was transiently transfected with either H2B-GFP or H2B-mCherry (acceptor population). Internal vesicles of donor cells were labeled with the membrane dye Vybrant DiD as described in the ‘Material and Methods’ section. Donor and acceptor cells were co-cultured for 16 hrs. Cells were then fixed and analyzed by flow cytometry. ( b ) Quantification by flow cytometry of DiD-positive acceptor cells upon ectopic expression of GFP-CDC42 V12, GFP-CDC42 T17N, RFP-IRSp53, RFP-IRS FP/AA, or GFP-VASP in the donor population. The percentage of DiD-positive acceptor cells in the total cell population was evaluated. Data represent the mean (±SEM), normalized to control cells arbitrarily set at 100%, of at least 4 independent experiments. *P

Techniques Used: Labeling, Expressing, Transfection, Construct, Cell Culture, Flow Cytometry, Cytometry

Eps8 positively regulates TNT formation and intercellular vesicle transfer via its bundling activity. ( a ) Representative confocal images showing intercellular connections upon ectopic expression of GFP-Eps8, GFP-Eps8Δcapping or GFP-Eps8Δbundling. Cells were fixed 14 hrs post-transfection, labeled with WGA-Alexa Fluor © -647 nm (grey) in order to detect TNTs and observed by confocal microscopy. Scale bar = 10 μM. ( b ) Quantification of TNT-connected cells upon ectopic expression of GFP-Eps8, GFP-Eps8Δcapping or GFP-Eps8Δbundling. The ratio of TNT-forming transfected cells/number of transfected cells was evaluated. ( c ) The donor population of CAD cells was transfected with either GFP-Eps8, GFP-Eps8Δcapping or GFP-Eps8Δbundling, and the acceptor population was transfected with H2B-mCherry. The co-culture was prepared as described in Fig. 3 . Cells were analyzed by flow cytometry to quantify the percentage of acceptor cells having received DiD-labeled vesicles from the donor population. Data represent the mean (±SEM), normalized to control cells arbitrarily set at 100%, of at least 3 independent experiments. ( b , c ) Data represent the mean (±SEM), normalized to control cells (GFP-vector transfected cells) arbitrarily set at 100%, of at least 3 independent experiments. *P
Figure Legend Snippet: Eps8 positively regulates TNT formation and intercellular vesicle transfer via its bundling activity. ( a ) Representative confocal images showing intercellular connections upon ectopic expression of GFP-Eps8, GFP-Eps8Δcapping or GFP-Eps8Δbundling. Cells were fixed 14 hrs post-transfection, labeled with WGA-Alexa Fluor © -647 nm (grey) in order to detect TNTs and observed by confocal microscopy. Scale bar = 10 μM. ( b ) Quantification of TNT-connected cells upon ectopic expression of GFP-Eps8, GFP-Eps8Δcapping or GFP-Eps8Δbundling. The ratio of TNT-forming transfected cells/number of transfected cells was evaluated. ( c ) The donor population of CAD cells was transfected with either GFP-Eps8, GFP-Eps8Δcapping or GFP-Eps8Δbundling, and the acceptor population was transfected with H2B-mCherry. The co-culture was prepared as described in Fig. 3 . Cells were analyzed by flow cytometry to quantify the percentage of acceptor cells having received DiD-labeled vesicles from the donor population. Data represent the mean (±SEM), normalized to control cells arbitrarily set at 100%, of at least 3 independent experiments. ( b , c ) Data represent the mean (±SEM), normalized to control cells (GFP-vector transfected cells) arbitrarily set at 100%, of at least 3 independent experiments. *P

Techniques Used: Activity Assay, Expressing, Transfection, Labeling, Whole Genome Amplification, Confocal Microscopy, Co-Culture Assay, Flow Cytometry, Cytometry, Plasmid Preparation

16) Product Images from "A far-red fluorescent chemogenetic reporter for in vivo molecular imaging"

Article Title: A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

Journal: bioRxiv

doi: 10.1101/2020.04.04.022145

frFAST forms a far-red fluorescent assembly with HPAR-3OM and enables to encode far-red fluorescence in cells. (a) frFAST principle and structure of HPAR-3OM (POI: protein of interest). (b) Absorption and emission spectra of the HPAR-3OM in absence (black) or presence (magenta) of frFAST. HPAR-3OM and frFAST concentrations were 2 μM and 40 μM, respectively, in pH 7.4 PBS (50 mM sodium phosphate, 150 mM NaCl). Spectra were recorded at 25°C. (c) Confocal micrographs of live HeLa and U2OS cells expressing various frFAST fusions. Cells were incubated with 10 μM HPAR-3OM for 15-30 seconds and then directly imaged using the following settings: Ex/Em 633/638-797 nm. (d) Confocal micrographs of live HeLa and U2OS cells co-expressing various EGFP, mCherry and frFAST fusions. Cells were incubated with 10 μM HPAR-3OM for 15-30 seconds and then directly imaged using the following settings: EGFP Ex/Em 488/493-599 nm, mCherry Ex/Em 543/560-598, frFAST Ex/Em 633/650-797 nm. (c,d) Mito: Mitochondrial targeting sequence from the subunit VIII of human cytochrome c oxidase, H2B: Histone 2B, MAP4: microtubule-associated protein 4, Lyn11: membrane localization signal. Scale bars 10 μm.
Figure Legend Snippet: frFAST forms a far-red fluorescent assembly with HPAR-3OM and enables to encode far-red fluorescence in cells. (a) frFAST principle and structure of HPAR-3OM (POI: protein of interest). (b) Absorption and emission spectra of the HPAR-3OM in absence (black) or presence (magenta) of frFAST. HPAR-3OM and frFAST concentrations were 2 μM and 40 μM, respectively, in pH 7.4 PBS (50 mM sodium phosphate, 150 mM NaCl). Spectra were recorded at 25°C. (c) Confocal micrographs of live HeLa and U2OS cells expressing various frFAST fusions. Cells were incubated with 10 μM HPAR-3OM for 15-30 seconds and then directly imaged using the following settings: Ex/Em 633/638-797 nm. (d) Confocal micrographs of live HeLa and U2OS cells co-expressing various EGFP, mCherry and frFAST fusions. Cells were incubated with 10 μM HPAR-3OM for 15-30 seconds and then directly imaged using the following settings: EGFP Ex/Em 488/493-599 nm, mCherry Ex/Em 543/560-598, frFAST Ex/Em 633/650-797 nm. (c,d) Mito: Mitochondrial targeting sequence from the subunit VIII of human cytochrome c oxidase, H2B: Histone 2B, MAP4: microtubule-associated protein 4, Lyn11: membrane localization signal. Scale bars 10 μm.

Techniques Used: Fluorescence, Expressing, Incubation, Sequencing

17) Product Images from "Local 3D matrix confinement determines division axis through cell shape"

Article Title: Local 3D matrix confinement determines division axis through cell shape

Journal: Oncotarget

doi: 10.18632/oncotarget.5848

The determination of the cell-division axis by cell shape is independent of cell-matrix interactions and matrix density A. Representative micrographs obtained from a high-magnification live-cell imaging video of an elongated HT1080 cell expressing H2B-mCherry, embedded in a collagen matrix. The collagen fibers are visualized by time-dependent reflection confocal microscopy. The deformation vector of the matrix quantified by PIV software was shown in blue arrows. Scale bar, 20 μm. B. The change in the magnitude of matrix deformation for matrix-embedded elongated and round β1-integrin-KD cells. The black arrow indicates prophase and telophase. C. Quantification of matrix deformation for elongated and round β1-integrin-KD cells during interphase and mitosis, indicating that matrix deformation is minimal during cell division. n = 5 for both phases. Data are represented as mean ± SEM. D. Comparison of the angle difference between the major axis and the division direction for HT1080 cells undergoing cell division in 3D matrices of varying densities, from 1 mg/ml to 6 mg/ml. The statistical analysis was performed using non-parametric analysis because the data does not pass the normality test.
Figure Legend Snippet: The determination of the cell-division axis by cell shape is independent of cell-matrix interactions and matrix density A. Representative micrographs obtained from a high-magnification live-cell imaging video of an elongated HT1080 cell expressing H2B-mCherry, embedded in a collagen matrix. The collagen fibers are visualized by time-dependent reflection confocal microscopy. The deformation vector of the matrix quantified by PIV software was shown in blue arrows. Scale bar, 20 μm. B. The change in the magnitude of matrix deformation for matrix-embedded elongated and round β1-integrin-KD cells. The black arrow indicates prophase and telophase. C. Quantification of matrix deformation for elongated and round β1-integrin-KD cells during interphase and mitosis, indicating that matrix deformation is minimal during cell division. n = 5 for both phases. Data are represented as mean ± SEM. D. Comparison of the angle difference between the major axis and the division direction for HT1080 cells undergoing cell division in 3D matrices of varying densities, from 1 mg/ml to 6 mg/ml. The statistical analysis was performed using non-parametric analysis because the data does not pass the normality test.

Techniques Used: Live Cell Imaging, Expressing, Confocal Microscopy, Plasmid Preparation, Software

3D-specific cell division phenotype does not interfere with mitotic progression and cell proliferation A. Representative micrographs of mitotic progression of an elongated HT1080 cell stably expressing LifeAct-EGFP and H2B-mCherry. B. Duration of the mitotic phase of cells in 3D matrix and on 2D substrates. There are 10 cells on 2D substrates and 13 cells in 3D collagen from three independent experiments. C. Division of a mother cell that underwent elongated cell division and of its two daughter cells. Red arrow indicates mother cell. Scale bar, 20 μm. D. and E. Lapse of time before the division of the first (D) and second (E) daughter cell after the mother cell underwent either round or elongated cell division. Differences were not significant. Scale bar, 20 μm. There are 10 round cells and 13 elongated cells from three independent experiments.
Figure Legend Snippet: 3D-specific cell division phenotype does not interfere with mitotic progression and cell proliferation A. Representative micrographs of mitotic progression of an elongated HT1080 cell stably expressing LifeAct-EGFP and H2B-mCherry. B. Duration of the mitotic phase of cells in 3D matrix and on 2D substrates. There are 10 cells on 2D substrates and 13 cells in 3D collagen from three independent experiments. C. Division of a mother cell that underwent elongated cell division and of its two daughter cells. Red arrow indicates mother cell. Scale bar, 20 μm. D. and E. Lapse of time before the division of the first (D) and second (E) daughter cell after the mother cell underwent either round or elongated cell division. Differences were not significant. Scale bar, 20 μm. There are 10 round cells and 13 elongated cells from three independent experiments.

Techniques Used: Stable Transfection, Expressing

18) Product Images from "Micropatterned substrates to promote and dissect reprogramming of human somatic cells"

Article Title: Micropatterned substrates to promote and dissect reprogramming of human somatic cells

Journal: bioRxiv

doi: 10.1101/111369

Micropatterned plates enable live tracking of reprogramming. A) Schematic showing the creation of μCP Well Plates. Gold-coated glass is stamped with initiator solution using PDMS mold and then reacted in PEG solution. Glass is then combined with a standard well plate that has had the bottom cut out and a double side adhesive attached. B) Coculture of two different cell types on a single μFeature. Brightfield only cells are fibroblasts. H2B-mCherry, Actin-GFP cells are PSCs. C) Representative images of the progression of a cell aggregate on a single μFeature through establishment. Cells were live stained using antibodies against TRA-1-60 (green) and CD44 (red).
Figure Legend Snippet: Micropatterned plates enable live tracking of reprogramming. A) Schematic showing the creation of μCP Well Plates. Gold-coated glass is stamped with initiator solution using PDMS mold and then reacted in PEG solution. Glass is then combined with a standard well plate that has had the bottom cut out and a double side adhesive attached. B) Coculture of two different cell types on a single μFeature. Brightfield only cells are fibroblasts. H2B-mCherry, Actin-GFP cells are PSCs. C) Representative images of the progression of a cell aggregate on a single μFeature through establishment. Cells were live stained using antibodies against TRA-1-60 (green) and CD44 (red).

Techniques Used: Staining

Reprogramming fibroblasts on micropatterned substrates causes erasure of somatic cell identity by increasing cell density and proliferation, and modulating YAP localization. A) Left: hPSCs expressing H2B-mCherry on the μCP Well Platform. Right: Model showing the erasure of somatic cell identity and the accompanying markers to an intermediate cell fate followed by the establishment of pluripotency. B) Representative images of the progression of a cell aggregate on a μCP Well Plate through a reprogramming time course. C) Cell density through reprogramming on micropatterned and unpatterned substrates. Micropatterned substrates had higher cell densities during erasure (D8 and D12) (n=49 technical replicates, mean ± 95% CI, Student’s two-tailed t-test, * p
Figure Legend Snippet: Reprogramming fibroblasts on micropatterned substrates causes erasure of somatic cell identity by increasing cell density and proliferation, and modulating YAP localization. A) Left: hPSCs expressing H2B-mCherry on the μCP Well Platform. Right: Model showing the erasure of somatic cell identity and the accompanying markers to an intermediate cell fate followed by the establishment of pluripotency. B) Representative images of the progression of a cell aggregate on a μCP Well Plate through a reprogramming time course. C) Cell density through reprogramming on micropatterned and unpatterned substrates. Micropatterned substrates had higher cell densities during erasure (D8 and D12) (n=49 technical replicates, mean ± 95% CI, Student’s two-tailed t-test, * p

Techniques Used: Expressing, Two Tailed Test

19) Product Images from "Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages"

Article Title: Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages

Journal: bioRxiv

doi: 10.1101/039289

Ebf is an independent master regulator of siphon muscle fate in the B7.5 lineage. (A-D) Close-up of ASM and first and second heart precursors (SHP; marked with “*”), derived from the B7.5 lineage, revealing Mrf mRNA (blue) and Bhlh-tun-1 mRNA (green). (A,B) Larvae electroporated with Mesp > H2B:mCherry;Mesp > nls:Cas9:nls;U6 > sgControlF+E and Tbx1 > LacZ (A) or Tbx1 > Ebf (B). In (B), note that expression of Mrf and Bhlh-tun-1 has expanded to the SHP, due to earlier Tbx1 > Ebf expression in the secondary TVCs. (C, D) Larvae electroporated with Mesp > H2B:mCherry;Mesp > nls:Cas9:nls;U6 > sgTbx1.303;U6 > sgTbx1.558 and Tbx1 > LacZ (C) or Tbx1 > Ebf (D). Note that in (C) there is a complete loss of Mrf or Bhlh-tun-1 expression, whereas in (D), Mrf and Bhlh-tun-1 show wild-type expression patterns in the ASM, and are also expressed in the SHP. Scale bars = 25μM. n=. All data was collected from a single technical replicate. (E) Schematic diagram comparing core regulatory interactions upstream of Mrf -driven differentiation and Notch -driven stemness in ASM and OSM. In black, documented shared expression of Hand-r in cells that give rise to, among other tissues, siphon muscles may be involved in activation of the core common regulators Tbx1/10 and Ebf , as indicated by green dashed arrows. Although both Ebf and Tbx1/10 impinge on Mrf expression, the distinct regulatory relationships in place in ASM vs. OSM are indicated by purple and brown arrows, respectively. The shared direct input from Ebf to Mrf in both ASM and OSM is indicated by the solid green arrow. In grey, Notch signaling downstream of Mrf activation has been established in the ASM ( Razy-Krajka et al . 2014 ) as the mechanism for cells to choose between stemness and differentiation, but has not yet been tested in the OSM.
Figure Legend Snippet: Ebf is an independent master regulator of siphon muscle fate in the B7.5 lineage. (A-D) Close-up of ASM and first and second heart precursors (SHP; marked with “*”), derived from the B7.5 lineage, revealing Mrf mRNA (blue) and Bhlh-tun-1 mRNA (green). (A,B) Larvae electroporated with Mesp > H2B:mCherry;Mesp > nls:Cas9:nls;U6 > sgControlF+E and Tbx1 > LacZ (A) or Tbx1 > Ebf (B). In (B), note that expression of Mrf and Bhlh-tun-1 has expanded to the SHP, due to earlier Tbx1 > Ebf expression in the secondary TVCs. (C, D) Larvae electroporated with Mesp > H2B:mCherry;Mesp > nls:Cas9:nls;U6 > sgTbx1.303;U6 > sgTbx1.558 and Tbx1 > LacZ (C) or Tbx1 > Ebf (D). Note that in (C) there is a complete loss of Mrf or Bhlh-tun-1 expression, whereas in (D), Mrf and Bhlh-tun-1 show wild-type expression patterns in the ASM, and are also expressed in the SHP. Scale bars = 25μM. n=. All data was collected from a single technical replicate. (E) Schematic diagram comparing core regulatory interactions upstream of Mrf -driven differentiation and Notch -driven stemness in ASM and OSM. In black, documented shared expression of Hand-r in cells that give rise to, among other tissues, siphon muscles may be involved in activation of the core common regulators Tbx1/10 and Ebf , as indicated by green dashed arrows. Although both Ebf and Tbx1/10 impinge on Mrf expression, the distinct regulatory relationships in place in ASM vs. OSM are indicated by purple and brown arrows, respectively. The shared direct input from Ebf to Mrf in both ASM and OSM is indicated by the solid green arrow. In grey, Notch signaling downstream of Mrf activation has been established in the ASM ( Razy-Krajka et al . 2014 ) as the mechanism for cells to choose between stemness and differentiation, but has not yet been tested in the OSM.

Techniques Used: Derivative Assay, Expressing, Activation Assay

Embryonic development of the trunk lateral cells (TLC) 5.5hpf to 8.5hpf. (A) Cartoon of divisions within the A7.6 lineage at 5.5hpf, 6hpf and 8.5hpf. Each cell is named according to the scheme developed by Conklin ( Conklin 1905 ). Boxes indicate the regions shown at each time-point in panels G-R. (B-F) Embryos electroporated with Hand-r > H2B:mCherry and MyT > unc76:YFP, which overlap exclusively in the A7.6 lineage. Note the faint expression of H2B:mCherry in two adjacent endoderm cells beginning at 5.5hpf, presumably derived from A7.5, the sister of A7.6, thus reflecting an occasional early onset of transgene expression in the mother A6.3. These A6.3-derived cells are marked with a Roman numeral “x” wherever they appear. This staining was accounted for in all observations, and did not interfere with our ability to identify A7.6-derived cells. Expression of MyT > GFP or of MyT mRNA in the a-line neural cells is marked with an “*”. (GR) Expression dynamics of Hand-related (G-K), MyT (L-P), or Ebf (Q,R) mRNA, with A7.6 lineage marked by MyT > H2B:mCherry. (S) Schematic diagram of all TLC divisions from 5hpf to 8.5hpf, with gene expression patterns mapped on to each cell and color-coded. Blue = hand-related; Red = MyT; Green = Ebf . Scale bars=25μm.
Figure Legend Snippet: Embryonic development of the trunk lateral cells (TLC) 5.5hpf to 8.5hpf. (A) Cartoon of divisions within the A7.6 lineage at 5.5hpf, 6hpf and 8.5hpf. Each cell is named according to the scheme developed by Conklin ( Conklin 1905 ). Boxes indicate the regions shown at each time-point in panels G-R. (B-F) Embryos electroporated with Hand-r > H2B:mCherry and MyT > unc76:YFP, which overlap exclusively in the A7.6 lineage. Note the faint expression of H2B:mCherry in two adjacent endoderm cells beginning at 5.5hpf, presumably derived from A7.5, the sister of A7.6, thus reflecting an occasional early onset of transgene expression in the mother A6.3. These A6.3-derived cells are marked with a Roman numeral “x” wherever they appear. This staining was accounted for in all observations, and did not interfere with our ability to identify A7.6-derived cells. Expression of MyT > GFP or of MyT mRNA in the a-line neural cells is marked with an “*”. (GR) Expression dynamics of Hand-related (G-K), MyT (L-P), or Ebf (Q,R) mRNA, with A7.6 lineage marked by MyT > H2B:mCherry. (S) Schematic diagram of all TLC divisions from 5hpf to 8.5hpf, with gene expression patterns mapped on to each cell and color-coded. Blue = hand-related; Red = MyT; Green = Ebf . Scale bars=25μm.

