Journal: Pharmaceutics

Article Title: A Novel Chitosan Nanosponge as a Vehicle for Transepidermal Drug Delivery

doi: 10.3390/pharmaceutics13091329

Figure Lengend Snippet: Amount of chitosan in chitosan nanosponges (CNS3Ks and CNS10Ks) with different ratios of chitosan as analyzed using a ninhydrin assay.

Article Snippet: The skin permeation effect of the model drug, i.e., NR, using CNS3K and CNS10K with different chitosan ratios, was measured using a Franz-type diffusion cell (FDC-6T; Logan Instruments, Somerset, NJ, USA) and fluorescence microscopy and handled according to guidelines of the Animal Care and Use Committee of Chungbuk National University (approval no. CA-20-27, 1 December 2020).

Techniques:

Journal: Frontiers in Oncology

Article Title: Novel Breast-Specific Long Non-coding RNA LINC00993 Acts as a Tumor Suppressor in Triple-Negative Breast Cancer

doi: 10.3389/fonc.2019.01325

Figure Lengend Snippet: LINC00993 suppresses the growth of triple-negative breast cancer (TNBC) cells in vitro . (A) Structure of LINC00993 expression plasmid for adenovirus. (B) LINC00993 expression adenovirus infection efficiency showed by fluorescence microscope in MDA-MB-231 cells. Original magnification, ×400. Scale bars, 50 μm. (C) Expression of LINC00993 detected by qRT-PCR. MDA-MB-231 cells were infected by adenovirus for 24 h, and RNA was extracted. (D) Image of clone formation assay. (E) Number of clones were counted 2 weeks after plantation. (F) MDA-MB-231 cells were planted into 24-well plates. Twenty-four hours later, adenovirus was added to each well. Three wells of cells were digested and counted every 24 h. (G) LINC00993 expression caused apoptosis shown by TUNEL assay. Green points reflected apoptosis, and we used DAPI to stain DNA. Positive control cells were treated with DNase I, negative control cells were collected without adding TUNEL reaction buffer. Original magnification, ×100. Scale bars, 50 μm. (H) Effect of LINC00993 on cell cycle detected by flow cytometry. (I) Flow cytometry cell cycle results shown in a bar plot. (J) Invasive ability tested by Transwell assay. Twenty thousand cells were plated in each well, cells were observed after 24 h of incubation. (K) Bar plot for the number of cells that migrated across the membrane. *** P < 0.001, based on Student's t -test. Data were presented as mean ± SEM.

Article Snippet: To analyze cell apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed with One Step TUNEL Apoptosis Assay Kit (FITC, Beyotime, China) according to the manufacturer's instructions.

Techniques: In Vitro, Expressing, Plasmid Preparation, Infection, Fluorescence, Microscopy, Quantitative RT-PCR, Tube Formation Assay, Clone Assay, TUNEL Assay, Staining, Positive Control, Negative Control, Flow Cytometry, Transwell Assay, Incubation, Membrane

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-d, Validation of mitotic exit in spindle-less cells by comparing H3-S10 dephosphorylation kinetics during unperturbed anaphase and spindle-less mitotic exit. a , Example images of wild-type HeLa cells during unperturbed anaphase fixed after time-lapse imaging, in metaphase, 6 min (maximally clustered), and 10 min after anaphase onset. Upper panel: chromatin labelled with H2B-mCherry, lower panel: H3-pS10 immunofluorescence. Single Z-slice shown. b , Quantification of H3-pS10 mean fluorescence and chromatin area in unperturbed mitosis, as shown in ( a ). Cell numbers: n = 9 (metaphase), n = 14 (3–6 min after anaphase onset), n = 8 (≥8 min after anaphase onset). Normalization to average value of metaphase time point. c , Example images of wild-type HeLa cells during spindle-less mitosis fixed after time-lapse imaging, in prometaphase (no flavopiridol), 10 min (maximally clustered), and 20 min after flavopiridol addition. Imaging as in ( a ). d , Quantification of H3-pS10 mean fluorescence and chromatin area in spindle-less mitosis, as shown in ( c ), demonstrates that histone 3-serine 10 was efficiently dephosphorylated in flavopiridol-induced mitotic exit and chromosomes cluster to a degree comparable to that of normal late anaphase. Cell numbers: n = 11 (nocodazole arrested pro-metaphase), n = 12 (10 min after flavopiridol addition), n = 13 (20 min after flavopiridol addition). Normalization to average value of prometaphase time point. e , Time-lapse microscopy of HeLa cell expressing IBB-EGFP and H2B-mCherry incubated in nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. f, Quantification of IBB-EGFP mean fluorescence within the chromosomal region, normalized to pre-flavopiridol time points, during spindle-less mitotic exit as in ( e ). n = 20 cells. g , Time-lapse microscopy of a HeLa cell expressing H2B-mCherry, progressing through reversine-induced mitotic exit in the absence of spindle. Yellow line indicates convex hull around chromosomes, single Z-slice shown. Time is relative to onset of clustering. h , Quantification of chromosome convex hull area of 11 cells as in ( g ). Individual cell curves were aligned based on half-maximum value of convex hull area. Normalization to average of first 4 time points. i , Live HeLa cell undergoing mitosis upon RNAi-mediated depletion of the spindle checkpoint protein Mad2 in the absence of a spindle. The cell line stably expresses H2B-mCherry and membrane marker AcGFP-Lap2β. Time relative to nuclear envelope breakdown (NEBD), single Z-slice shown. Representative example of 14 cells shown. Bars indicate mean in ( b, d ), lines and shaded areas indicate mean ± SD in ( f, h ). Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Biomarker Discovery, De-Phosphorylation Assay, Imaging, Immunofluorescence, Fluorescence, Time-lapse Microscopy, Expressing, Incubation, Stable Transfection, Membrane, Marker