Techniques Used: Thin Layer Chromatography, Expressing, Derivative Assay, Staining

Effect of Ebf or Tbx1/10 gain-of-function on expression of Mrf (A-D) 24hpf larvae electroporated with LexO(A7.6) > > H2B:mCherry and LexAop > LacZ (A); LexAop > Ebf (B); LexAop > Tbx1/10 (C); or LexAop > Ebf; LexAop > Tbx1/10 (D). (E) Boxplot showing proportion of larvae in each condition in which we observed ectopic Mrf expression, with sample sizes indicated. n= 41 for LacZ; n=47 for Ebf; n=64 for Tbx1/10; n=84 for Ebf + Tbx1/10.
Figure Legend Snippet: Effect of Ebf or Tbx1/10 gain-of-function on expression of Mrf (A-D) 24hpf larvae electroporated with LexO(A7.6) > > H2B:mCherry and LexAop > LacZ (A); LexAop > Ebf (B); LexAop > Tbx1/10 (C); or LexAop > Ebf; LexAop > Tbx1/10 (D). (E) Boxplot showing proportion of larvae in each condition in which we observed ectopic Mrf expression, with sample sizes indicated. n= 41 for LacZ; n=47 for Ebf; n=64 for Tbx1/10; n=84 for Ebf + Tbx1/10.

Techniques Used: Expressing

Effects of Tbx1/10 gain-of-function and loss-of-function on expression of Ebf (B-E) and effect of Ebf gain-of-function and loss-of-function on Tbx1/10 at 16hpf (F-I). (A) Cartoon showing whole-embryonic context of the anterior TLC at 16hpf. (B-E) Close-up of anterior TLC population at 16hpf, electroporated with LexO(A7.6) > H2B:mCherry and LexAop > LacZ (B); LexAop > Tbx1/10 (C); LexAop > nls:Cas9:nls;U6 > sgControlF+E (D); or LexAop > nls:Cas9:nls;U6 > sgTbx1.303;U6 > sgTbx1.558 (E). (F-I) Close-up of anterior TLC in lóhpf embryos electroporated with LexO(A7.6) > H2B:mCherry and LexAop > LacZ (F); LexAop > Ebf (G); LexAop > nls:Cas9:nls;U6 > sgControlF+E (H); or LexAop > nls:Cas9:nls;U6 > sgTbx1.303;U6 > sgTbx1.558 (I). Scale bars = 10μm. Total n are pooled from two biological replicates of batch-electroporation of zygotes. n=61 for (B); n=62 for (C); n=77 for (D); n=66 for (E).
Figure Legend Snippet: Effects of Tbx1/10 gain-of-function and loss-of-function on expression of Ebf (B-E) and effect of Ebf gain-of-function and loss-of-function on Tbx1/10 at 16hpf (F-I). (A) Cartoon showing whole-embryonic context of the anterior TLC at 16hpf. (B-E) Close-up of anterior TLC population at 16hpf, electroporated with LexO(A7.6) > H2B:mCherry and LexAop > LacZ (B); LexAop > Tbx1/10 (C); LexAop > nls:Cas9:nls;U6 > sgControlF+E (D); or LexAop > nls:Cas9:nls;U6 > sgTbx1.303;U6 > sgTbx1.558 (E). (F-I) Close-up of anterior TLC in lóhpf embryos electroporated with LexO(A7.6) > H2B:mCherry and LexAop > LacZ (F); LexAop > Ebf (G); LexAop > nls:Cas9:nls;U6 > sgControlF+E (H); or LexAop > nls:Cas9:nls;U6 > sgTbx1.303;U6 > sgTbx1.558 (I). Scale bars = 10μm. Total n are pooled from two biological replicates of batch-electroporation of zygotes. n=61 for (B); n=62 for (C); n=77 for (D); n=66 for (E).

Techniques Used: Expressing, Thin Layer Chromatography, Electroporation

Embryonic development of the trunk lateral cells (TLC) 8.5hpf to 16hpf. First cartoon in A, and panels B and F are the same data shown in Fig. 2K and R, to emphasize continuity of A10.48 cell and to show the anterior TLC in the whole-embryo context. Panels B-K show only the anterior TLC, which are boxed in the cartoons in A. (B-K) Close-up of anterior TLC (descendants of A10.48/47 cell pair only) marked with LexO(A7.6) > > H2B:mCherry showing expression of Hand-r (B-E), Ebf (F-I) and Tbx1/10 (J, K) at 8.5hpf, lOhpf, 13hpf, and 16hpf. Arrowheads mark the A10.48 cell; large white arrows mark A11.96; small white arrows mark the daughters of A11.96, named A12.192/191. Scale bars = 25 μm. (E) Schematic diagram of cell divisions and gene expression in the anterior TLC 10-16hpf. Blue = hand-related; Green = Ebf; Red = Tbx1/10; lighter versions of each color indicates lower levels of gene expression.
Figure Legend Snippet: Embryonic development of the trunk lateral cells (TLC) 8.5hpf to 16hpf. First cartoon in A, and panels B and F are the same data shown in Fig. 2K and R, to emphasize continuity of A10.48 cell and to show the anterior TLC in the whole-embryo context. Panels B-K show only the anterior TLC, which are boxed in the cartoons in A. (B-K) Close-up of anterior TLC (descendants of A10.48/47 cell pair only) marked with LexO(A7.6) > > H2B:mCherry showing expression of Hand-r (B-E), Ebf (F-I) and Tbx1/10 (J, K) at 8.5hpf, lOhpf, 13hpf, and 16hpf. Arrowheads mark the A10.48 cell; large white arrows mark A11.96; small white arrows mark the daughters of A11.96, named A12.192/191. Scale bars = 25 μm. (E) Schematic diagram of cell divisions and gene expression in the anterior TLC 10-16hpf. Blue = hand-related; Green = Ebf; Red = Tbx1/10; lighter versions of each color indicates lower levels of gene expression.

Techniques Used: Thin Layer Chromatography, Expressing

Development of the OSM precursors after larval hatching. (A-D) Larvae electroporated with LexO(A7.6) > > H2B:mCherry;Isl > unc76:GFP showing initiation of Isl > unc76:GFP expression in the OSMP at 16hpf (A), followed by cell divisions, anterior migration of the OSMP, and ring formation around the stomodeum by 24hpf (B-D). Boxed regions in A-D are shown close-up in A’-D’. (E-H) Close-up of OSMP in 18-24hpf larvae labeled with LexO(A7.6) > > H2B:mCh and with Mrf (blue) and Orphan-bHLH-1 (green) mRNA revealed by in situ hybridization. (I) Complete schematic diagram of the development of the A7.6 lineage, from the time of its birth at 5hpf, until differentiation of the OSM around 24hpf, with gene expression dynamics labeled and mapped on clonally. (J) For comparison, a simplified schematic diagram of key steps in ASM development within the B7.5 lineage based on ( Davidson and Levine 2003 ; Stolfi et al . 2010 ; Wang et al . 2013 ).
Figure Legend Snippet: Development of the OSM precursors after larval hatching. (A-D) Larvae electroporated with LexO(A7.6) > > H2B:mCherry;Isl > unc76:GFP showing initiation of Isl > unc76:GFP expression in the OSMP at 16hpf (A), followed by cell divisions, anterior migration of the OSMP, and ring formation around the stomodeum by 24hpf (B-D). Boxed regions in A-D are shown close-up in A’-D’. (E-H) Close-up of OSMP in 18-24hpf larvae labeled with LexO(A7.6) > > H2B:mCh and with Mrf (blue) and Orphan-bHLH-1 (green) mRNA revealed by in situ hybridization. (I) Complete schematic diagram of the development of the A7.6 lineage, from the time of its birth at 5hpf, until differentiation of the OSM around 24hpf, with gene expression dynamics labeled and mapped on clonally. (J) For comparison, a simplified schematic diagram of key steps in ASM development within the B7.5 lineage based on ( Davidson and Levine 2003 ; Stolfi et al . 2010 ; Wang et al . 2013 ).

Techniques Used: Expressing, Migration, Labeling, In Situ Hybridization

The Gal80-repressible LexA/LexAop binary transgenic system efficiently and specifically labels the A7.6 blastomeric lineage of Ciona robusta . (A) Confocal image of 24hpf larva showing expression of Handr > H2B:mCh and Isl > unc76:GFP. OSM are mCherry+/GFP+ while ASM are mCherry-/GFP+. Endoderm is marked with “*”. (B) 24hpf larva expressing Hand-r > LHG;LexAop > H2B:mCherry and Isl > unc76:GFP, showing that Handr > LHG;LexAop > unc76:GFP fully recapitulates expression of Handr > H2B:mCherry. (C) Single confocal slice of 24hpf larva expressing Hand-r > LHG;LexAop > H2B:MCherry and the endodermal marker Nkx2-1 > CD4:GFP, showing that Hand-r > LHG;LexAop > H2B:mCherry is expressed throughout the endoderm. (D) Single confocal slice of 24hpf larva showing that expression of Nkx2-l > Gal80 abolishes expression of Hand-r > LHG;LexAop > H2B:mCherry in the endoderm only.
Figure Legend Snippet: The Gal80-repressible LexA/LexAop binary transgenic system efficiently and specifically labels the A7.6 blastomeric lineage of Ciona robusta . (A) Confocal image of 24hpf larva showing expression of Handr > H2B:mCh and Isl > unc76:GFP. OSM are mCherry+/GFP+ while ASM are mCherry-/GFP+. Endoderm is marked with “*”. (B) 24hpf larva expressing Hand-r > LHG;LexAop > H2B:mCherry and Isl > unc76:GFP, showing that Handr > LHG;LexAop > unc76:GFP fully recapitulates expression of Handr > H2B:mCherry. (C) Single confocal slice of 24hpf larva expressing Hand-r > LHG;LexAop > H2B:MCherry and the endodermal marker Nkx2-1 > CD4:GFP, showing that Hand-r > LHG;LexAop > H2B:mCherry is expressed throughout the endoderm. (D) Single confocal slice of 24hpf larva showing that expression of Nkx2-l > Gal80 abolishes expression of Hand-r > LHG;LexAop > H2B:mCherry in the endoderm only.

Techniques Used: Transgenic Assay, Expressing, Marker

Tissue-specific knockdown of Ebf or Tbx1/10 in the A7.6 lineage leads to loss of OSM. (A-C) 26hpf larvae electroporated with LexO(A7.6) > H2B:mCherry;LexAop > nls > Cas9:nls and U6 > sgControlF+E (A), U6 > sgTbx1.303;U6 > sgTbx1.558 (B), or U6 > sgEbf.774 (C). (D) Boxplot showing the proportion of larvae in which LexO(A7.6) > H2B:mCherry and Mrf mRNA were both expressed in the OSM, with sample sizes indicated. The total n are pooled from two biological replicates of batch-electroporation of zygotes: n=37 for sgControl; n=77 for sgEbf; n=43 for sgTbx1/10. Scale bars = 25μm.
Figure Legend Snippet: Tissue-specific knockdown of Ebf or Tbx1/10 in the A7.6 lineage leads to loss of OSM. (A-C) 26hpf larvae electroporated with LexO(A7.6) > H2B:mCherry;LexAop > nls > Cas9:nls and U6 > sgControlF+E (A), U6 > sgTbx1.303;U6 > sgTbx1.558 (B), or U6 > sgEbf.774 (C). (D) Boxplot showing the proportion of larvae in which LexO(A7.6) > H2B:mCherry and Mrf mRNA were both expressed in the OSM, with sample sizes indicated. The total n are pooled from two biological replicates of batch-electroporation of zygotes: n=37 for sgControl; n=77 for sgEbf; n=43 for sgTbx1/10. Scale bars = 25μm.

Techniques Used: Electroporation

20) Product Images from "GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence"

Article Title: GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence

Journal: Scientific Reports

doi: 10.1038/s41598-021-04127-5

In vivo tumour growth dynamics in GFAP-modulated tumours. ( a ) Schematic overview of the experimental setup. U251-MG GFAP-modulated cell clones expressing H2B-mNeonGreen were implanted in the brain of NSG mice under a CIW. Time-lapse intravital imaging was performed through a CIW to study the tumour growth dynamics of each tumour type. ( b ) Representative 3D reconstructed tile-scans showing distinct tumours generated by different GFAP-modulated clones. Two clones engineered with different CRISPR-Cas9 sgRNAs are presented (CTL1, GFAPδ-KO2 and GFAPα-KO2 from CRISPR set A and CTL3, GFAPδ-KO3 and GFAPα-KO4 from CRISPR set B). Scale bar = 500 µm ( c ) Quantification of tumour density for each indicated tumour type. n = 6 (CTLs), n = 5 (GFAPδ-KO), and n = 6 (GFAPα-KO) mice. All tumours were imaged when they had filled half the imaging window (endpoint), which was between 13 and 35 days after the cranial window implantion, except for one CTL1 tumour which reached endpoint after 72 days. Black dots represent clones from CRISPR set A (CTL1, GFAPδ-KO2, GFAPα-KO2) and white dots represent clones from CRISPR set B (CTL3, GFAPδ-KO3, GFAPα-KO4) .The data is shown as mean ± S.E.M, *p
Figure Legend Snippet: In vivo tumour growth dynamics in GFAP-modulated tumours. ( a ) Schematic overview of the experimental setup. U251-MG GFAP-modulated cell clones expressing H2B-mNeonGreen were implanted in the brain of NSG mice under a CIW. Time-lapse intravital imaging was performed through a CIW to study the tumour growth dynamics of each tumour type. ( b ) Representative 3D reconstructed tile-scans showing distinct tumours generated by different GFAP-modulated clones. Two clones engineered with different CRISPR-Cas9 sgRNAs are presented (CTL1, GFAPδ-KO2 and GFAPα-KO2 from CRISPR set A and CTL3, GFAPδ-KO3 and GFAPα-KO4 from CRISPR set B). Scale bar = 500 µm ( c ) Quantification of tumour density for each indicated tumour type. n = 6 (CTLs), n = 5 (GFAPδ-KO), and n = 6 (GFAPα-KO) mice. All tumours were imaged when they had filled half the imaging window (endpoint), which was between 13 and 35 days after the cranial window implantion, except for one CTL1 tumour which reached endpoint after 72 days. Black dots represent clones from CRISPR set A (CTL1, GFAPδ-KO2, GFAPα-KO2) and white dots represent clones from CRISPR set B (CTL3, GFAPδ-KO3, GFAPα-KO4) .The data is shown as mean ± S.E.M, *p

Techniques Used: In Vivo, Clone Assay, Expressing, Mouse Assay, Imaging, Generated, CRISPR

Modification of GFAP isoform expression affects macroscopic growth patterns in organotypic brain slice cultures. ( a ) Schematic of experimental set-up: H2B-mNeonGreen expressing control (CTL), GFAPδ-KO and GFAPα-KO cell clones are injected in organotypic brain slices together with an H2B-mCherry expressing internal control (I-CTL) and co-cultured for one week. After fixation, whole-mount immunofluorescent staining, and clearing, confocal images are used to create a 3D reconstruction of the invasion patterns. ( b ) Representative image of I-CTL1 (magenta) and CTL1 (green) cells within the organotypic brain slice model. Invading cells are mainly found around the mouse brain vasculature (laminin, cyan). Laminin deposits in the tumour core can be used to distinguish stationary cells from cells invading the tissue, indicated with the orange dotted line. ( c ) Schematic depicting the method used to quantify the distribution of nuclei in the organotypic brain slices. ( d ) Distribution of nuclei of all I-CTL and CTL cells in the organotypic brain slices (n = 16 independent experiments, 4 different clones). ( e ) Representative images of invasion pattern of GFAPδ-KO clone 1 and I-CTL 1. ( f ) Distribution of nuclei of all GFAPδ-KO and I-CTL cells in the organotypic brain slices (n = 18 independent experiments, 4 different clones). ( g ) Representative image of the invasion pattern of GFAPα-KO clone 2 and I-CTL 1. ( h ) Distribution of nuclei of all GFAPα-KO cells and I-CTL cells in the organotypic brain slices (n = 20 independent experiments, 4 different clones). ( i ) Quantification of the percentage of invaded cells per condition, n = 16 (CTLs), n = 18 (GFAPδ-KO), and n = 20 (GFAPα-KO) injected organotypic brain slices derived from 4 different clones (CRISPR set A and B) per condition. Significance was determined using a two-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 100 µm. The data is shown as mean ± S.E.M, *p
Figure Legend Snippet: Modification of GFAP isoform expression affects macroscopic growth patterns in organotypic brain slice cultures. ( a ) Schematic of experimental set-up: H2B-mNeonGreen expressing control (CTL), GFAPδ-KO and GFAPα-KO cell clones are injected in organotypic brain slices together with an H2B-mCherry expressing internal control (I-CTL) and co-cultured for one week. After fixation, whole-mount immunofluorescent staining, and clearing, confocal images are used to create a 3D reconstruction of the invasion patterns. ( b ) Representative image of I-CTL1 (magenta) and CTL1 (green) cells within the organotypic brain slice model. Invading cells are mainly found around the mouse brain vasculature (laminin, cyan). Laminin deposits in the tumour core can be used to distinguish stationary cells from cells invading the tissue, indicated with the orange dotted line. ( c ) Schematic depicting the method used to quantify the distribution of nuclei in the organotypic brain slices. ( d ) Distribution of nuclei of all I-CTL and CTL cells in the organotypic brain slices (n = 16 independent experiments, 4 different clones). ( e ) Representative images of invasion pattern of GFAPδ-KO clone 1 and I-CTL 1. ( f ) Distribution of nuclei of all GFAPδ-KO and I-CTL cells in the organotypic brain slices (n = 18 independent experiments, 4 different clones). ( g ) Representative image of the invasion pattern of GFAPα-KO clone 2 and I-CTL 1. ( h ) Distribution of nuclei of all GFAPα-KO cells and I-CTL cells in the organotypic brain slices (n = 20 independent experiments, 4 different clones). ( i ) Quantification of the percentage of invaded cells per condition, n = 16 (CTLs), n = 18 (GFAPδ-KO), and n = 20 (GFAPα-KO) injected organotypic brain slices derived from 4 different clones (CRISPR set A and B) per condition. Significance was determined using a two-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 100 µm. The data is shown as mean ± S.E.M, *p

Techniques Used: Modification, Expressing, Slice Preparation, Clone Assay, Injection, Cell Culture, Staining, Derivative Assay, CRISPR

21) Product Images from "Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies"

Article Title: Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

Journal: bioRxiv

doi: 10.1101/2021.04.16.440240

Test of nanobodies concentration gradient. Lysates from S2R+ cells transfected with H2B-mCherry-VHH05 or H2B-mCherry-127D01 were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA with a concentration gradient from 1:100, 1:1000, 1:10000, 1:100000, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody followed by anti-Mouse IgG HRP. Two conditions of long-time exposure (60 s) and short time exposure (20 s) were set for signal reading.
Figure Legend Snippet: Test of nanobodies concentration gradient. Lysates from S2R+ cells transfected with H2B-mCherry-VHH05 or H2B-mCherry-127D01 were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA with a concentration gradient from 1:100, 1:1000, 1:10000, 1:100000, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody followed by anti-Mouse IgG HRP. Two conditions of long-time exposure (60 s) and short time exposure (20 s) were set for signal reading.

Techniques Used: Concentration Assay, Transfection, SDS Page, Western Blot

Schematic representation of the constructs and confocal images in S2R+ cells. (A and B) Transcriptional elements promoter and SV40 of the expression vectors, the different protein coding modules are represented as colored filled boxes. (C and D) Individual transfection results of VHH05 and 127D01 vectors in S2R+ cells. Confocal images show the distribution of fluorescent proteins in the cell. (E) Confocal images of co-transfection of NbVHH05-GFP with different cell compartments mCherry-VHH05 vectors showed the co-localization signal of GFP and mCherry. (F) Confocal images of co-transfection of Nb127D01-GFP with different cell compartments. mCherry-127D01 vectors showed the co-localization signal of GFP and mCherry. (G) Vectors information of NbVHH05-mCherry and mito-GFP-VHH05. Confocal images of co-transfection ofthese two vectors showed the co-localization signal of GFP and mCherry. (H) Vector information of Nb127D01-mCherry and mito-GFP-127D01. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry.
Figure Legend Snippet: Schematic representation of the constructs and confocal images in S2R+ cells. (A and B) Transcriptional elements promoter and SV40 of the expression vectors, the different protein coding modules are represented as colored filled boxes. (C and D) Individual transfection results of VHH05 and 127D01 vectors in S2R+ cells. Confocal images show the distribution of fluorescent proteins in the cell. (E) Confocal images of co-transfection of NbVHH05-GFP with different cell compartments mCherry-VHH05 vectors showed the co-localization signal of GFP and mCherry. (F) Confocal images of co-transfection of Nb127D01-GFP with different cell compartments. mCherry-127D01 vectors showed the co-localization signal of GFP and mCherry. (G) Vectors information of NbVHH05-mCherry and mito-GFP-VHH05. Confocal images of co-transfection ofthese two vectors showed the co-localization signal of GFP and mCherry. (H) Vector information of Nb127D01-mCherry and mito-GFP-127D01. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry.

Techniques Used: Construct, Expressing, Transfection, Cotransfection, Plasmid Preparation

Nanobody-based system for altering localization of NanoTagged proteins. (A and B) Diagram showing the vectors used for the secreted protein trapping method. NbVHH05/Nb127D01 fused to mCherry contains KDEL and BiP signal peptide and is driven by the actin5C promoter. (C) Four independent cell transfection experiments were performed. In 1 and 3, only GFP-VHH05 or GFP-127D01 was transfected. In 2 and 4, NbVHH05-mCherry-KDEL with GFP-VHH05, or Nb127D01-mCherry-KDEL with GFP-127D01, were co-transfected. Images show the GFP and mCherry signal 48 hours after transfection. Nuclei are stained with DAPI. (D) Immunoblots of GFP and tubulin in cell lysates from transfections 1-4. (E and F) Diagram showing the vectors used for cytoplasmic protein trapping. NbVHH05/Nb127D01 is fused to GFP and CD8, and driven by actin5C promoter. Target proteins are mCherry containing VHH05 or 127D01 at the C-terminus and mito signal at the N-terminus. (G) Results of co-transfection of CD8-NbVHH05-GFP/mito-mCherry-VHH05 and CD8-Nb127D01-GFP/mito-mCherry-127D01 in S2R+ cells. DAPI, GFP, mCherry and merge channels show protein expression levels with antibody stainings.
Figure Legend Snippet: Nanobody-based system for altering localization of NanoTagged proteins. (A and B) Diagram showing the vectors used for the secreted protein trapping method. NbVHH05/Nb127D01 fused to mCherry contains KDEL and BiP signal peptide and is driven by the actin5C promoter. (C) Four independent cell transfection experiments were performed. In 1 and 3, only GFP-VHH05 or GFP-127D01 was transfected. In 2 and 4, NbVHH05-mCherry-KDEL with GFP-VHH05, or Nb127D01-mCherry-KDEL with GFP-127D01, were co-transfected. Images show the GFP and mCherry signal 48 hours after transfection. Nuclei are stained with DAPI. (D) Immunoblots of GFP and tubulin in cell lysates from transfections 1-4. (E and F) Diagram showing the vectors used for cytoplasmic protein trapping. NbVHH05/Nb127D01 is fused to GFP and CD8, and driven by actin5C promoter. Target proteins are mCherry containing VHH05 or 127D01 at the C-terminus and mito signal at the N-terminus. (G) Results of co-transfection of CD8-NbVHH05-GFP/mito-mCherry-VHH05 and CD8-Nb127D01-GFP/mito-mCherry-127D01 in S2R+ cells. DAPI, GFP, mCherry and merge channels show protein expression levels with antibody stainings.

Techniques Used: Transfection, Staining, Western Blot, Cotransfection, Expressing

Test of potential interaction between VHH05 and 127D01. (A) Fluorescence confocal results showed no co-localization signal. Co-transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2R+ cells. (B) Western blots indicate no cross interaction between the two systems. Lysates from S2R+ cells transfected with different types tagged vectors (as in Figure 1 ) or a mock control plasmid were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-Mouse IgG HRP.
Figure Legend Snippet: Test of potential interaction between VHH05 and 127D01. (A) Fluorescence confocal results showed no co-localization signal. Co-transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2R+ cells. (B) Western blots indicate no cross interaction between the two systems. Lysates from S2R+ cells transfected with different types tagged vectors (as in Figure 1 ) or a mock control plasmid were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-Mouse IgG HRP.

Techniques Used: Fluorescence, Cotransfection, Western Blot, Transfection, Plasmid Preparation, SDS Page

VHH05 and 127D01 NanoTag sequences and their corresponding nanobodies, and use of nanobodies as chromobodies. (A and C) VHH05 and 127D01 were inserted at the N-terminus, internally or at the C-terminus of a protein of interest (POI). GSG denotes the linker, M is the start codon, and Stop is the stop codon. (B and D) Nanobody sequences of NbVHH05 and Nb127D01. Bolded and underlined CDR1-3 correspond to complementarity-determining regions (CDRs). (E) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-VHH05-H2B-mCherry into S2R+ cells. H2B is a nuclear protein. (F) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-CD8-VHH05-mCherry into S2R+ cells. CD8 is a cell membrane protein (G) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-mito-mCherry-VHH05 into S2R+ cells. Mito-mCherry-VHH05 contains a localization signal peptide for mitochondrial outer membrane targeting. (H, I and J) Experiments are as in E, F and G, except that pAW-actin5C-Nb127D01-GFP and pAW-actin5C-127D01-H2B-mCherry were co-transfected.
Figure Legend Snippet: VHH05 and 127D01 NanoTag sequences and their corresponding nanobodies, and use of nanobodies as chromobodies. (A and C) VHH05 and 127D01 were inserted at the N-terminus, internally or at the C-terminus of a protein of interest (POI). GSG denotes the linker, M is the start codon, and Stop is the stop codon. (B and D) Nanobody sequences of NbVHH05 and Nb127D01. Bolded and underlined CDR1-3 correspond to complementarity-determining regions (CDRs). (E) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-VHH05-H2B-mCherry into S2R+ cells. H2B is a nuclear protein. (F) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-CD8-VHH05-mCherry into S2R+ cells. CD8 is a cell membrane protein (G) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-mito-mCherry-VHH05 into S2R+ cells. Mito-mCherry-VHH05 contains a localization signal peptide for mitochondrial outer membrane targeting. (H, I and J) Experiments are as in E, F and G, except that pAW-actin5C-Nb127D01-GFP and pAW-actin5C-127D01-H2B-mCherry were co-transfected.

Techniques Used: Cotransfection, Transfection

22) Product Images from "Transfer of disrupted-in-schizophrenia 1 aggregates between neuronal-like cells occurs in tunnelling nanotubes and is promoted by dopamine"

Article Title: Transfer of disrupted-in-schizophrenia 1 aggregates between neuronal-like cells occurs in tunnelling nanotubes and is promoted by dopamine

Journal: Open Biology

doi: 10.1098/rsob.160328

DISC1 aggregates can transfer between neuronal cells through TNTs and the transfer is influenced by TNT formation. ( a ) Representative confocal images of GFP-DISC1 transfected cells (in green) co-cultured with H2B-mCherry transfected cells (nuclei in red). Plasma membrane was stained using WGA-rhodamine (in red). Three-dimensional reconstructions (insets) show the TNTs connecting two cells that contain several DISC1 aggregates (green). Scale bars, 10 µm. ( b ) Representative confocal images of CAD cells co-transfected with GFP-DISC1 and with RFP-Myo10 or RFP-VASP (donor cells) co-cultured with H2B-mCherry transfected cells (acceptor cells). ( c ) Percentage of acceptor cells containing GFP-DISC1 aggregates in ( b ) from three independent experiments. (* p
Figure Legend Snippet: DISC1 aggregates can transfer between neuronal cells through TNTs and the transfer is influenced by TNT formation. ( a ) Representative confocal images of GFP-DISC1 transfected cells (in green) co-cultured with H2B-mCherry transfected cells (nuclei in red). Plasma membrane was stained using WGA-rhodamine (in red). Three-dimensional reconstructions (insets) show the TNTs connecting two cells that contain several DISC1 aggregates (green). Scale bars, 10 µm. ( b ) Representative confocal images of CAD cells co-transfected with GFP-DISC1 and with RFP-Myo10 or RFP-VASP (donor cells) co-cultured with H2B-mCherry transfected cells (acceptor cells). ( c ) Percentage of acceptor cells containing GFP-DISC1 aggregates in ( b ) from three independent experiments. (* p

Techniques Used: Transfection, Cell Culture, Staining, Whole Genome Amplification

Transfer of DISC1 aggregates between neuronal cells is cell-contact dependent. ( a ) Schematic depicting the co-culture system used to quantify transfer of DISC1 aggregates using flow cytometry. Neuronal donor CAD cells were transfected with GFP-DISC1 for 12 h then co-cultured with acceptor H2B-mCherry transfected cells for 15 h. Cells were then analysed by flow cytometry to quantify the number of double positive cells (i.e. rate of transfer). ( b ) Percentage of double positive cells scored by flow cytometry after three independent co-culture experiments described in ( a ) in control, co-culture, supernatant and filter conditions (see Material and methods). In the co-culture conditions 2.5% of double positive cells were scored, whereas when cell contact was abolished (filter and supernatant conditions) the rate of transfer was drastically decreased. Data show mean ± s.e.m (** p
Figure Legend Snippet: Transfer of DISC1 aggregates between neuronal cells is cell-contact dependent. ( a ) Schematic depicting the co-culture system used to quantify transfer of DISC1 aggregates using flow cytometry. Neuronal donor CAD cells were transfected with GFP-DISC1 for 12 h then co-cultured with acceptor H2B-mCherry transfected cells for 15 h. Cells were then analysed by flow cytometry to quantify the number of double positive cells (i.e. rate of transfer). ( b ) Percentage of double positive cells scored by flow cytometry after three independent co-culture experiments described in ( a ) in control, co-culture, supernatant and filter conditions (see Material and methods). In the co-culture conditions 2.5% of double positive cells were scored, whereas when cell contact was abolished (filter and supernatant conditions) the rate of transfer was drastically decreased. Data show mean ± s.e.m (** p

Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry, Transfection, Cell Culture

23) Product Images from "PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules"

Article Title: PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules

Journal: bioRxiv

doi: 10.1101/2022.01.04.474939

Depletion of PLEKHA5 delays mitotic progression in asynchronous HeLa cells. (A–B) Mitotic progression analysis in asynchronous HeLa cells by time-lapse, live-cell imaging of H2B-mCherry. Asynchronous HeLa cells stably expressing H2B-mCherry were transfected with an siRNA targeting PLEKHA5 or a control siRNA. Thirty h after transfection, cells were imaged at 4-min intervals for 10–12 h. (A) Still images from a time-lapse movie of the nucleus (H2B-mCherry) in asynchronous HeLa cells. The time from nuclear envelope breakdown (NEBD) to anaphase onset (chromosomal segregation) allows for quantitation of mitosis progression in single cells. (B) Quantification of mitosis progression in asynchronous HeLa cells, with time from NEBD to anaphase plotted as cumulative frequency (left) and scatter plot (right) (n=50–51 cells). (C–D) G2/M transition analysis in asynchronous HeLa cells by time-lapse, live-cell imaging of H2B-mCherry and EGFP-PCNA. Asynchronous HeLa cells stably expressing H2B-mCherry and EGFP-PCNA were transfected with siPLEKHA5 or a control siRNA. Thirty h after transfection, cells were imaged at 10-min intervals for 30–32 h. (C) Stills from a time-lapse movie of the nuclear region in a single cell. The time between disappearance of PCNA puncta and NEBD allows for quantitation of delays in the G2/M transition in single cells. (D) Quantification of G2/M transition in asynchronous HeLa cells, with time from PCNA foci dissolution to NEBD plotted as cumulative frequency (left) and scatter plot (right) (n=69–70 cells). Mann-Whitney U test: n.s. not significant; ** p
Figure Legend Snippet: Depletion of PLEKHA5 delays mitotic progression in asynchronous HeLa cells. (A–B) Mitotic progression analysis in asynchronous HeLa cells by time-lapse, live-cell imaging of H2B-mCherry. Asynchronous HeLa cells stably expressing H2B-mCherry were transfected with an siRNA targeting PLEKHA5 or a control siRNA. Thirty h after transfection, cells were imaged at 4-min intervals for 10–12 h. (A) Still images from a time-lapse movie of the nucleus (H2B-mCherry) in asynchronous HeLa cells. The time from nuclear envelope breakdown (NEBD) to anaphase onset (chromosomal segregation) allows for quantitation of mitosis progression in single cells. (B) Quantification of mitosis progression in asynchronous HeLa cells, with time from NEBD to anaphase plotted as cumulative frequency (left) and scatter plot (right) (n=50–51 cells). (C–D) G2/M transition analysis in asynchronous HeLa cells by time-lapse, live-cell imaging of H2B-mCherry and EGFP-PCNA. Asynchronous HeLa cells stably expressing H2B-mCherry and EGFP-PCNA were transfected with siPLEKHA5 or a control siRNA. Thirty h after transfection, cells were imaged at 10-min intervals for 30–32 h. (C) Stills from a time-lapse movie of the nuclear region in a single cell. The time between disappearance of PCNA puncta and NEBD allows for quantitation of delays in the G2/M transition in single cells. (D) Quantification of G2/M transition in asynchronous HeLa cells, with time from PCNA foci dissolution to NEBD plotted as cumulative frequency (left) and scatter plot (right) (n=69–70 cells). Mann-Whitney U test: n.s. not significant; ** p

Techniques Used: Live Cell Imaging, Stable Transfection, Expressing, Transfection, Quantitation Assay, MANN-WHITNEY

24) Product Images from "Opposing roles for JNK and Aurora A in regulating the association of WDR62 with spindle microtubules"

Article Title: Opposing roles for JNK and Aurora A in regulating the association of WDR62 with spindle microtubules

Journal: Journal of Cell Science

doi: 10.1242/jcs.157537

WDR62 associates with microtubules during interphase and mitosis. (A) WDR62–mCherry and tubulin–GFP were coexpressed in AD293 cells and their association with astral microtubules during mitotic entry was revealed by live-cell fluorescence
Figure Legend Snippet: WDR62 associates with microtubules during interphase and mitosis. (A) WDR62–mCherry and tubulin–GFP were coexpressed in AD293 cells and their association with astral microtubules during mitotic entry was revealed by live-cell fluorescence

Techniques Used: Fluorescence

WDR62 recruits JNK1 to the spindle pole. (A) GFP-tagged JNK1 or JNK2 were coexpressed with mCherry-tagged WDR62 or histone 2B (H2B), as a negative control. The cytoplasmic or nuclear localization of JNK isoforms was evaluated in interphase. (B) Nuclear
Figure Legend Snippet: WDR62 recruits JNK1 to the spindle pole. (A) GFP-tagged JNK1 or JNK2 were coexpressed with mCherry-tagged WDR62 or histone 2B (H2B), as a negative control. The cytoplasmic or nuclear localization of JNK isoforms was evaluated in interphase. (B) Nuclear

Techniques Used: Negative Control

25) Product Images from "Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies"

Article Title: Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

Journal: eLife

doi: 10.7554/eLife.74326

Test of potential interaction between VHH05 and 127D01. ( A ) Fluorescence confocal results showed no co-localization signal. Co-transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2 R + cells. ( B ) Western blots indicate no cross-interaction between the two systems. Lysates from S2 R + cells transfected with different types tagged vectors (as in Figure 1 ) or a mock control plasmid were analyzed by SDS-PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-mouse IgG HRP. Raw data of Western blot for Figure 2—figure supplement 3b .
Figure Legend Snippet: Test of potential interaction between VHH05 and 127D01. ( A ) Fluorescence confocal results showed no co-localization signal. Co-transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2 R + cells. ( B ) Western blots indicate no cross-interaction between the two systems. Lysates from S2 R + cells transfected with different types tagged vectors (as in Figure 1 ) or a mock control plasmid were analyzed by SDS-PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-mouse IgG HRP. Raw data of Western blot for Figure 2—figure supplement 3b .

Techniques Used: Fluorescence, Cotransfection, Western Blot, Transfection, Plasmid Preparation, SDS Page

Nanobody-based system for altering localization of NanoTagged proteins. ( A and B ) Diagram showing the vectors used for cytoplasmic protein trapping. NbVHH05/Nb127D01 is fused to GFP and CD8, and driven by actin5C promoter. Target proteins are mCherry containing VHH05-tag or 127D01-tag at the C-terminus and mito signal at the N-terminus. ( C ) Results of transfection of mito-mCherry-NanoTag or co-transfection of CD8-NbVHH05-GFP/mito-mCherry-VHH05 and CD8-Nb127D01-GFP/mito-mCherry-127D01 in S2 R + cells. GFP, mCherry, and merged channels show protein expression levels with antibody staining.
Figure Legend Snippet: Nanobody-based system for altering localization of NanoTagged proteins. ( A and B ) Diagram showing the vectors used for cytoplasmic protein trapping. NbVHH05/Nb127D01 is fused to GFP and CD8, and driven by actin5C promoter. Target proteins are mCherry containing VHH05-tag or 127D01-tag at the C-terminus and mito signal at the N-terminus. ( C ) Results of transfection of mito-mCherry-NanoTag or co-transfection of CD8-NbVHH05-GFP/mito-mCherry-VHH05 and CD8-Nb127D01-GFP/mito-mCherry-127D01 in S2 R + cells. GFP, mCherry, and merged channels show protein expression levels with antibody staining.

Techniques Used: Transfection, Cotransfection, Expressing, Staining

Test of nanobody concentration gradient. Lysates from S2 R + cells transfected with H2B-mCherry-VHH05 or H2B-mCherry-127D01 were analyzed by SDS-PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA with a concentration gradient from 1:100, 1:1000, 1:10,000, 1:100,000, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody followed by anti-mouse IgG HRP. Two conditions of long-time exposure (60 s) and short-time exposure (20 s) were set for signal reading. Raw data of Western blot for Figure 3—figure supplement 1 .
Figure Legend Snippet: Test of nanobody concentration gradient. Lysates from S2 R + cells transfected with H2B-mCherry-VHH05 or H2B-mCherry-127D01 were analyzed by SDS-PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA with a concentration gradient from 1:100, 1:1000, 1:10,000, 1:100,000, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody followed by anti-mouse IgG HRP. Two conditions of long-time exposure (60 s) and short-time exposure (20 s) were set for signal reading. Raw data of Western blot for Figure 3—figure supplement 1 .

Techniques Used: Concentration Assay, Transfection, SDS Page, Western Blot

Schematic representation of the constructs and confocal images in S2R+ cells. ( A and B ) Transcriptional elements promoter and SV40 of the expression vectors, the different protein coding modules are represented as colored filled boxes. ( C and D ) Individual transfection results of VHH05 and 127D01 vectors in S2R+ cells. Confocal images show the distribution of fluorescent proteins in the cell. DAPI staining shows the nuclei. ( E ) Confocal images of co-transfection of NbVHH05-GFP with different cell compartments mCherry-VHH05 vectors showed the co-localization signal of GFP and mCherry. ( F ) Confocal images of co-transfection of Nb127D01-GFP with different cell compartments. mCherry-127D01 vectors showed the co-localization signal of GFP and mCherry. ( G ) Vectors information of NbVHH05-mCherry and mito-GFP-VHH05. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry. ( H ) Vector information of Nb127D01-mCherry and mito-GFP-127D01. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry. Scale bars: 25 μm.
Figure Legend Snippet: Schematic representation of the constructs and confocal images in S2R+ cells. ( A and B ) Transcriptional elements promoter and SV40 of the expression vectors, the different protein coding modules are represented as colored filled boxes. ( C and D ) Individual transfection results of VHH05 and 127D01 vectors in S2R+ cells. Confocal images show the distribution of fluorescent proteins in the cell. DAPI staining shows the nuclei. ( E ) Confocal images of co-transfection of NbVHH05-GFP with different cell compartments mCherry-VHH05 vectors showed the co-localization signal of GFP and mCherry. ( F ) Confocal images of co-transfection of Nb127D01-GFP with different cell compartments. mCherry-127D01 vectors showed the co-localization signal of GFP and mCherry. ( G ) Vectors information of NbVHH05-mCherry and mito-GFP-VHH05. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry. ( H ) Vector information of Nb127D01-mCherry and mito-GFP-127D01. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry. Scale bars: 25 μm.

Techniques Used: Construct, Expressing, Transfection, Staining, Cotransfection, Plasmid Preparation

26) Product Images from "Absolute quantification of cohesin, CTCF and their regulators in human cells"

Article Title: Absolute quantification of cohesin, CTCF and their regulators in human cells

Journal: bioRxiv

doi: 10.1101/560425

Fluorescence correlation spectroscopy of cohesin subunits and regulators. FCS measurements to estimate the concentration of endogenously GFP-tagged proteins in the nucleus/chromatin and cytoplasm of G1, G2 and prometaphase cells. (A) FCS measurements were taken at different positions (3 in the nucleus/chromatin, 2 in the cytoplasm) in cells in G1 (top), G2 (middle) or prometaphase (bottom), whereby photon counts were recorded for 30 s at each position. Shown are example images for SCC1-EGFP H2B-mCherry in the GFP (left), DNA (middle) and transmission channels. Scale bar: 10 μm. (B) After data fitting and quality control, the GFP-based protein concentrations within the two cellular compartments were obtained. Shown are violin plots (solid lines) indicating the probability density of the protein concentrations determined from each FCS measurement (dots). Note that the EGFP-sororin cell line displayed a mitotic defect, raising the possibility that EGFP-sororin may be hypomorphic.
Figure Legend Snippet: Fluorescence correlation spectroscopy of cohesin subunits and regulators. FCS measurements to estimate the concentration of endogenously GFP-tagged proteins in the nucleus/chromatin and cytoplasm of G1, G2 and prometaphase cells. (A) FCS measurements were taken at different positions (3 in the nucleus/chromatin, 2 in the cytoplasm) in cells in G1 (top), G2 (middle) or prometaphase (bottom), whereby photon counts were recorded for 30 s at each position. Shown are example images for SCC1-EGFP H2B-mCherry in the GFP (left), DNA (middle) and transmission channels. Scale bar: 10 μm. (B) After data fitting and quality control, the GFP-based protein concentrations within the two cellular compartments were obtained. Shown are violin plots (solid lines) indicating the probability density of the protein concentrations determined from each FCS measurement (dots). Note that the EGFP-sororin cell line displayed a mitotic defect, raising the possibility that EGFP-sororin may be hypomorphic.

Techniques Used: Fluorescence, Spectroscopy, Concentration Assay, Transmission Assay

27) Product Images from "CoA synthase regulates mitotic fidelity via CBP-mediated acetylation"

Article Title: CoA synthase regulates mitotic fidelity via CBP-mediated acetylation

Journal: Nature Communications

doi: 10.1038/s41467-018-03422-6

COASY knockdown induced multinucleation and prolonged mitosis. a , b COASY knockdown by siRNA in MDA-MB231 triggered cobble-stone morphological change ( a ) and multinucleation ( b ). MDA-MB-231 cells transfected with control or COASY siRNA for 72 h were stained with DAPI (nuclei) and Alexa Fluor 488 phalloidin (F-actin). Scale bars, 50 mm ( a ), 20 mm ( b ). c The percentage of multi-nucleated A549 cells after transfection with control or two independent COASY siRNAs for 72 h. For each sample, more than 150 cells were examined by immunofluorescence microscopy. d Live cell time-lapse imaging showed that COASY knockdown extended mitosis and induced cytokinesis failure. A549 cells expressing histone 2B (H2B)-mCherry (nucleus marker) were transfected with control or COASY siRNA for 24 h before live cell imaging. Scale bars, 10 mm. e COASY knockdown increased the time in mitosis as determined by the time of nuclear envelope breakdown (NEBD) to anaphase onset (in min). For each sample, more than 35 cells were examined by live cell imaging. f COASY knockdown increased percentage of cytokinesis failure during mitosis. For each sample, more than 35 cells were examined by live cell imaging. g Western blots show that COASY knockdown extend the time of mitosis by using cyclin B1 expression as a mitotic marker. A549 cells were synchronized by thymidine-nocodazole block and released in fresh media. The samples were then harvested every 20 min and analyzed by western blots. Bars show standard error of the mean. ** p
Figure Legend Snippet: COASY knockdown induced multinucleation and prolonged mitosis. a , b COASY knockdown by siRNA in MDA-MB231 triggered cobble-stone morphological change ( a ) and multinucleation ( b ). MDA-MB-231 cells transfected with control or COASY siRNA for 72 h were stained with DAPI (nuclei) and Alexa Fluor 488 phalloidin (F-actin). Scale bars, 50 mm ( a ), 20 mm ( b ). c The percentage of multi-nucleated A549 cells after transfection with control or two independent COASY siRNAs for 72 h. For each sample, more than 150 cells were examined by immunofluorescence microscopy. d Live cell time-lapse imaging showed that COASY knockdown extended mitosis and induced cytokinesis failure. A549 cells expressing histone 2B (H2B)-mCherry (nucleus marker) were transfected with control or COASY siRNA for 24 h before live cell imaging. Scale bars, 10 mm. e COASY knockdown increased the time in mitosis as determined by the time of nuclear envelope breakdown (NEBD) to anaphase onset (in min). For each sample, more than 35 cells were examined by live cell imaging. f COASY knockdown increased percentage of cytokinesis failure during mitosis. For each sample, more than 35 cells were examined by live cell imaging. g Western blots show that COASY knockdown extend the time of mitosis by using cyclin B1 expression as a mitotic marker. A549 cells were synchronized by thymidine-nocodazole block and released in fresh media. The samples were then harvested every 20 min and analyzed by western blots. Bars show standard error of the mean. ** p

Techniques Used: Multiple Displacement Amplification, Transfection, Staining, Immunofluorescence, Microscopy, Imaging, Expressing, Marker, Live Cell Imaging, Western Blot, Blocking Assay

28) Product Images from "Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies"

Article Title: Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

Journal: bioRxiv

doi: 10.1101/2021.04.16.440240

Test of nanobody concentration gradient. Lysates from S2R+ cells transfected with H2B-mCherry-VHH05 or H2B-mCherry-127D01 were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA with a concentration gradient from 1:100, 1:1000, 1:10000, 1:100000, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody followed by anti-Mouse IgG HRP. Two conditions of long-time exposure (60 s) and short time exposure (20 s) were set for signal reading.
Figure Legend Snippet: Test of nanobody concentration gradient. Lysates from S2R+ cells transfected with H2B-mCherry-VHH05 or H2B-mCherry-127D01 were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA with a concentration gradient from 1:100, 1:1000, 1:10000, 1:100000, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody followed by anti-Mouse IgG HRP. Two conditions of long-time exposure (60 s) and short time exposure (20 s) were set for signal reading.

Techniques Used: Concentration Assay, Transfection, SDS Page, Western Blot

Nanobody-based system for altering localization of NanoTagged proteins. ( A and B ) Diagram showing the vectors used for cytoplasmic protein trapping. NbVHH05/Nb127D01 is fused to GFP and CD8, and driven by actin5C promoter. Target proteins are mCherry containing VHH05 or 127D01 at the C-terminus and mito signal at the N-terminus. ( C ) Results of transfection of mito- mCherry-NanoTag or co-transfection of CD8-NbVHH05-GFP/mito-mCherry-VHH05 and CD8-Nb127D01-GFP/mito-mCherry-127D01 in S2R+ cells. GFP, mCherry and merge channels show protein expression levels with antibody staining.
Figure Legend Snippet: Nanobody-based system for altering localization of NanoTagged proteins. ( A and B ) Diagram showing the vectors used for cytoplasmic protein trapping. NbVHH05/Nb127D01 is fused to GFP and CD8, and driven by actin5C promoter. Target proteins are mCherry containing VHH05 or 127D01 at the C-terminus and mito signal at the N-terminus. ( C ) Results of transfection of mito- mCherry-NanoTag or co-transfection of CD8-NbVHH05-GFP/mito-mCherry-VHH05 and CD8-Nb127D01-GFP/mito-mCherry-127D01 in S2R+ cells. GFP, mCherry and merge channels show protein expression levels with antibody staining.

Techniques Used: Transfection, Cotransfection, Expressing, Staining

VHH05 and 127D01 NanoTag sequences and their corresponding nanobodies, and use of nanobodies as chromobodies. ( A and C ) VHH05 and 127D01 were inserted at the N-terminus, internally or at the C-terminus of a protein of interest (POI). GSG denotes the linker, M is the start codon, and Stop is the stop codon. ( B and D ) Nanobody sequences of NbVHH05 and Nb127D01. Bolded and underlined CDR1-3 correspond to complementarity-determining regions (CDRs). ( E ) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-VHH05-H2B- mCherry into S2R+ cells. H2B is a nuclear protein. ( F ) Co-transfection of pAW- actin5C-NbVHH05-GFP and pAW-actin5C-CD8-VHH05-mCherry into S2R+ cells. CD8 is a cell membrane protein ( G ) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-mito-mCherry-VHH05 into S2R+ cells. Mito-mCherry-VHH05 contains a localization signal peptide for mitochondrial outer membrane targeting. ( H , I and J ) Experiments are as in E, F and G, except that pAW-actin5C-Nb127D01-GFP and pAW-actin5C-127D01-H2B-mCherry were co-transfected.
Figure Legend Snippet: VHH05 and 127D01 NanoTag sequences and their corresponding nanobodies, and use of nanobodies as chromobodies. ( A and C ) VHH05 and 127D01 were inserted at the N-terminus, internally or at the C-terminus of a protein of interest (POI). GSG denotes the linker, M is the start codon, and Stop is the stop codon. ( B and D ) Nanobody sequences of NbVHH05 and Nb127D01. Bolded and underlined CDR1-3 correspond to complementarity-determining regions (CDRs). ( E ) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-VHH05-H2B- mCherry into S2R+ cells. H2B is a nuclear protein. ( F ) Co-transfection of pAW- actin5C-NbVHH05-GFP and pAW-actin5C-CD8-VHH05-mCherry into S2R+ cells. CD8 is a cell membrane protein ( G ) Co-transfection of pAW-actin5C-NbVHH05-GFP and pAW-actin5C-mito-mCherry-VHH05 into S2R+ cells. Mito-mCherry-VHH05 contains a localization signal peptide for mitochondrial outer membrane targeting. ( H , I and J ) Experiments are as in E, F and G, except that pAW-actin5C-Nb127D01-GFP and pAW-actin5C-127D01-H2B-mCherry were co-transfected.

Techniques Used: Cotransfection, Transfection

Schematic representation of the constructs and confocal images in S2R+ cells. ( A and B ) Transcriptional elements promoter and SV40 of the expression vectors, the different protein coding modules are represented as colored filled boxes. ( C and D ) Individual transfection results of VHH05 and 127D01 vectors in S2R+ cells. Confocal images show the distribution of fluorescent proteins in the cell. ( E ) Confocal images of co-transfection of NbVHH05-GFP with different cell compartments mCherry-VHH05 vectors showed the co-localization signal of GFP and mCherry. ( F ) Confocal images of co-transfection of Nb127D01-GFP with different cell compartments. mCherry-127D01 vectors showed the co-localization signal of GFP and mCherry. ( G ) Vectors information of NbVHH05-mCherry and mito-GFP-VHH05. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry. ( H ) Vector information of Nb127D01-mCherry and mito- GFP-127D01. Confocal images of co-transfection of these two vectors showed the co- localization signal of GFP and mCherry.
Figure Legend Snippet: Schematic representation of the constructs and confocal images in S2R+ cells. ( A and B ) Transcriptional elements promoter and SV40 of the expression vectors, the different protein coding modules are represented as colored filled boxes. ( C and D ) Individual transfection results of VHH05 and 127D01 vectors in S2R+ cells. Confocal images show the distribution of fluorescent proteins in the cell. ( E ) Confocal images of co-transfection of NbVHH05-GFP with different cell compartments mCherry-VHH05 vectors showed the co-localization signal of GFP and mCherry. ( F ) Confocal images of co-transfection of Nb127D01-GFP with different cell compartments. mCherry-127D01 vectors showed the co-localization signal of GFP and mCherry. ( G ) Vectors information of NbVHH05-mCherry and mito-GFP-VHH05. Confocal images of co-transfection of these two vectors showed the co-localization signal of GFP and mCherry. ( H ) Vector information of Nb127D01-mCherry and mito- GFP-127D01. Confocal images of co-transfection of these two vectors showed the co- localization signal of GFP and mCherry.

Techniques Used: Construct, Expressing, Transfection, Cotransfection, Plasmid Preparation

Nanobody-based system for altering localization of NanoTagged proteins. ( A and B ) Diagram showing the vectors used for the secreted protein trapping method. NbVHH05/Nb127D01 fused to mCherry contains KDEL and BiP signal peptide and is driven by the actin5C promoter. ( C ) Four independent cell transfection experiments were performed. In 1 and 3, only GFP-VHH05 or GFP-127D01 was transfected. In 2 and 4, NbVHH05-mCherry-KDEL with GFP-VHH05, or Nb127D01- mCherry-KDEL with GFP-127D01, were co-transfected. Images show the GFP and mCherry signal 48 hours after transfection. Nuclei are stained with DAPI. ( D ) Immunoblots of GFP and tubulin in cell lysates from transfections 1-4.
Figure Legend Snippet: Nanobody-based system for altering localization of NanoTagged proteins. ( A and B ) Diagram showing the vectors used for the secreted protein trapping method. NbVHH05/Nb127D01 fused to mCherry contains KDEL and BiP signal peptide and is driven by the actin5C promoter. ( C ) Four independent cell transfection experiments were performed. In 1 and 3, only GFP-VHH05 or GFP-127D01 was transfected. In 2 and 4, NbVHH05-mCherry-KDEL with GFP-VHH05, or Nb127D01- mCherry-KDEL with GFP-127D01, were co-transfected. Images show the GFP and mCherry signal 48 hours after transfection. Nuclei are stained with DAPI. ( D ) Immunoblots of GFP and tubulin in cell lysates from transfections 1-4.

Techniques Used: Transfection, Staining, Western Blot

Test of potential interaction between VHH05 and 127D01. ( A ) Fluorescence confocal results showed no co-localization signal. Co- transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2R+ cells. ( B ) Western blots indicate no cross interaction between the two systems. Lysates from S2R+ cells transfected with different types tagged vectors (as in Figure 1 ) or a mock control plasmid were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-Mouse IgG HRP.
Figure Legend Snippet: Test of potential interaction between VHH05 and 127D01. ( A ) Fluorescence confocal results showed no co-localization signal. Co- transfection of Nb127D01-GFP and mito-mCherry-VHH05 or H2B-mCherry-VHH05, NbVHH05-GFP and mito-mCherry-127D01 or H2B-mCherry-127D01 in S2R+ cells. ( B ) Western blots indicate no cross interaction between the two systems. Lysates from S2R+ cells transfected with different types tagged vectors (as in Figure 1 ) or a mock control plasmid were analyzed by SDS–PAGE and western blotting. The blot was developed with NbVHH05-ALFA and Nb127D01-ALFA, followed by NbALFA-HRP or a mouse anti-tubulin primary antibody, and followed by anti-Mouse IgG HRP.

Techniques Used: Fluorescence, Cotransfection, Western Blot, Transfection, Plasmid Preparation, SDS Page

29) Product Images from "Heterologous calcium-dependent inactivation of Orai1 by neighboring TRPV1 channels modulates cell migration and wound healing"

Article Title: Heterologous calcium-dependent inactivation of Orai1 by neighboring TRPV1 channels modulates cell migration and wound healing

Journal: Communications Biology

doi: 10.1038/s42003-019-0338-1

Developing and testing the Orai1-GCaMP3 sensor. a Representative confocal images of the fluorescence obtained with Orai1-GCaMP3 (blue) and histone 2B fused to mCherry as nuclear marker (yellow) expressed in HEK293 cells obtained from at least 3 independent transfections. Left panels show the 3D ( x , y , and z axes) confocal projections from Orai1-GCaMP3 to illustrate its plasma membrane localization. Scale bar 5 µM. b Western blot analysis from HEK293 cells expressing the Orai1-GCaMP3 sensor and Orai1-GFP (representative blot from 3 independent blots). Notice the 55 kDa band corresponding to endogenous Orai1 and the 79 kDa band from Orai1-GFP and the Orai1-GCaMP3 sensor. c Cell population calcium measurements using a spectrofluorometer with HEK293 cells expressing Orai1-GFP + STIM1-DsRed (yellow) or Orai1-GCaMP3 + STIM1-DsRed (green) or measuring the endogenous SOCE (black). Measurements obtained from at least 5 independent transfections. Calcium increments induced by the application of thapsigargin (TG) in free extracellular calcium and SOCE measured after re-addition of 2 mM calcium to the extracellular solution. d Area under the curve (AUC) for fluorescence obtained after re-addition of 2 mM calcium (calcium influx) with endogenous Orai1 (gray), Orai1-GFP (yellow), and Orai1-GCaMP3 (green). e Representative confocal images of the puncta formation after TG application in cells expressing Orai1-GCaMP3 (blue) and STIM1-DsRed (yellow). Images shown before the application of thapsigargin (pre-TG) and after (post-TG) for the equatorial plane (middle) of the cell (EP) and the cortical plane (CP, the plane closest to the Petri dish bottom). Scale bar 5 µM. f Co-localization index (PC, Pearson’s correlation coefficient) for Orai1-GCaMP3 + STIM1-DsRed before and after TG. g Co-localization index (PCC) for Orai1-GFP + STIM1-DsRed before and after TG. In all cases, data shows the mean ± standard deviation from at least 6 independent transfections. Asterisks show p values of
Figure Legend Snippet: Developing and testing the Orai1-GCaMP3 sensor. a Representative confocal images of the fluorescence obtained with Orai1-GCaMP3 (blue) and histone 2B fused to mCherry as nuclear marker (yellow) expressed in HEK293 cells obtained from at least 3 independent transfections. Left panels show the 3D ( x , y , and z axes) confocal projections from Orai1-GCaMP3 to illustrate its plasma membrane localization. Scale bar 5 µM. b Western blot analysis from HEK293 cells expressing the Orai1-GCaMP3 sensor and Orai1-GFP (representative blot from 3 independent blots). Notice the 55 kDa band corresponding to endogenous Orai1 and the 79 kDa band from Orai1-GFP and the Orai1-GCaMP3 sensor. c Cell population calcium measurements using a spectrofluorometer with HEK293 cells expressing Orai1-GFP + STIM1-DsRed (yellow) or Orai1-GCaMP3 + STIM1-DsRed (green) or measuring the endogenous SOCE (black). Measurements obtained from at least 5 independent transfections. Calcium increments induced by the application of thapsigargin (TG) in free extracellular calcium and SOCE measured after re-addition of 2 mM calcium to the extracellular solution. d Area under the curve (AUC) for fluorescence obtained after re-addition of 2 mM calcium (calcium influx) with endogenous Orai1 (gray), Orai1-GFP (yellow), and Orai1-GCaMP3 (green). e Representative confocal images of the puncta formation after TG application in cells expressing Orai1-GCaMP3 (blue) and STIM1-DsRed (yellow). Images shown before the application of thapsigargin (pre-TG) and after (post-TG) for the equatorial plane (middle) of the cell (EP) and the cortical plane (CP, the plane closest to the Petri dish bottom). Scale bar 5 µM. f Co-localization index (PC, Pearson’s correlation coefficient) for Orai1-GCaMP3 + STIM1-DsRed before and after TG. g Co-localization index (PCC) for Orai1-GFP + STIM1-DsRed before and after TG. In all cases, data shows the mean ± standard deviation from at least 6 independent transfections. Asterisks show p values of

Techniques Used: Fluorescence, Marker, Transfection, Western Blot, Expressing, Periodic Counter-current Chromatography, Standard Deviation

30) Product Images from "Transfer of disrupted-in-schizophrenia 1 aggregates between neuronal-like cells occurs in tunnelling nanotubes and is promoted by dopamine"

Article Title: Transfer of disrupted-in-schizophrenia 1 aggregates between neuronal-like cells occurs in tunnelling nanotubes and is promoted by dopamine

Journal: Open Biology

doi: 10.1098/rsob.160328

DISC1 aggregates can transfer between neuronal cells through TNTs and the transfer is influenced by TNT formation. ( a ) Representative confocal images of GFP-DISC1 transfected cells (in green) co-cultured with H2B-mCherry transfected cells (nuclei in red). Plasma membrane was stained using WGA-rhodamine (in red). Three-dimensional reconstructions (insets) show the TNTs connecting two cells that contain several DISC1 aggregates (green). Scale bars, 10 µm. ( b ) Representative confocal images of CAD cells co-transfected with GFP-DISC1 and with RFP-Myo10 or RFP-VASP (donor cells) co-cultured with H2B-mCherry transfected cells (acceptor cells). ( c ) Percentage of acceptor cells containing GFP-DISC1 aggregates in ( b ) from three independent experiments. (* p
Figure Legend Snippet: DISC1 aggregates can transfer between neuronal cells through TNTs and the transfer is influenced by TNT formation. ( a ) Representative confocal images of GFP-DISC1 transfected cells (in green) co-cultured with H2B-mCherry transfected cells (nuclei in red). Plasma membrane was stained using WGA-rhodamine (in red). Three-dimensional reconstructions (insets) show the TNTs connecting two cells that contain several DISC1 aggregates (green). Scale bars, 10 µm. ( b ) Representative confocal images of CAD cells co-transfected with GFP-DISC1 and with RFP-Myo10 or RFP-VASP (donor cells) co-cultured with H2B-mCherry transfected cells (acceptor cells). ( c ) Percentage of acceptor cells containing GFP-DISC1 aggregates in ( b ) from three independent experiments. (* p

Techniques Used: Transfection, Cell Culture, Staining, Whole Genome Amplification

Transfer of DISC1 aggregates between neuronal cells is cell-contact dependent. ( a ) Schematic depicting the co-culture system used to quantify transfer of DISC1 aggregates using flow cytometry. Neuronal donor CAD cells were transfected with GFP-DISC1 for 12 h then co-cultured with acceptor H2B-mCherry transfected cells for 15 h. Cells were then analysed by flow cytometry to quantify the number of double positive cells (i.e. rate of transfer). ( b ) Percentage of double positive cells scored by flow cytometry after three independent co-culture experiments described in ( a ) in control, co-culture, supernatant and filter conditions (see Material and methods). In the co-culture conditions 2.5% of double positive cells were scored, whereas when cell contact was abolished (filter and supernatant conditions) the rate of transfer was drastically decreased. Data show mean ± s.e.m (** p
Figure Legend Snippet: Transfer of DISC1 aggregates between neuronal cells is cell-contact dependent. ( a ) Schematic depicting the co-culture system used to quantify transfer of DISC1 aggregates using flow cytometry. Neuronal donor CAD cells were transfected with GFP-DISC1 for 12 h then co-cultured with acceptor H2B-mCherry transfected cells for 15 h. Cells were then analysed by flow cytometry to quantify the number of double positive cells (i.e. rate of transfer). ( b ) Percentage of double positive cells scored by flow cytometry after three independent co-culture experiments described in ( a ) in control, co-culture, supernatant and filter conditions (see Material and methods). In the co-culture conditions 2.5% of double positive cells were scored, whereas when cell contact was abolished (filter and supernatant conditions) the rate of transfer was drastically decreased. Data show mean ± s.e.m (** p

Techniques Used: Co-Culture Assay, Flow Cytometry, Cytometry, Transfection, Cell Culture

31) Product Images from "Absolute quantification of cohesin, CTCF and their regulators in human cells"

Article Title: Absolute quantification of cohesin, CTCF and their regulators in human cells

Journal: bioRxiv

doi: 10.1101/560425

Fluorescence correlation spectroscopy of cohesin subunits and regulators. FCS measurements to estimate the concentration of endogenously GFP-tagged proteins in the nucleus/chromatin and cytoplasm of G1, G2 and prometaphase cells. (A) FCS measurements were taken at different positions (3 in the nucleus/chromatin, 2 in the cytoplasm) in cells in G1 (top), G2 (middle) or prometaphase (bottom), whereby photon counts were recorded for 30 s at each position. Shown are example images for SCC1-EGFP H2B-mCherry in the GFP (left), DNA (middle) and transmission channels. Scale bar: 10 μm. (B) After data fitting and quality control, the GFP-based protein concentrations within the two cellular compartments were obtained. Shown are violin plots (solid lines) indicating the probability density of the protein concentrations determined from each FCS measurement (dots). Note that the EGFP-sororin cell line displayed a mitotic defect, raising the possibility that EGFP-sororin may be hypomorphic.
Figure Legend Snippet: Fluorescence correlation spectroscopy of cohesin subunits and regulators. FCS measurements to estimate the concentration of endogenously GFP-tagged proteins in the nucleus/chromatin and cytoplasm of G1, G2 and prometaphase cells. (A) FCS measurements were taken at different positions (3 in the nucleus/chromatin, 2 in the cytoplasm) in cells in G1 (top), G2 (middle) or prometaphase (bottom), whereby photon counts were recorded for 30 s at each position. Shown are example images for SCC1-EGFP H2B-mCherry in the GFP (left), DNA (middle) and transmission channels. Scale bar: 10 μm. (B) After data fitting and quality control, the GFP-based protein concentrations within the two cellular compartments were obtained. Shown are violin plots (solid lines) indicating the probability density of the protein concentrations determined from each FCS measurement (dots). Note that the EGFP-sororin cell line displayed a mitotic defect, raising the possibility that EGFP-sororin may be hypomorphic.

Techniques Used: Fluorescence, Spectroscopy, Concentration Assay, Transmission Assay

32) Product Images from "Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis"

Article Title: Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis

Journal: bioRxiv

doi: 10.1101/2020.06.24.164988

PMMA culture device supports the generation of 3D biomimetic skeletal muscle tissues. A , Computer generated depiction of the PMMA mold design. The top part containing eight pairs of vertical posts is shown in black. Eight holes positioned equidistant between each pair of posts allows for gas and media exchange. Two larger sized vertical posts at either end of the top part serve to fix the top and bottom portion (translucent white) together and ensure vertical posts are properly positioned. The bottom portion is affixed to a microscopy grade glass. B , Images of remodeled C2C12 muscle tissues at 7 days of differentiation anchored to the end of the posts captured to provide a view looking up into a well in which the top and bottom parts are fashioned together (left; 4X objective) or from the side looking at the top part to visualize 6 pairs of posts with tissues at the bottom. C , Schematic workflow used to raise 3D skeletal muscle tissues in vitro . D-E , Representative confocal microscopy longitudinal ( D , whole mount, flattened stack, 40X water immersion objective) and transverse ( E , cryosection, single snap, 40X water immersion objective) images of multinucleated myotubes within a 14 day old muscle tissue immunostained for sacomeric alpha-actinin (SAA, green) and counterstained with Hoechst 33342 to visualize nuclei (blue). The cross section of the tissue was measured to be 0.17 ± 0.03 mm 2 . F , Timeseries of nuclear motion withing developing 3D skeletal muscle tissue 10 days after differentiation, demonstrating the possibility of high resolution imaging during living tissue formation. Lifeact-GFP (green) and H2B-mCherry (red) was stably introduced into AB1167 cells.
Figure Legend Snippet: PMMA culture device supports the generation of 3D biomimetic skeletal muscle tissues. A , Computer generated depiction of the PMMA mold design. The top part containing eight pairs of vertical posts is shown in black. Eight holes positioned equidistant between each pair of posts allows for gas and media exchange. Two larger sized vertical posts at either end of the top part serve to fix the top and bottom portion (translucent white) together and ensure vertical posts are properly positioned. The bottom portion is affixed to a microscopy grade glass. B , Images of remodeled C2C12 muscle tissues at 7 days of differentiation anchored to the end of the posts captured to provide a view looking up into a well in which the top and bottom parts are fashioned together (left; 4X objective) or from the side looking at the top part to visualize 6 pairs of posts with tissues at the bottom. C , Schematic workflow used to raise 3D skeletal muscle tissues in vitro . D-E , Representative confocal microscopy longitudinal ( D , whole mount, flattened stack, 40X water immersion objective) and transverse ( E , cryosection, single snap, 40X water immersion objective) images of multinucleated myotubes within a 14 day old muscle tissue immunostained for sacomeric alpha-actinin (SAA, green) and counterstained with Hoechst 33342 to visualize nuclei (blue). The cross section of the tissue was measured to be 0.17 ± 0.03 mm 2 . F , Timeseries of nuclear motion withing developing 3D skeletal muscle tissue 10 days after differentiation, demonstrating the possibility of high resolution imaging during living tissue formation. Lifeact-GFP (green) and H2B-mCherry (red) was stably introduced into AB1167 cells.

Techniques Used: Generated, Microscopy, In Vitro, Confocal Microscopy, Imaging, Stable Transfection

33) Product Images from "Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo"

Article Title: Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo

Journal: Scientific Reports

doi: 10.1038/s41598-019-40959-y

TNF-α stimulated nuclear translocation of NF-κB in HUVECs. HUVECs transfected with GFP-RelA and H2B-mCherry were stimulated with 10 ng/mL TNF-α (a – c ). A time series of GFP-RelA shows strong nuclear concentration at 30 minutes and empty nuclei at 0 and 360 minutes (a) , the population mean of approximate 600 single cell measurements (b) , projected view of the entire cell population (c) . Immunohistochemistry of non-transfected HUVECs treated with 10 ng/mL TNF-α fixed and stained (p65-AF488) at different time points point normalized to unstimulated cells (*p
Figure Legend Snippet: TNF-α stimulated nuclear translocation of NF-κB in HUVECs. HUVECs transfected with GFP-RelA and H2B-mCherry were stimulated with 10 ng/mL TNF-α (a – c ). A time series of GFP-RelA shows strong nuclear concentration at 30 minutes and empty nuclei at 0 and 360 minutes (a) , the population mean of approximate 600 single cell measurements (b) , projected view of the entire cell population (c) . Immunohistochemistry of non-transfected HUVECs treated with 10 ng/mL TNF-α fixed and stained (p65-AF488) at different time points point normalized to unstimulated cells (*p

Techniques Used: Translocation Assay, Transfection, Concentration Assay, Immunohistochemistry, Staining

Schematic diagram of the experimental pipeline: Step 1: Perfusion system including flow chamber, two windkessels, medium reservoir and a peristaltic pump. Cells within the flow chamber were recorded with the fluorescence microscope using LED lamps. Step 2: The raw image of H2B-mCherry and GFP-RelA were processed by enhancing the contrast, correcting non-uniform illumination and removing noise with a median filter. Step 3: H2B-mCherry image was made binary and the nuclei were numbered. Step 4: The numbered nuclei were tracked throughout all time frames. Step 5: The coordinates from the tracked nuclei were used to calculate the nuclear GFP-RelA intensity in the corrected GFP-RelA image. Step 6: nuclear GFP-RelA intensity in each cell was normalized by the time average GFP-RelA intensity. The population mean of the normalized nuclear GFP-RelA intensity including standard deviation was plotted as the result.
Figure Legend Snippet: Schematic diagram of the experimental pipeline: Step 1: Perfusion system including flow chamber, two windkessels, medium reservoir and a peristaltic pump. Cells within the flow chamber were recorded with the fluorescence microscope using LED lamps. Step 2: The raw image of H2B-mCherry and GFP-RelA were processed by enhancing the contrast, correcting non-uniform illumination and removing noise with a median filter. Step 3: H2B-mCherry image was made binary and the nuclei were numbered. Step 4: The numbered nuclei were tracked throughout all time frames. Step 5: The coordinates from the tracked nuclei were used to calculate the nuclear GFP-RelA intensity in the corrected GFP-RelA image. Step 6: nuclear GFP-RelA intensity in each cell was normalized by the time average GFP-RelA intensity. The population mean of the normalized nuclear GFP-RelA intensity including standard deviation was plotted as the result.

Techniques Used: Fluorescence, Microscopy, Standard Deviation

34) Product Images from "MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis"

Article Title: MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

Journal: Translational Oncology

doi: 10.1016/j.tranon.2017.04.002

Effect of Chk1 inhibitors on mitotic progression after X-irradiation. The effect of the two Chk1 inhibitors on cell fate after X-irradiation was evaluated using live-cell imaging. EMT6 cells stably expressing histone H2B fused to mCherry and α-tubulin fused to EYFP (EMT6/H2B-R/Tub-G cells) were X-irradiated at 2.5 Gy and incubated with or without Chk1 inhibitors for 24 h. Live-cell imaging was conducted from 3 to 24-h post-irradiation. (A) Representative images of EMT6/H2B-R/Tub-G cells undergoing mitosis. Upper panels represent the merged images of histone H2B fused to mCherry (red) and differential interference contrast (DIC; gray). Lower panels represent the fluorescent images of α-tubulin fused to EYFP (green). The scale bar represents 10 μm. (B) The fate of individual cells after treatment was evaluated by analyzing the appearance of red-fluorescent nuclei and green-fluorescent microtubules at each time point. Each horizontal bar represents one cell (n = 20). White, interphase; black, mitotic phase; magenta, micronucleus; red, multilobular nucleus; green, multipolar mitosis; blue, mitosis failure. (C) The scatter plot indicates the duration from the start of observations to the onset of the first mitotic event. (D) The scatter plot shows the duration of the mitotic phase. The bars represent mean values. * P
Figure Legend Snippet: Effect of Chk1 inhibitors on mitotic progression after X-irradiation. The effect of the two Chk1 inhibitors on cell fate after X-irradiation was evaluated using live-cell imaging. EMT6 cells stably expressing histone H2B fused to mCherry and α-tubulin fused to EYFP (EMT6/H2B-R/Tub-G cells) were X-irradiated at 2.5 Gy and incubated with or without Chk1 inhibitors for 24 h. Live-cell imaging was conducted from 3 to 24-h post-irradiation. (A) Representative images of EMT6/H2B-R/Tub-G cells undergoing mitosis. Upper panels represent the merged images of histone H2B fused to mCherry (red) and differential interference contrast (DIC; gray). Lower panels represent the fluorescent images of α-tubulin fused to EYFP (green). The scale bar represents 10 μm. (B) The fate of individual cells after treatment was evaluated by analyzing the appearance of red-fluorescent nuclei and green-fluorescent microtubules at each time point. Each horizontal bar represents one cell (n = 20). White, interphase; black, mitotic phase; magenta, micronucleus; red, multilobular nucleus; green, multipolar mitosis; blue, mitosis failure. (C) The scatter plot indicates the duration from the start of observations to the onset of the first mitotic event. (D) The scatter plot shows the duration of the mitotic phase. The bars represent mean values. * P

Techniques Used: Irradiation, Live Cell Imaging, Stable Transfection, Expressing, Incubation

35) Product Images from "Hematopoietic PBX-interacting protein is a substrate and an inhibitor of the APC/C–Cdc20 complex and regulates mitosis by stabilizing cyclin B1"

Article Title: Hematopoietic PBX-interacting protein is a substrate and an inhibitor of the APC/C–Cdc20 complex and regulates mitosis by stabilizing cyclin B1

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.006733

Loss of HPIP expression delays cell division. A , HPIP knockdown by various HPIP-specific shRNAs ( shHPIP-1 , shHPIP-2 , and shHPIP-3 ) in HeLa cells was analyzed by Western blotting. B , cell proliferation upon HPIP knockdown in HeLa cells was analyzed by WST-1 assay. C , representative time-lapse live cell images of HPIP-depleted HeLa cells (magnification, 20×). D , quantification data of C . A total of 20 cells were analyzed for each sample ( n = 20). E , flow cytometry (FACS) analysis showing HeLa cells at various stages of cell cycle (percentage) upon HPIP knockdown. F , representative time-lapse live cell fluorescent images of either siCtrl or siHPIP-treated HeLa–H2B/tubulin cells that are synchronized by DT block at the S phase followed by release into fresh medium and captured at indicated time points. Green , H2B–EGFP; red , α-Tubulin–mCherry (magnification, 20×). EGFP , enhanced green fluorescence protein. G , quantification data of F . A total of 13 cells were analyzed for each sample ( n = 13). H , HeLa cells transfected with either shCtrl or shHPIP were fixed and stained with H3 Pho-S10 antibody and propidium iodide and then subjected to flow cytometry analysis. The mitotic cells were boxed . The mitotic indices are indicated as percentages of total cell population. The quantified results are presented as means ± S.D. using Student's t test. **, p
Figure Legend Snippet: Loss of HPIP expression delays cell division. A , HPIP knockdown by various HPIP-specific shRNAs ( shHPIP-1 , shHPIP-2 , and shHPIP-3 ) in HeLa cells was analyzed by Western blotting. B , cell proliferation upon HPIP knockdown in HeLa cells was analyzed by WST-1 assay. C , representative time-lapse live cell images of HPIP-depleted HeLa cells (magnification, 20×). D , quantification data of C . A total of 20 cells were analyzed for each sample ( n = 20). E , flow cytometry (FACS) analysis showing HeLa cells at various stages of cell cycle (percentage) upon HPIP knockdown. F , representative time-lapse live cell fluorescent images of either siCtrl or siHPIP-treated HeLa–H2B/tubulin cells that are synchronized by DT block at the S phase followed by release into fresh medium and captured at indicated time points. Green , H2B–EGFP; red , α-Tubulin–mCherry (magnification, 20×). EGFP , enhanced green fluorescence protein. G , quantification data of F . A total of 13 cells were analyzed for each sample ( n = 13). H , HeLa cells transfected with either shCtrl or shHPIP were fixed and stained with H3 Pho-S10 antibody and propidium iodide and then subjected to flow cytometry analysis. The mitotic cells were boxed . The mitotic indices are indicated as percentages of total cell population. The quantified results are presented as means ± S.D. using Student's t test. **, p

Techniques Used: Expressing, Western Blot, WST-1 Assay, Flow Cytometry, FACS, Blocking Assay, Fluorescence, Transfection, Staining

HPIP binds and inhibits APC/C–Cdc20 activity through IR motif. A and B , HeLa cells stably transfected with either shCtrl or shHPIP were lysed, and cell lysates were subjected to co-IP by APC3 ( A ) or MAD2 ( B ) followed by Western blotting as indicated. C , conservation of IR motif in HPIP among various species. D , HeLa cells transfected with wtHPIP, mtHPIP–D4, or mtHPIP–IR were subjected to co-IP with APC3 antibody followed by Western blotting as indicated. E , HeLa cells transfected with wtHPIP, mtHPIP–D4, or mtHPIP–IR were subjected to double thymidine synchronization followed by release at the indicated time points and blotted as indicated. An asterisk denotes expression of the indicated proteins at peak in the specified time period. F , representative time-lapse live cell fluorescent images of GFP–HPIP, GFP–mtHPIP–D4, or GFP–mtHPIP–IR transfected HeLa–H2B–mCherry ( red ) cells that are synchronized by DT block at the S phase followed by release into fresh medium and captured at indicated time points (magnification, 20×). G , quantification data of F . The quantified results are presented as means ± S.D. using Student's t test. *, p
Figure Legend Snippet: HPIP binds and inhibits APC/C–Cdc20 activity through IR motif. A and B , HeLa cells stably transfected with either shCtrl or shHPIP were lysed, and cell lysates were subjected to co-IP by APC3 ( A ) or MAD2 ( B ) followed by Western blotting as indicated. C , conservation of IR motif in HPIP among various species. D , HeLa cells transfected with wtHPIP, mtHPIP–D4, or mtHPIP–IR were subjected to co-IP with APC3 antibody followed by Western blotting as indicated. E , HeLa cells transfected with wtHPIP, mtHPIP–D4, or mtHPIP–IR were subjected to double thymidine synchronization followed by release at the indicated time points and blotted as indicated. An asterisk denotes expression of the indicated proteins at peak in the specified time period. F , representative time-lapse live cell fluorescent images of GFP–HPIP, GFP–mtHPIP–D4, or GFP–mtHPIP–IR transfected HeLa–H2B–mCherry ( red ) cells that are synchronized by DT block at the S phase followed by release into fresh medium and captured at indicated time points (magnification, 20×). G , quantification data of F . The quantified results are presented as means ± S.D. using Student's t test. *, p

Techniques Used: Activity Assay, Stable Transfection, Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Blocking Assay

36) Product Images from "Dynamics and regulation of nuclear import and nuclear movements of HIV-1 complexes"

Article Title: Dynamics and regulation of nuclear import and nuclear movements of HIV-1 complexes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006570

The kinetics of nuclear import of HIV-1 virions with VSV-G or HIV-1 envelope is similar and is not altered by expression of POM121-mCherry. (A) Schematic of a POM121-mCherry expressing HeLa cell infected with a VSV-G pseudotyped virion (top panel), a POM121-mCherry expressing TZM-bl cell, which express high levels of CD4 and CCR5, infected with a virion containing HIV-1 envelope (middle panel), or an H2B-mCherry expressing HeLa cell infected with a VSV-G pseudotyped virion (bottom panel). Example deconvolved images of cells expressing POM121-mCherry or H2B-mCherry are shown to the right of the schematics. Scale bar, 5 μm. (B-D) The time of nuclear import (B) , time in cytoplasm (C) , and NE residence time (D) for each viral complex that entered the nucleus is shown. Viral complexes were detected manually from analysis of 10-hr long movies initiated 10 min after infection (1 frame/3 min). The nuclear import events for the VSV-G pseudotyped virions infected in POM121-mCherry expressing cells are replotted from Fig 6 for comparison. Numbers below sample name in (B) indicate the number of nuclear import events analyzed. For (B-D) , the average values ± SD are shown above each sample; black lines indicate median values; n.s, not significant ( P > 0.05; Mann-Whitney test).
Figure Legend Snippet: The kinetics of nuclear import of HIV-1 virions with VSV-G or HIV-1 envelope is similar and is not altered by expression of POM121-mCherry. (A) Schematic of a POM121-mCherry expressing HeLa cell infected with a VSV-G pseudotyped virion (top panel), a POM121-mCherry expressing TZM-bl cell, which express high levels of CD4 and CCR5, infected with a virion containing HIV-1 envelope (middle panel), or an H2B-mCherry expressing HeLa cell infected with a VSV-G pseudotyped virion (bottom panel). Example deconvolved images of cells expressing POM121-mCherry or H2B-mCherry are shown to the right of the schematics. Scale bar, 5 μm. (B-D) The time of nuclear import (B) , time in cytoplasm (C) , and NE residence time (D) for each viral complex that entered the nucleus is shown. Viral complexes were detected manually from analysis of 10-hr long movies initiated 10 min after infection (1 frame/3 min). The nuclear import events for the VSV-G pseudotyped virions infected in POM121-mCherry expressing cells are replotted from Fig 6 for comparison. Numbers below sample name in (B) indicate the number of nuclear import events analyzed. For (B-D) , the average values ± SD are shown above each sample; black lines indicate median values; n.s, not significant ( P > 0.05; Mann-Whitney test).

Techniques Used: Expressing, Infection, MANN-WHITNEY

37) Product Images from "RhoA- and Ran-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes"

Article Title: RhoA- and Ran-induced antagonistic forces underlie symmetry breaking and spindle rotation in mouse oocytes

Journal: bioRxiv

doi: 10.1101/2020.10.20.348045

Cortical actomyosin polarization reorganizes during anaphase II (live experiments). (A) Live fluorescence imaging of an activated metaphase II oocyte undergoing spindle rotation. The oocyte has been injected with a combination of the H2b-mCherry DNA marker and the eGFP-UtrCH F-actin marker (S8 Movie). The images represent maximum z-projections of multiple confocal planes and the symbols highlight remarkable events occurring during the second meiotic division. (B) 2D maps of the polarized domain showing variation over time of the cortex distance to DNA clusters (color-coded top panel) and the cortical F-actin levels (bottom panel). The maps are extracted from the images shown on the right. The isodistance lines are used as landmarks to delimit region of the cortex equidistant from the DNA clusters. The dashed lines separate the best focus plane for each DNA cluster. (C) 1D representation of the data shown in panel B (see Methods). The graphs show variation over time of the DNA clusters distance to the cortex (top panel, d In and d Out ) and the cortical F-actin levels in each polarized domain (see bottom panel, F In and F Out ). The variation of spindle rotation angle α is also represented on both graphs. The diagrams on the left illustrate the measurements described above. All scale bars represent a length of 10 μm.
Figure Legend Snippet: Cortical actomyosin polarization reorganizes during anaphase II (live experiments). (A) Live fluorescence imaging of an activated metaphase II oocyte undergoing spindle rotation. The oocyte has been injected with a combination of the H2b-mCherry DNA marker and the eGFP-UtrCH F-actin marker (S8 Movie). The images represent maximum z-projections of multiple confocal planes and the symbols highlight remarkable events occurring during the second meiotic division. (B) 2D maps of the polarized domain showing variation over time of the cortex distance to DNA clusters (color-coded top panel) and the cortical F-actin levels (bottom panel). The maps are extracted from the images shown on the right. The isodistance lines are used as landmarks to delimit region of the cortex equidistant from the DNA clusters. The dashed lines separate the best focus plane for each DNA cluster. (C) 1D representation of the data shown in panel B (see Methods). The graphs show variation over time of the DNA clusters distance to the cortex (top panel, d In and d Out ) and the cortical F-actin levels in each polarized domain (see bottom panel, F In and F Out ). The variation of spindle rotation angle α is also represented on both graphs. The diagrams on the left illustrate the measurements described above. All scale bars represent a length of 10 μm.

Techniques Used: Fluorescence, Imaging, Injection, Marker

The polarized cortex exerts attraction forces on chromatid clusters. (A) Simplified diagram of the RanGTP gradient, emanating from the chromosomes, and leading to the cortical accumulation of F-actin through the activation of the Cdc42, N-WASP and Arp2/3 pathway. The proximity of the chromosomes promotes cortical F-actin polarity, which in turn attracts the chromosomes to the cortex. (B) Fixed F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) imaging of metaphase II arrested oocytes expressing dominant negative forms of the Ran and Cdc42 GTPase (respectively RanT24N and Cdc42T17N). Control oocytes have been injected with water. (C) Line graphs: averaged ± s.d. cortical F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) fluorescence intensities as a function of the cortex distance to DNA clusters. The fluorescence intensities are normalized to the mean of each profile. Quantifications have been performed on 10 controls, 11 RanT24N and 15 Cdc42T17N injected metaphase II oocytes, gathered from 2 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes injected with the H2b-mCherry DNA marker alone (control) or a combination of H2b-mCherry and a Cdc42 dominant negative form (Cdc42T17N, group #1 and #2) (S9 Movie). The Cdc42T17N injected oocytes present two characteristic phenotypes, with a first group of gametes extruding a small PBII (as compared to controls) and a second group whose spindle relocates towards the center of the cell. (E) The graph shows variation over time of the distance between the oocyte centroid and the DNA clusters mid-point. The grey profiles represent controls (H2b-mCherry alone) while the light and dark blue profiles represent Cdc42T17N injected oocytes (group #1 and #2, respectively). The red line shows a linear fit, from 0 to 60 min, of the Cdc42T17N group #2. (F) Distribution of the distance between the oocyte centroid and the DNA clusters mid-point after 120 min of recording. Quantifications have been performed on 34 control and 23 Cdc42T17N injected oocytes, gathered respectively from 9 and 3 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Cdc42T17N injected oocyte (S10 Movie). The presented images are extracted from timepoint t = 15 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.3 μm.min −1 vector. All scale bars represent a length of 10 μm.
Figure Legend Snippet: The polarized cortex exerts attraction forces on chromatid clusters. (A) Simplified diagram of the RanGTP gradient, emanating from the chromosomes, and leading to the cortical accumulation of F-actin through the activation of the Cdc42, N-WASP and Arp2/3 pathway. The proximity of the chromosomes promotes cortical F-actin polarity, which in turn attracts the chromosomes to the cortex. (B) Fixed F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) imaging of metaphase II arrested oocytes expressing dominant negative forms of the Ran and Cdc42 GTPase (respectively RanT24N and Cdc42T17N). Control oocytes have been injected with water. (C) Line graphs: averaged ± s.d. cortical F-actin (phalloidin staining) and myosin-II (pMLC2 immuno-staining) fluorescence intensities as a function of the cortex distance to DNA clusters. The fluorescence intensities are normalized to the mean of each profile. Quantifications have been performed on 10 controls, 11 RanT24N and 15 Cdc42T17N injected metaphase II oocytes, gathered from 2 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes injected with the H2b-mCherry DNA marker alone (control) or a combination of H2b-mCherry and a Cdc42 dominant negative form (Cdc42T17N, group #1 and #2) (S9 Movie). The Cdc42T17N injected oocytes present two characteristic phenotypes, with a first group of gametes extruding a small PBII (as compared to controls) and a second group whose spindle relocates towards the center of the cell. (E) The graph shows variation over time of the distance between the oocyte centroid and the DNA clusters mid-point. The grey profiles represent controls (H2b-mCherry alone) while the light and dark blue profiles represent Cdc42T17N injected oocytes (group #1 and #2, respectively). The red line shows a linear fit, from 0 to 60 min, of the Cdc42T17N group #2. (F) Distribution of the distance between the oocyte centroid and the DNA clusters mid-point after 120 min of recording. Quantifications have been performed on 34 control and 23 Cdc42T17N injected oocytes, gathered respectively from 9 and 3 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Cdc42T17N injected oocyte (S10 Movie). The presented images are extracted from timepoint t = 15 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.3 μm.min −1 vector. All scale bars represent a length of 10 μm.

Techniques Used: Activation Assay, Staining, Immunostaining, Imaging, Expressing, Dominant Negative Mutation, Injection, Fluorescence, Marker, Plasmid Preparation

The central spindle/RhoA pathway is required for spindle rotation. (A) Simplified diagram of the central spindle pathway leading to the activation of the RhoA GTPase and the assembly of the cytokinesis ring. (B) Fixed RhoA (immuno-staining) imaging of differentially staged oocytes undergoing spindle rotation. The top panel represents single confocal planes while the bottom panels represent maximum z-projections of multiple confocal planes. The inserted diagrams in the top panel illustrate the progressive closure of the cytokinesis ring (C) Inhibiting the central spindle pathway, using the Bi-2536 (PLK1 inhibitor), prevents the cortical recruitment of RhoA (see white arrow) and the emission of the PBII. The left images show the RhoA localization in fixed oocytes treated with or without the Bi-2536. The right histograms show the PBII emission rate from 81 control and 105 Bi-2536 treated oocytes, both gathered from 4 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes treated with or without the Bi-2536 (S6 Movie). The oocytes have been injected with the H2b-mCherry DNA marker. (E) Averaged variation over time ± s.d. of the spindle rotation angle α in oocytes treated with or without Bi-2536. (F) Box plots: distribution of early (0-20 min) and late (100-120 min) spindle rotation angle α in 70 control and 35 Bi-2536 treated oocytes, gathered respectively from 9 and 6 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Bi-2536 treated oocyte (S7 Movie). The presented images are extracted from timepoint t = 48 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.25 μm.min −1 vector. Box plots in F extend from the first (Q1) to the third (Q3) quartile (where Q3–Q1 is the interquartile range (IQR)); whiskers are Q1 or Q3 ± 1.5 × IQR; horizontal lines represent the median; and black squares represent the mean. Statistics in F were obtained using a two-sided Mann–Whitney test. The exact p values are shown directly above the graphs. All scale bars represent a length of 10 μm.
Figure Legend Snippet: The central spindle/RhoA pathway is required for spindle rotation. (A) Simplified diagram of the central spindle pathway leading to the activation of the RhoA GTPase and the assembly of the cytokinesis ring. (B) Fixed RhoA (immuno-staining) imaging of differentially staged oocytes undergoing spindle rotation. The top panel represents single confocal planes while the bottom panels represent maximum z-projections of multiple confocal planes. The inserted diagrams in the top panel illustrate the progressive closure of the cytokinesis ring (C) Inhibiting the central spindle pathway, using the Bi-2536 (PLK1 inhibitor), prevents the cortical recruitment of RhoA (see white arrow) and the emission of the PBII. The left images show the RhoA localization in fixed oocytes treated with or without the Bi-2536. The right histograms show the PBII emission rate from 81 control and 105 Bi-2536 treated oocytes, both gathered from 4 independent experiments. (D) Live fluorescence and DIC imaging of activated oocytes treated with or without the Bi-2536 (S6 Movie). The oocytes have been injected with the H2b-mCherry DNA marker. (E) Averaged variation over time ± s.d. of the spindle rotation angle α in oocytes treated with or without Bi-2536. (F) Box plots: distribution of early (0-20 min) and late (100-120 min) spindle rotation angle α in 70 control and 35 Bi-2536 treated oocytes, gathered respectively from 9 and 6 independent experiments. (G) Particle image velocimetry (PIV) measurements, performed on DIC movies, quantifying cytoplasmic flows in a control and a Bi-2536 treated oocyte (S7 Movie). The presented images are extracted from timepoint t = 48 min and show results as a vector field whose color code indicates the speed of tracked particles. The arrow in the top left corner shows the length and the color of 0.25 μm.min −1 vector. Box plots in F extend from the first (Q1) to the third (Q3) quartile (where Q3–Q1 is the interquartile range (IQR)); whiskers are Q1 or Q3 ± 1.5 × IQR; horizontal lines represent the median; and black squares represent the mean. Statistics in F were obtained using a two-sided Mann–Whitney test. The exact p values are shown directly above the graphs. All scale bars represent a length of 10 μm.

Techniques Used: Activation Assay, Immunostaining, Imaging, Fluorescence, Injection, Marker, Plasmid Preparation, MANN-WHITNEY

Stochastic symmetry breaking underlies spindle rotation. (A) Live fluorescence and DIC imaging of activated metaphase II oocytes undergoing spindle rotation. The oocytes have been injected either with the H2b-mCherry DNA marker alone (main DIC time series, S1 Movie) or with a combination of H2b-mCherry and the eGFP-MAP4 microtubule marker (inserted spindle time series, S2 Movie). The white arrows show membrane invaginations leading to PBII closure, except in inserted spindle image 20 min where it indicates the central spindle position. (B) High temporal resolution montage of the main DIC time series presented in panel A. The symbols highlight remarkable events occurring during the second meiotic division. (C) Automated 3D segmentation and tracking procedure to monitor the position of the DNA clusters within the shell of the oocyte (see Methods and S4 Movie). (D) Diagram showing the method used to quantify DNA clusters distance d and the spindle rotation angle α . (E) Variation over time of DNA clusters distance d in a selected oocyte (main graph). The red line shows the linear fit used to register in time oocyte population (inserted graphs). (F) Variation over time of spindle rotation angle α in a selected oocyte. The red curve shows the logistic fit used to extract rotation parameters. The initial time of rotation (t i rotation) is defined as the time when the rotation reaches 5% of the total fitted rotation. (G) Two examples of H2b-mCherry injected oocytes illustrating the stochastic triggering of spindle rotation (see S5 Movie). The white arrows indicate the approximate start of the rotation. (H) Distribution of the t i rotation for a control population of 70 oocytes gathered from 9 independent experiments (main graph). Probability density function (pdf) of the t i rotation distribution used to determine three equiprobable categories of increasing t i rotation (inserted graph). (I) Averaged variation over time ± s.d. of the DNA clusters distance d (top graph) and spindle rotation angle α (bottom graph) averaged per category as defined in panel H. All scale bars represent a length of 10 μm.
Figure Legend Snippet: Stochastic symmetry breaking underlies spindle rotation. (A) Live fluorescence and DIC imaging of activated metaphase II oocytes undergoing spindle rotation. The oocytes have been injected either with the H2b-mCherry DNA marker alone (main DIC time series, S1 Movie) or with a combination of H2b-mCherry and the eGFP-MAP4 microtubule marker (inserted spindle time series, S2 Movie). The white arrows show membrane invaginations leading to PBII closure, except in inserted spindle image 20 min where it indicates the central spindle position. (B) High temporal resolution montage of the main DIC time series presented in panel A. The symbols highlight remarkable events occurring during the second meiotic division. (C) Automated 3D segmentation and tracking procedure to monitor the position of the DNA clusters within the shell of the oocyte (see Methods and S4 Movie). (D) Diagram showing the method used to quantify DNA clusters distance d and the spindle rotation angle α . (E) Variation over time of DNA clusters distance d in a selected oocyte (main graph). The red line shows the linear fit used to register in time oocyte population (inserted graphs). (F) Variation over time of spindle rotation angle α in a selected oocyte. The red curve shows the logistic fit used to extract rotation parameters. The initial time of rotation (t i rotation) is defined as the time when the rotation reaches 5% of the total fitted rotation. (G) Two examples of H2b-mCherry injected oocytes illustrating the stochastic triggering of spindle rotation (see S5 Movie). The white arrows indicate the approximate start of the rotation. (H) Distribution of the t i rotation for a control population of 70 oocytes gathered from 9 independent experiments (main graph). Probability density function (pdf) of the t i rotation distribution used to determine three equiprobable categories of increasing t i rotation (inserted graph). (I) Averaged variation over time ± s.d. of the DNA clusters distance d (top graph) and spindle rotation angle α (bottom graph) averaged per category as defined in panel H. All scale bars represent a length of 10 μm.

Techniques Used: Fluorescence, Imaging, Injection, Marker

38) Product Images from "Mechanics regulate human embryonic stem cell self-organization to specify mesoderm"

Article Title: Mechanics regulate human embryonic stem cell self-organization to specify mesoderm

Journal: bioRxiv

doi: 10.1101/2020.02.10.943076

Compliant substrates promote hESC self-organization into “gastrulation-like” nodes. A) Cartoon of hESCs seeded as large colonies on compliant (2700 Pa) polyacrylamide hydrogels and stimulated with BMP4. B) Time-lapse brightfield (bottom) and immunofluorescent (top) images of an hESC colony stimulated with BMP4. Nuclei were visualized using H2B-mCherry (cyan). Arrows indicate “gastrulation-like” nodes. Scale bar = 500 μm. C) Representative spinning-disk confocal images of the top and bottom of the hESC colony 48 h following BMP4 stimulation showing T(brachyury) expression (mNeonGreen; green; right) and nuclei (DAPI; cyan; left) within the “gastrulation-like” nodes. The rectangle shown on the colony cartoon in panel A indicates the region of the hESC colony where the images were taken. Scale bar = 20 μm. D) Line graph quantifying the number of “gastrulation-like” nodes formed per hESC colony between 24 and 48 h after BMP4 stimulation. Each data point represents the mean number of nodes per colony ± SEM for n = 9 colonies from three independent experiments. E) Line graph quantifying the size of the “gastrulation-like” nodes formed between 24 and 48 h following BMP4 stimulation. Each data point represents the mean node size ± SEM of all the nodes identified in n = 9 colonies from three independent experiments. F) Representative immunofluorescent images of nuclei (DAPI; cyan; far left), T (mNeonGreen; green; middle-left), E-cadherin (Alexa568; magenta; middle-right), and composite (merge; far right) in the “gastrulation-like” nodes 48 h after BMP4 stimulation. The rectangle on the colony cartoon indicates the region within the colony where the images were taken. Scale bar = 100 μm. G) Representative immunofluorescent images of nuclei (DAPI; cyan; far left), T (mNeonGreen; green; middle-left), Slug (Alexa568; magenta; middle-right), and composite (merge; far right) in the “gastrulation-like” nodes 48 h after BMP4 stimulation. The rectangle on the colony cartoon indicates the region within the colony where the images were taken. Scale bar = 100 μm. H) Schematic representation of the “gastrulation-like” phenotype observed in the BMP4-stimulated hESC colonies plated on compliant hydrogels, compared to gastrulation in the developing embryo. Cross-sections along the dashed lines are depicted below the hESC colony and the developing embryo schematics. GN = gastrulation node. PS = primitive streak. EMT = epithelial to mesenchymal transition.
Figure Legend Snippet: Compliant substrates promote hESC self-organization into “gastrulation-like” nodes. A) Cartoon of hESCs seeded as large colonies on compliant (2700 Pa) polyacrylamide hydrogels and stimulated with BMP4. B) Time-lapse brightfield (bottom) and immunofluorescent (top) images of an hESC colony stimulated with BMP4. Nuclei were visualized using H2B-mCherry (cyan). Arrows indicate “gastrulation-like” nodes. Scale bar = 500 μm. C) Representative spinning-disk confocal images of the top and bottom of the hESC colony 48 h following BMP4 stimulation showing T(brachyury) expression (mNeonGreen; green; right) and nuclei (DAPI; cyan; left) within the “gastrulation-like” nodes. The rectangle shown on the colony cartoon in panel A indicates the region of the hESC colony where the images were taken. Scale bar = 20 μm. D) Line graph quantifying the number of “gastrulation-like” nodes formed per hESC colony between 24 and 48 h after BMP4 stimulation. Each data point represents the mean number of nodes per colony ± SEM for n = 9 colonies from three independent experiments. E) Line graph quantifying the size of the “gastrulation-like” nodes formed between 24 and 48 h following BMP4 stimulation. Each data point represents the mean node size ± SEM of all the nodes identified in n = 9 colonies from three independent experiments. F) Representative immunofluorescent images of nuclei (DAPI; cyan; far left), T (mNeonGreen; green; middle-left), E-cadherin (Alexa568; magenta; middle-right), and composite (merge; far right) in the “gastrulation-like” nodes 48 h after BMP4 stimulation. The rectangle on the colony cartoon indicates the region within the colony where the images were taken. Scale bar = 100 μm. G) Representative immunofluorescent images of nuclei (DAPI; cyan; far left), T (mNeonGreen; green; middle-left), Slug (Alexa568; magenta; middle-right), and composite (merge; far right) in the “gastrulation-like” nodes 48 h after BMP4 stimulation. The rectangle on the colony cartoon indicates the region within the colony where the images were taken. Scale bar = 100 μm. H) Schematic representation of the “gastrulation-like” phenotype observed in the BMP4-stimulated hESC colonies plated on compliant hydrogels, compared to gastrulation in the developing embryo. Cross-sections along the dashed lines are depicted below the hESC colony and the developing embryo schematics. GN = gastrulation node. PS = primitive streak. EMT = epithelial to mesenchymal transition.

Techniques Used: Expressing

39) Product Images from "In vitro characterization of the human segmentation clock"

Article Title: In vitro characterization of the human segmentation clock

Journal: Nature

doi: 10.1038/s41586-019-1885-9

a , Scheme showing the insertion of a constitutively expressed pCAG-H2B-mCherry nuclear label in the safe harbor AAVS1 locus in a HES7-Achilles human iPSC background. b , Diffusion (μm 2 /min) for individual human HES7-Achilles cells automatically tracked over a period of 24 hours. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. n=76 c , Distribution of pairwise instantaneous phase shifts between individual oscillating human HES7-Achilles cells, binned by instantaneous distance between pairs of cells. P-values for the pair-wise Kolmogorov-Smirnov test are as follows: 0.6407, 0.1811, 0.1340, 0.1428, 0.6784, 0.8171. n=1000. d , Distribution of phases along the unit circle at early, middle, and late timepoints. Each dot represents one cell. n=144 cells. e , Illustration of phase determination: representative raw HES7-Achilles fluorescence profile for an automatically tracked cell (left) and corresponding processed signal along with the inferred phase from Hilbert transform (right). f , Heatmap of HES7-Achilles fluorescence intensity over time in automatically tracked cells. Each line represents one cell. n=144 cells. g , Histogram of the time (hours since the onset of imaging) at cell division for manually tracked human HES7-Achilles cells. n=67 h , Left: Immunofluorescence staining for phosphorylated histone H3 (Ser10) in human iPSC-derived PSM cells treated with either vehicle control (DMSO) or 5μM Aphidicolin for 24 or 48 hours, starting on day 2 of differentiation. n=5. Scale bar = 100μm. Right: Quantification of phosphorylated histone H3 (Ser10) nuclei as a percent of total nuclei. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. i , Scatter plot showing the cell cycle time in human iPSC-derived PSM cells cultured in CLFBR medium. Mean±SD. n=26. j , Scatter plot showing the cell cycle time in human iPSC-derived PSM cells cultured in CLFBR medium. Mean±SD. n=26. k , Normalized HES7-Achilles fluorescence intensity profiles for 3 individual human iPSC-derived PSM cells pre-treated with 5μM Aphidicolin for 24 hours. n=6 independent experiments. l , Kuramoto order parameter over 20 hours on day 3 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 5μM Aphidicolin for 24 hours. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=45 cells (Control), 48 cells (Aphi). m , Comparison of the Kuramoto order parameter for oscillating HES7-Achilles treated with vehicle control (DMSO) or 5μM Aphidicolin. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. Paired two-sided t-test p=0.348. n=45 cells (Control), 48 cells (Aphi). n , qRT-PCR for Notch target genes HES7, NRARP and LFNG in human iPSC-derived PSM cells treated with vehicle control (DMSO) or 25μM DAPT on day 2 of differentiation. Mean±SD. n=3. o , Example of HES7-Achilles fluorescence intensity in a small region of interest (ROI) over a period of 45 hours in cells treated with DMSO (vehicle control) or the γ-secretase inhibitor DAPT (25μM) in CLFBR medium. n=16 independent experiments p , Representative example of Hes7-Achilles fluorescence intensity profiles for mESC-derived PSM cells treated with vehicle control (DMSO) or 25μM DAPT. n=13 independent experiments q , Kuramoto order parameter over 20 hours on day 2 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 25μM DAPT. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=131 cells (Control), 110 cells (DAPT). r , Representative immunofluorescence staining for YAP, F-actin (phalloidin) and DAPI nuclear stain in isolated human PSM-like cells treated with either DMSO or Latrunculin A (350nM). Scale bar = 50 μm. n=4 independent experiments. s , ChIP-qPCR fold enrichment of the LFNG and HES7 promoters in chromatin pulled down with an antibody against NOTCH1 relative to isotype IgG controls. Mean ±SD. iPSC control n=4, all other conditions n=3. t , Mean HES7-Achilles fluorescence intensity for isolated human cells cultured with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. n=18 cells. u , Scatter plot showing the HES7-Achilles oscillatory period for isolated human cells cultured with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Mean ±SD. n= 47 (LatA), 22 (LatA + DAPT) v , Kuramoto order parameter over 18 hours on day 2 of differentiation for human HES7-Achilles cells treated with DMSO, LatA alone or LatA in combination with DAPT. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=53 cells (Control), 18 cells (LatA), 18 cells (LatA + DAPT). w , Comparison of the Kuramoto order parameter in confluent HES7-Achilles cells vs. isolated cells treated with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. Paired one-way ANOVA with Bonferroni correction: Confluent control vs. LatA p=1.16e −6 , Confluent control vs. LatA + DAPT p=6.8e −13 , LatA vs. LatA + DAPT p=0.304. n=53 cells (Control), 18 cells (LatA), 18 cells (LatA + DAPT).
Figure Legend Snippet: a , Scheme showing the insertion of a constitutively expressed pCAG-H2B-mCherry nuclear label in the safe harbor AAVS1 locus in a HES7-Achilles human iPSC background. b , Diffusion (μm 2 /min) for individual human HES7-Achilles cells automatically tracked over a period of 24 hours. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. n=76 c , Distribution of pairwise instantaneous phase shifts between individual oscillating human HES7-Achilles cells, binned by instantaneous distance between pairs of cells. P-values for the pair-wise Kolmogorov-Smirnov test are as follows: 0.6407, 0.1811, 0.1340, 0.1428, 0.6784, 0.8171. n=1000. d , Distribution of phases along the unit circle at early, middle, and late timepoints. Each dot represents one cell. n=144 cells. e , Illustration of phase determination: representative raw HES7-Achilles fluorescence profile for an automatically tracked cell (left) and corresponding processed signal along with the inferred phase from Hilbert transform (right). f , Heatmap of HES7-Achilles fluorescence intensity over time in automatically tracked cells. Each line represents one cell. n=144 cells. g , Histogram of the time (hours since the onset of imaging) at cell division for manually tracked human HES7-Achilles cells. n=67 h , Left: Immunofluorescence staining for phosphorylated histone H3 (Ser10) in human iPSC-derived PSM cells treated with either vehicle control (DMSO) or 5μM Aphidicolin for 24 or 48 hours, starting on day 2 of differentiation. n=5. Scale bar = 100μm. Right: Quantification of phosphorylated histone H3 (Ser10) nuclei as a percent of total nuclei. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. i , Scatter plot showing the cell cycle time in human iPSC-derived PSM cells cultured in CLFBR medium. Mean±SD. n=26. j , Scatter plot showing the cell cycle time in human iPSC-derived PSM cells cultured in CLFBR medium. Mean±SD. n=26. k , Normalized HES7-Achilles fluorescence intensity profiles for 3 individual human iPSC-derived PSM cells pre-treated with 5μM Aphidicolin for 24 hours. n=6 independent experiments. l , Kuramoto order parameter over 20 hours on day 3 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 5μM Aphidicolin for 24 hours. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=45 cells (Control), 48 cells (Aphi). m , Comparison of the Kuramoto order parameter for oscillating HES7-Achilles treated with vehicle control (DMSO) or 5μM Aphidicolin. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. Paired two-sided t-test p=0.348. n=45 cells (Control), 48 cells (Aphi). n , qRT-PCR for Notch target genes HES7, NRARP and LFNG in human iPSC-derived PSM cells treated with vehicle control (DMSO) or 25μM DAPT on day 2 of differentiation. Mean±SD. n=3. o , Example of HES7-Achilles fluorescence intensity in a small region of interest (ROI) over a period of 45 hours in cells treated with DMSO (vehicle control) or the γ-secretase inhibitor DAPT (25μM) in CLFBR medium. n=16 independent experiments p , Representative example of Hes7-Achilles fluorescence intensity profiles for mESC-derived PSM cells treated with vehicle control (DMSO) or 25μM DAPT. n=13 independent experiments q , Kuramoto order parameter over 20 hours on day 2 of differentiation for human HES7-Achilles cells treated with vehicle control (DMSO) or 25μM DAPT. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=131 cells (Control), 110 cells (DAPT). r , Representative immunofluorescence staining for YAP, F-actin (phalloidin) and DAPI nuclear stain in isolated human PSM-like cells treated with either DMSO or Latrunculin A (350nM). Scale bar = 50 μm. n=4 independent experiments. s , ChIP-qPCR fold enrichment of the LFNG and HES7 promoters in chromatin pulled down with an antibody against NOTCH1 relative to isotype IgG controls. Mean ±SD. iPSC control n=4, all other conditions n=3. t , Mean HES7-Achilles fluorescence intensity for isolated human cells cultured with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. n=18 cells. u , Scatter plot showing the HES7-Achilles oscillatory period for isolated human cells cultured with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Mean ±SD. n= 47 (LatA), 22 (LatA + DAPT) v , Kuramoto order parameter over 18 hours on day 2 of differentiation for human HES7-Achilles cells treated with DMSO, LatA alone or LatA in combination with DAPT. Synchronization threshold is shown as the mean±SD of the Kuramoto order parameter for same dataset, but with randomized phases. n=53 cells (Control), 18 cells (LatA), 18 cells (LatA + DAPT). w , Comparison of the Kuramoto order parameter in confluent HES7-Achilles cells vs. isolated cells treated with either 350nM Latrunculin A alone or in combination with 25μM DAPT. Middle hinge corresponds to median, lower and upper hinges correspond to 1 st and 3 rd quartiles, lower and upper whiskers correspond to minimum and maximum. Paired one-way ANOVA with Bonferroni correction: Confluent control vs. LatA p=1.16e −6 , Confluent control vs. LatA + DAPT p=6.8e −13 , LatA vs. LatA + DAPT p=0.304. n=53 cells (Control), 18 cells (LatA), 18 cells (LatA + DAPT).

Techniques Used: Diffusion-based Assay, Fluorescence, Imaging, Immunofluorescence, Staining, Derivative Assay, Cell Culture, Quantitative RT-PCR, Isolation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

40) Product Images from "KIF24 controls the clustering of supernumerary centrosomes in pancreatic ductal adenocarcinoma cells"

Article Title: KIF24 controls the clustering of supernumerary centrosomes in pancreatic ductal adenocarcinoma cells

Journal: bioRxiv

doi: 10.1101/2022.03.16.484562

KIF24 depletion restores impaired mitotic events in Panc1 cells (A) Frames from live cell imaging of Panc1 or Kif24-3 cells stably expressing H2B-mCherry. Scale bar, 10 µm. (B) Time from nuclear envelope breakdown (NEB) to anaphase onset in indicated cells was measured. n = 119 (Panc1_H2B-mCherry), 135 (Kif24-3_H2B-mCherry). (C) The percentage of cells that entered and stayed > 180 min in mitosis was determined. The average of three independent experiments is shown; > 40 cells were scored each time. (B, C) All data are shown as mean ± SD. two-tailed Student’s t -test. **, p
Figure Legend Snippet: KIF24 depletion restores impaired mitotic events in Panc1 cells (A) Frames from live cell imaging of Panc1 or Kif24-3 cells stably expressing H2B-mCherry. Scale bar, 10 µm. (B) Time from nuclear envelope breakdown (NEB) to anaphase onset in indicated cells was measured. n = 119 (Panc1_H2B-mCherry), 135 (Kif24-3_H2B-mCherry). (C) The percentage of cells that entered and stayed > 180 min in mitosis was determined. The average of three independent experiments is shown; > 40 cells were scored each time. (B, C) All data are shown as mean ± SD. two-tailed Student’s t -test. **, p

Techniques Used: Live Cell Imaging, Stable Transfection, Expressing, Two Tailed Test

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    Addgene inc h2b mcherry
    Sp1 regulates mitotic progression. (A,B) Live cell imaging of mAID-Sp1; <t>H2B-mCherry</t> cells following the indicated treatments. While images were taken every three minutes, the above image sequence represents images taken every nine minutes to best highlight the differences between the treatments. Time = h:min. Scale bar = 5 μm. (C) Time (m) from nuclear envelope breakdown to G 1 . 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.016, unpaired t -test‥ (D) Time (m) from nuclear envelope breakdown to anaphase. 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.042, unpaired t -test‥ (E) Schematic outlining the experimental strategy for (F). (F) Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 μm. (F) Quantification of (E). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.0037, unpaired t -test. All images are representative.
    H2b Mcherry, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The depletion of NuSAP or Kid attenuates the amplitude and velocity of centromere movements. ( a ) Representative images of the 3D view using xz and yz projections in the control, NuSAP- or Kid-depleted synchronized metaphase <t>mCherry-H2B</t> <t>HeLa</t> cells. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of 3D centromere tracks, colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in the control, NuSAP- or Kid-depleted metaphase cells. Error bars represent +s.e.m. * P
    Stable H2b Mcherry Hela Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc hsp70l loxp mcherry stop loxp h2b gfp cryaa cerulean plasmid 24334
    The efficiency and effects of the CETI-PIC3 system are preserved in subsequent generations. (A) Embryos from an F3 generation of the CETI-PIC3 line were crossed to the Tg(ins:cre) line. β cell-specific induction of <t>H2B-GFP</t> was observed and Tnfa staining was enriched within β cells. (B) qRT-PCR analysis demonstrates increase in tnfa transcript levels in this generation of embryos. n =3. * P
    Hsp70l Loxp Mcherry Stop Loxp H2b Gfp Cryaa Cerulean Plasmid 24334, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sp1 regulates mitotic progression. (A,B) Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every three minutes, the above image sequence represents images taken every nine minutes to best highlight the differences between the treatments. Time = h:min. Scale bar = 5 μm. (C) Time (m) from nuclear envelope breakdown to G 1 . 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.016, unpaired t -test‥ (D) Time (m) from nuclear envelope breakdown to anaphase. 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.042, unpaired t -test‥ (E) Schematic outlining the experimental strategy for (F). (F) Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 μm. (F) Quantification of (E). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.0037, unpaired t -test. All images are representative.

    Journal: bioRxiv

    Article Title: Transcription factor Sp1 regulates mitotic fidelity through Aurora B kinase-mediated condensin I localization). The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data:

    doi: 10.1101/2020.06.19.158030

    Figure Lengend Snippet: Sp1 regulates mitotic progression. (A,B) Live cell imaging of mAID-Sp1; H2B-mCherry cells following the indicated treatments. While images were taken every three minutes, the above image sequence represents images taken every nine minutes to best highlight the differences between the treatments. Time = h:min. Scale bar = 5 μm. (C) Time (m) from nuclear envelope breakdown to G 1 . 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.016, unpaired t -test‥ (D) Time (m) from nuclear envelope breakdown to anaphase. 40 cells counted per treatment. n = 2. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.042, unpaired t -test‥ (E) Schematic outlining the experimental strategy for (F). (F) Fluorescent detection of DAPI-stained chromosomes in mAID-Sp1 cells that were arrested in metaphase with MG132. Misaligned (white arrow) chromosomes are completely distinguishable from the metaphase plate. Scale bar = 1 μm. (F) Quantification of (E). Minimum 30 cells counted per treatment. n = 3. Black circles represent the mean of each biological replicate. Error bars represent s.e.m. p = 0.0037, unpaired t -test. All images are representative.

    Article Snippet: H2B-mCherry was a gift from Robert Benezra (Addgene plasmid #20972) and pLENTI CMV GFP NEO was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid #17447) ( ) pBabe Blast osTIR1-9myc was a gift from Andrew Holland (Addgene plasmid #80073).

    Techniques: Live Cell Imaging, Sequencing, Staining

    APC/C dysfunction results in CIN buffering and adaptation to extreme CIN. A and B, RPE1/H2B-mCherry cells were arrested in mitosis using 40 nmol/L STLC, collected by shake-off, and released into media containing 3 μmol/L proTAME or DMSO (t = 0h). Stacks were acquired every 3 minutes to determine anaphase onset ( A ) and the presence of lagging chromosomes ( B ; error bars, mean ± 95% CI). Sample images from movies are shown, and the arrow indicates a lagging chromosome in a cell without proTAME. Representative images are shown (scale bar, 10 μm). C, Degradation kinetics of cyclin A2-Venus fluorescence quantified from unsynchronized single cells as they progress through mitosis. 1.5 μmol/L proTAME or DMSO was added 2 hours before imaging. Total cell fluorescence was quantified and normalized to the level at NEBD. Curves end at anaphase onset ( n = 24 cells per condition; error bars indicate SD; ***, P

    Journal: Cancer discovery

    Article Title: APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability

    doi: 10.1158/2159-8290.CD-16-0645

    Figure Lengend Snippet: APC/C dysfunction results in CIN buffering and adaptation to extreme CIN. A and B, RPE1/H2B-mCherry cells were arrested in mitosis using 40 nmol/L STLC, collected by shake-off, and released into media containing 3 μmol/L proTAME or DMSO (t = 0h). Stacks were acquired every 3 minutes to determine anaphase onset ( A ) and the presence of lagging chromosomes ( B ; error bars, mean ± 95% CI). Sample images from movies are shown, and the arrow indicates a lagging chromosome in a cell without proTAME. Representative images are shown (scale bar, 10 μm). C, Degradation kinetics of cyclin A2-Venus fluorescence quantified from unsynchronized single cells as they progress through mitosis. 1.5 μmol/L proTAME or DMSO was added 2 hours before imaging. Total cell fluorescence was quantified and normalized to the level at NEBD. Curves end at anaphase onset ( n = 24 cells per condition; error bars indicate SD; ***, P

    Article Snippet: H2B-mCherry was introduced by lentiviral delivery (Addgene plasmid 21217, a gift from Mark Mercola).

    Techniques: Fluorescence, Imaging

    APC/C subunit mutational status affects CIN in cancer cells. A and B, H2B-mCherry was introduced in H2030, U251, and SW480 cell lines and NEBD–anaphase duration ( A ) as well as the frequency of anaphase segregation errors ( B ) with and without proTAME was determined by time-lapse fluorescence microscopy. Stacks were acquired every 3 minutes, and an example of segregation error is shown for each cell line (scale bar, 10 μm; in A, bars, mean ± 95% CI of a representative experiment; in B, P values from Fisher exact test). C, Experimental procedure used to generate CRISPR/Cas9 edited H2030 and HT29 cells used for plots D to I. In each case, a single clone was infected (lentiCRISPR/CDC27 for H2030) or transfected (espCas9(1.1)/CDC23 + ssDNA donor) in a well of a 96-well plate before the colony reached confluency. Following transfection or transduction, the colony was dispersed by limiting dilution into 96-well plates. Clones were then screened for heterozygous disruption of CDC27 in H2030 cells or correction of the heterozygous E245 nonsense mutation in HT29 cells. Nonedited clones identified during screening were used as controls. Phenotypic analysis of all newly derived cell lines was performed following minimal clonal expansion to limit phenotypic diversity that may be acquired due to ongoing CIN. D, Lollipop plot of CDC27 showing only truncating mutations reported in TCGA and the location of the guide RNA used to disrupt CDC27 in H2030 cells. The clone isolated contained a heterozygous 35-bp deletion creating the truncation I442Sfs*15. E, NEBD–anaphase duration was determined for the H2030 clones using phase–contrast time-lapse microscopy (3 minutes/frame; bars, average ± 95% CI). F, The frequency of anaphase lagging chromosomes was determined on fixed cells by indirect IF microscopy (*, P

    Journal: Cancer discovery

    Article Title: APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability

    doi: 10.1158/2159-8290.CD-16-0645

    Figure Lengend Snippet: APC/C subunit mutational status affects CIN in cancer cells. A and B, H2B-mCherry was introduced in H2030, U251, and SW480 cell lines and NEBD–anaphase duration ( A ) as well as the frequency of anaphase segregation errors ( B ) with and without proTAME was determined by time-lapse fluorescence microscopy. Stacks were acquired every 3 minutes, and an example of segregation error is shown for each cell line (scale bar, 10 μm; in A, bars, mean ± 95% CI of a representative experiment; in B, P values from Fisher exact test). C, Experimental procedure used to generate CRISPR/Cas9 edited H2030 and HT29 cells used for plots D to I. In each case, a single clone was infected (lentiCRISPR/CDC27 for H2030) or transfected (espCas9(1.1)/CDC23 + ssDNA donor) in a well of a 96-well plate before the colony reached confluency. Following transfection or transduction, the colony was dispersed by limiting dilution into 96-well plates. Clones were then screened for heterozygous disruption of CDC27 in H2030 cells or correction of the heterozygous E245 nonsense mutation in HT29 cells. Nonedited clones identified during screening were used as controls. Phenotypic analysis of all newly derived cell lines was performed following minimal clonal expansion to limit phenotypic diversity that may be acquired due to ongoing CIN. D, Lollipop plot of CDC27 showing only truncating mutations reported in TCGA and the location of the guide RNA used to disrupt CDC27 in H2030 cells. The clone isolated contained a heterozygous 35-bp deletion creating the truncation I442Sfs*15. E, NEBD–anaphase duration was determined for the H2030 clones using phase–contrast time-lapse microscopy (3 minutes/frame; bars, average ± 95% CI). F, The frequency of anaphase lagging chromosomes was determined on fixed cells by indirect IF microscopy (*, P

    Article Snippet: H2B-mCherry was introduced by lentiviral delivery (Addgene plasmid 21217, a gift from Mark Mercola).

    Techniques: Fluorescence, Microscopy, CRISPR, Infection, Transfection, Transduction, Clone Assay, Mutagenesis, Derivative Assay, Isolation, Time-lapse Microscopy

    Identification of APC/C subunits in a genome-wide siRNA screen for CIN survival. A, Chromosome segregation error rates determined by FISH in postmitotic daughter cells. RPE1 cells were treated with reversine for 2 hours, then mitotic cells were collected by shake-off and allowed to reattach in the presence of reversine, before fixation. Graph represents the average rate measured for chromosomes 6, 7, 8, and 10 (bars, average ± 95% CI). B, HCT116 wild-type and p53 −/− isogenic lines were imaged for 72 hours by live-cell imaging. Cell density for each drug concentration was normalized to DMSO for wild-type and p53 −/− cell separately. Fold difference in cell density of HCT116 p53 −/− relative to wild-type is displayed for each drug concentration. C, Clonogenic assay using isogenic HCT116 cells grown for 10 days in the presence or absence or reversine, as indicated. D, Genome-wide RNAi screen for synthetic viability with MPS1 inhibition. RPE1 cells were synchronized in G 0 –G 1 by contact inhibition, trypsinized, and reverse-transfected at low density in triplicate to allow uniform passage through mitosis. Cells were exposed to 250 nmol/L reversine for 96 hours and fixed. Automated image acquisition and analysis were performed for various parameters, and Z-scores were derived based on median plate normalization for each siRNA pool. E, DAPI staining of fixed cells 48 hours following the indicated siRNA treatments with 250 nmol/L reversine where indicated. F, Chromosome segregation error rates measured by FISH as in A. Cells were transfected with a nontargeting or a CDC16 siRNA pool, and 48 hours later, cells were synchronized with a single thymidine block. Reversine was added 10 hours after thymidine release, prior to mitotic entry. Mitotic cells were collected at 12 hours by shake-off, allowed to reattach on coverslips still in the presence of reversine, and fixed (bars, average ± 95% CI). G, Time-lapse fluorescence microscopy of RPE1 cells expressing H2B-mCherry imaged every 3 minutes, 1 hour following the addition of 350 nmol/L reversine +/− proTAME (pT), as indicated. The duration from NEBD to metaphase and metaphase to anaphase is shown for cells in which all chromosomes congressed to form a metaphase plate. Each row corresponds to a single cell ( n > 60 cells each; bars, mean ± 95% CI). H, Sample images of segregation errors scored in G. Maximum intensity projections are shown for timeframes immediately preceding and following anaphase onset. Shown are examples of a correct division (no error), an example of a lagging chromosome following proper congression at metaphase (middle), and an example where anaphase occurred before all chromosomes congressed to the metaphase plate (congression defect, hence NEBD–metaphase could not be determined). Scale bar, 10 μm.

    Journal: Cancer discovery

    Article Title: APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability

    doi: 10.1158/2159-8290.CD-16-0645

    Figure Lengend Snippet: Identification of APC/C subunits in a genome-wide siRNA screen for CIN survival. A, Chromosome segregation error rates determined by FISH in postmitotic daughter cells. RPE1 cells were treated with reversine for 2 hours, then mitotic cells were collected by shake-off and allowed to reattach in the presence of reversine, before fixation. Graph represents the average rate measured for chromosomes 6, 7, 8, and 10 (bars, average ± 95% CI). B, HCT116 wild-type and p53 −/− isogenic lines were imaged for 72 hours by live-cell imaging. Cell density for each drug concentration was normalized to DMSO for wild-type and p53 −/− cell separately. Fold difference in cell density of HCT116 p53 −/− relative to wild-type is displayed for each drug concentration. C, Clonogenic assay using isogenic HCT116 cells grown for 10 days in the presence or absence or reversine, as indicated. D, Genome-wide RNAi screen for synthetic viability with MPS1 inhibition. RPE1 cells were synchronized in G 0 –G 1 by contact inhibition, trypsinized, and reverse-transfected at low density in triplicate to allow uniform passage through mitosis. Cells were exposed to 250 nmol/L reversine for 96 hours and fixed. Automated image acquisition and analysis were performed for various parameters, and Z-scores were derived based on median plate normalization for each siRNA pool. E, DAPI staining of fixed cells 48 hours following the indicated siRNA treatments with 250 nmol/L reversine where indicated. F, Chromosome segregation error rates measured by FISH as in A. Cells were transfected with a nontargeting or a CDC16 siRNA pool, and 48 hours later, cells were synchronized with a single thymidine block. Reversine was added 10 hours after thymidine release, prior to mitotic entry. Mitotic cells were collected at 12 hours by shake-off, allowed to reattach on coverslips still in the presence of reversine, and fixed (bars, average ± 95% CI). G, Time-lapse fluorescence microscopy of RPE1 cells expressing H2B-mCherry imaged every 3 minutes, 1 hour following the addition of 350 nmol/L reversine +/− proTAME (pT), as indicated. The duration from NEBD to metaphase and metaphase to anaphase is shown for cells in which all chromosomes congressed to form a metaphase plate. Each row corresponds to a single cell ( n > 60 cells each; bars, mean ± 95% CI). H, Sample images of segregation errors scored in G. Maximum intensity projections are shown for timeframes immediately preceding and following anaphase onset. Shown are examples of a correct division (no error), an example of a lagging chromosome following proper congression at metaphase (middle), and an example where anaphase occurred before all chromosomes congressed to the metaphase plate (congression defect, hence NEBD–metaphase could not be determined). Scale bar, 10 μm.

    Article Snippet: H2B-mCherry was introduced by lentiviral delivery (Addgene plasmid 21217, a gift from Mark Mercola).

    Techniques: Genome Wide, Fluorescence In Situ Hybridization, Live Cell Imaging, Concentration Assay, Clonogenic Assay, Inhibition, Transfection, Derivative Assay, Staining, Blocking Assay, Fluorescence, Microscopy, Expressing

    The depletion of NuSAP or Kid attenuates the amplitude and velocity of centromere movements. ( a ) Representative images of the 3D view using xz and yz projections in the control, NuSAP- or Kid-depleted synchronized metaphase mCherry-H2B HeLa cells. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of 3D centromere tracks, colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in the control, NuSAP- or Kid-depleted metaphase cells. Error bars represent +s.e.m. * P

    Journal: Nature Communications

    Article Title: NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force

    doi: 10.1038/ncomms10597

    Figure Lengend Snippet: The depletion of NuSAP or Kid attenuates the amplitude and velocity of centromere movements. ( a ) Representative images of the 3D view using xz and yz projections in the control, NuSAP- or Kid-depleted synchronized metaphase mCherry-H2B HeLa cells. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of 3D centromere tracks, colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in the control, NuSAP- or Kid-depleted metaphase cells. Error bars represent +s.e.m. * P

    Article Snippet: Stable H2B-mCherry HeLa cells were generated using the pcDNA3-H2B-mCherry vector from Addgene (#20972 (ref. )) and cultured in the presence of 0.2 mg ml−1 G418 (Sigma).

    Techniques:

    The effects of NuSAP and Kid depletion on the amplitude and velocity of centromere oscillation in monopolar cells are correlated. ( a ) Representative images of the 3D view using xz and yz projections in the control, NuSAP- or Kid-depleted monastrol-treated monopolar HeLa cells stably expressing mCherry-H2B. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of the 3D centromere tracks colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in the control, NuSAP- or Kid-depleted monopolar cells. Error bars represent +s.e.m. * P

    Journal: Nature Communications

    Article Title: NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force

    doi: 10.1038/ncomms10597

    Figure Lengend Snippet: The effects of NuSAP and Kid depletion on the amplitude and velocity of centromere oscillation in monopolar cells are correlated. ( a ) Representative images of the 3D view using xz and yz projections in the control, NuSAP- or Kid-depleted monastrol-treated monopolar HeLa cells stably expressing mCherry-H2B. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of the 3D centromere tracks colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in the control, NuSAP- or Kid-depleted monopolar cells. Error bars represent +s.e.m. * P

    Article Snippet: Stable H2B-mCherry HeLa cells were generated using the pcDNA3-H2B-mCherry vector from Addgene (#20972 (ref. )) and cultured in the presence of 0.2 mg ml−1 G418 (Sigma).

    Techniques: Stable Transfection, Expressing

    NuSAP and Kid regulate centromere movement at interpolar microtubules. ( a ) Representative images of the 3D view using xz and yz projections in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted synchronized metaphase HeLa cells stably expressing mCherry-H2B. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of the 3D centromere tracks colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted metaphase cells. Error bars represent +s.e.m. * P

    Journal: Nature Communications

    Article Title: NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force

    doi: 10.1038/ncomms10597

    Figure Lengend Snippet: NuSAP and Kid regulate centromere movement at interpolar microtubules. ( a ) Representative images of the 3D view using xz and yz projections in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted synchronized metaphase HeLa cells stably expressing mCherry-H2B. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of the 3D centromere tracks colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted metaphase cells. Error bars represent +s.e.m. * P

    Article Snippet: Stable H2B-mCherry HeLa cells were generated using the pcDNA3-H2B-mCherry vector from Addgene (#20972 (ref. )) and cultured in the presence of 0.2 mg ml−1 G418 (Sigma).

    Techniques: Stable Transfection, Expressing

    NuSAP tunes the polar ejection force with Kid at interpolar microtubules in monopolar cells. ( a ) Representative images of the 3D view using xz and yz projections in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted monastrol-treated monopolar HeLa cells stably expressing mCherry-H2B. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of 3D centromere tracks colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted monopolar cells. Error bars represent +s.e.m. * P

    Journal: Nature Communications

    Article Title: NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force

    doi: 10.1038/ncomms10597

    Figure Lengend Snippet: NuSAP tunes the polar ejection force with Kid at interpolar microtubules in monopolar cells. ( a ) Representative images of the 3D view using xz and yz projections in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted monastrol-treated monopolar HeLa cells stably expressing mCherry-H2B. Centromeres were marked with GFP-CENPA and centrosomes with GFP-centrin. Scale bar, 5 μm. ( b ) Representative images of 3D centromere tracks colour-coded for time and velocity. ( c , d ) Bar chart and histogram representing the average ( c ) and the distribution ( d ) of the amplitude of centromere oscillation in Nuf2-, NuSAP/Nuf2- or Kid/Nuf2-depleted monopolar cells. Error bars represent +s.e.m. * P

    Article Snippet: Stable H2B-mCherry HeLa cells were generated using the pcDNA3-H2B-mCherry vector from Addgene (#20972 (ref. )) and cultured in the presence of 0.2 mg ml−1 G418 (Sigma).

    Techniques: Stable Transfection, Expressing

    The efficiency and effects of the CETI-PIC3 system are preserved in subsequent generations. (A) Embryos from an F3 generation of the CETI-PIC3 line were crossed to the Tg(ins:cre) line. β cell-specific induction of H2B-GFP was observed and Tnfa staining was enriched within β cells. (B) qRT-PCR analysis demonstrates increase in tnfa transcript levels in this generation of embryos. n =3. * P

    Journal: Disease Models & Mechanisms

    Article Title: A novel Cre-enabled tetracycline-inducible transgenic system for tissue-specific cytokine expression in the zebrafish: CETI-PIC3

    doi: 10.1242/dmm.042556

    Figure Lengend Snippet: The efficiency and effects of the CETI-PIC3 system are preserved in subsequent generations. (A) Embryos from an F3 generation of the CETI-PIC3 line were crossed to the Tg(ins:cre) line. β cell-specific induction of H2B-GFP was observed and Tnfa staining was enriched within β cells. (B) qRT-PCR analysis demonstrates increase in tnfa transcript levels in this generation of embryos. n =3. * P

    Article Snippet: The ubiquitin promoter sequence ( ) and the sequence for the loxP-mCherry-stop-loxP cassette (Addgene plasmid # 24334 ) were similarly PCR amplified and cloned into the pInducer lentiviral vector construct between the MCS and the rtTA site.

    Techniques: Staining, Quantitative RT-PCR

    The transcript levels of cytokines are increased and tissue-specific induction of cytokines is achieved within the induced ins-CETI-PIC3 model system. (A) Schematic for the experimental procedure. All treatments were performed at 72 h post-fertilization (hpf). For B, C and D, RNA was subsequently isolated from the whole bodies of at least 15 embryos in each clutch and all analysis was performed on whole-body samples. (B) Dose response to 0, 0.5, 1 and 2.5 µg/ml doxycycline is shown for tnfa , ifng1 and il1b , and H2B-GFP . (C) Time response to 5 µg/ml doxycycline is shown for up to 12 h after treatment with doxycycline. All experiments for D and E were performed in ins-CETI-PIC3 experimental embryos and clutch-mate control Tg(ins:cre) or CETI-PIC3 embryos treated with 5 µg/ml doxycycline for 48 h. (D) The levels of tnfa , ifng1 and il1b , and H2B-GFP are all increased after doxycycline induction in the ins-CETI-PIC3 embryos compared to Tg(ins:cre) and CETI-PIC3 control embryos. n =4. * P

    Journal: Disease Models & Mechanisms

    Article Title: A novel Cre-enabled tetracycline-inducible transgenic system for tissue-specific cytokine expression in the zebrafish: CETI-PIC3

    doi: 10.1242/dmm.042556

    Figure Lengend Snippet: The transcript levels of cytokines are increased and tissue-specific induction of cytokines is achieved within the induced ins-CETI-PIC3 model system. (A) Schematic for the experimental procedure. All treatments were performed at 72 h post-fertilization (hpf). For B, C and D, RNA was subsequently isolated from the whole bodies of at least 15 embryos in each clutch and all analysis was performed on whole-body samples. (B) Dose response to 0, 0.5, 1 and 2.5 µg/ml doxycycline is shown for tnfa , ifng1 and il1b , and H2B-GFP . (C) Time response to 5 µg/ml doxycycline is shown for up to 12 h after treatment with doxycycline. All experiments for D and E were performed in ins-CETI-PIC3 experimental embryos and clutch-mate control Tg(ins:cre) or CETI-PIC3 embryos treated with 5 µg/ml doxycycline for 48 h. (D) The levels of tnfa , ifng1 and il1b , and H2B-GFP are all increased after doxycycline induction in the ins-CETI-PIC3 embryos compared to Tg(ins:cre) and CETI-PIC3 control embryos. n =4. * P

    Article Snippet: The ubiquitin promoter sequence ( ) and the sequence for the loxP-mCherry-stop-loxP cassette (Addgene plasmid # 24334 ) were similarly PCR amplified and cloned into the pInducer lentiviral vector construct between the MCS and the rtTA site.

    Techniques: Isolation

    Design of the CETI-PIC3 line. (A) The genetic construct of the CETI-PIC3 line. A tetracycline-on (Tet-on) system is used to induce the three cytokines of interest. An H2B-GFP cassette is placed downstream of the cytokines as a marker to visually show that the cytokines have been induced. (B) This model takes advantage of Cre-lox systems to induce cytokines in a tissue-specific manner. (C) Any tissue-specific promoter driving a Cre cassette can be used to induce tissue-specific inflammation in this model after excision of the stop codon downstream of the RFP cassette. (D) When CET-PIC3 fish are crossed to a tissue-specific Cre and subsequently treated with doxycycline, there is dose-dependent induction of the cytokines in a tissue-specific manner. (E) Survival curve for wild-type (WT) embryos treated with different doses of doxycycline (0-50 µg/ml) for different time periods (0-72 h). (F) Survival curve for CETI-PIC3 embryos treated with different doses of doxycycline (0-50 µg/ml) for different time periods (0-72 h). There is no difference in survival for WT versus CETI-PIC3 doxycycline-treated embryos. n =10 embryos per dose/time. In all figures, data are mean±s.e.m.

    Journal: Disease Models & Mechanisms

    Article Title: A novel Cre-enabled tetracycline-inducible transgenic system for tissue-specific cytokine expression in the zebrafish: CETI-PIC3

    doi: 10.1242/dmm.042556

    Figure Lengend Snippet: Design of the CETI-PIC3 line. (A) The genetic construct of the CETI-PIC3 line. A tetracycline-on (Tet-on) system is used to induce the three cytokines of interest. An H2B-GFP cassette is placed downstream of the cytokines as a marker to visually show that the cytokines have been induced. (B) This model takes advantage of Cre-lox systems to induce cytokines in a tissue-specific manner. (C) Any tissue-specific promoter driving a Cre cassette can be used to induce tissue-specific inflammation in this model after excision of the stop codon downstream of the RFP cassette. (D) When CET-PIC3 fish are crossed to a tissue-specific Cre and subsequently treated with doxycycline, there is dose-dependent induction of the cytokines in a tissue-specific manner. (E) Survival curve for wild-type (WT) embryos treated with different doses of doxycycline (0-50 µg/ml) for different time periods (0-72 h). (F) Survival curve for CETI-PIC3 embryos treated with different doses of doxycycline (0-50 µg/ml) for different time periods (0-72 h). There is no difference in survival for WT versus CETI-PIC3 doxycycline-treated embryos. n =10 embryos per dose/time. In all figures, data are mean±s.e.m.

    Article Snippet: The ubiquitin promoter sequence ( ) and the sequence for the loxP-mCherry-stop-loxP cassette (Addgene plasmid # 24334 ) were similarly PCR amplified and cloned into the pInducer lentiviral vector construct between the MCS and the rtTA site.

    Techniques: Construct, Marker, Fluorescence In Situ Hybridization