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Chromosome organization during spindle-less mitotic exit. 3D-video of HeLa cell expressing H2B-mCherry imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Yellow line indicates convex hull around chromosomes. b , Quantification of convex hull area of 16 cells as in ( a ), and inter-chromosomal area. c, HeLa cell expressing GEMs and H2B-mCherry, during spindle-less mitotic exit as in ( a ). Dashed lines indicate chromosomal area; single Z-slices shown. d, GEM particle count within chromosomal area as in ( c ), normalized to pre-flavopiridol time points (green). Chromosome convex hull area, normalized to pre-flavopiridol time points (magenta). n = 35 cells. Lines and shaded areas represent mean ± SD, dashed vertical lines indicate flavopiridol addition. Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Expressing

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, b, Effect of actin depolymerization on chromosome clustering. a , Live HeLa cells stably expressing actin-EGFP and H2B-mCherry were imaged in absence (control) or presence of latrunculin B. Nocodazole was present in both conditions. b , Quantification of chromosome convex hull area during a flavopiridol induced mitotic exit in the presence of nocodazole, in the absence (control) and presence of latrunculin B as shown in ( a ), normalized to average area pre-flavopiridol addition. Cell numbers: n = 13 (control), n = 22 (latrunculin B). c , Timing of chromosome clustering relative to nuclear envelopment. Live mitotic HeLa cell expressing H2B-mCherry and the inner nuclear envelope protein AcGFP-LAP2β were imaged in presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. d, Quantification of chromosome area by convex hull (yellow line in H2B channel in ( c )) and quantification of AcGFP-LAP2β accumulation at the surface of the chromatin region, within a rim of 1.6 μm width (yellow ROI in Lap2β channel in ( c )). n = 21 cells. e-h , Kinetochore tracking in cells progressing through spindle-less mitotic exit. e , Live mitotic HeLa cell stably expressing H2B-mCherry and CENP-A-EGFP imaged in the presence of nocodazole before and after flavopiridol addition, time-lapse = 10 s; Z-projection. f , 240 s long kinetochore trajectories starting 240 s before and at 400 after flavopiridol addition. g , Representative mean squared displacement (MSD) analyses of kinetochore tracks (CENP-A–EGFP) of nocodazole-treated mitotic HeLa cells before and after flavopiridol addition. h , Diffusion coefficients derived from mean square displacement (MSD) analyses of trajectories as in ( f ). Significance was tested by a two-tailed ratio paired t-test (****P = 2.9 × 10 −11 ). n = 13 cells. Bars and lines indicate mean, shaded areas indicate ± SD, dashed vertical lines refer to flavopiridol addition. Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Stable Transfection, Expressing, Control, Diffusion-based Assay, Derivative Assay, Two Tailed Test

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: Live cell microscopy of wild-type HeLa cells stably expressing H2B-mCherry synchronized to metaphase by MG132 treatment. a , Upper panel: negative control treated with dimethyl sulfoxide (DMSO) solvent, lower panel: mitotic exit induced through acute flavopiridol addition at t = 0 min. Chromosome arms extend out of the metaphase plate before flavopiridol addition, but densely cluster on the metaphase plate 8 min after flavopiridol addition. b , Quantification of chromosome convex hull area of 24 control and 25 flavopiridol treated cells, as in ( a ). c , Upper panel: acute mitotic spindle depolymerization by addition of nocodazole at t = 2 min leads to chromosome movement out of the metaphase plate; lower panel: mitotic exit was induced by flavopiridol (0 min), briefly before nocodazole-mediated spindle disassembly (2 min), preventing movement of chromosomes out of the metaphase plate. d , Chromosome convex hull area measurements of 27 nocodazole and 23 flavopiridol and nocodazole treated cells, as shown in ( c ). Values normalized to average of all frames prior to first drug treatment. Showing single Z-slices, lines and shaded areas indicate mean ± SD, dashed vertical lines indicate drug addition, scale bars 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Microscopy, Stable Transfection, Expressing, Negative Control, Solvent, Control

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-c, Effect of BAF depletion on chromosome clustering. a , Live mitotic HeLa cells stably expressing H2B-mCherry and GEMs were imaged 72 h after siRNA transfection, in the presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. White dashed lines indicate chromosome areas, single Z-slice shown. b , Quantification of chromosome convex hull area, normalized to pre-flavopiridol addition. c , GEM particle count within chromosomal area normalized to pre-flavopiridol addition. n = 23 cells (siBAF), n = 23 cells (siControl). d , Immunoblot analysis of BAF and actin 72 h after siRNA transfection, showing one of two biological replicates. For gel source data, see . e , Localization of BAF-EGFP in live mitotic HeLa cell imaged in the presence of nocodazole; flavopiridol was added at t = 0 s to induce mitotic exit. f , Quantification of chromosome convex hull area and BAF-EGFP accumulation at the surface of the chromatin region as in ( e ), normalized to pre-flavopiridol. n = 21 cells. g , Localization of Ki-67, EGFP tagged on endogenous loci in live HeLa cell progressing from metaphase to anaphase (anaphase onset = 0 min), DNA was stained with SiR-Hoechst, Z-projection. h, Quantification of chromosome convex hull area and EGFP-Ki-67 mean fluorescence on chromosomes, normalized to pre-anaphase, for 41 cells as in ( g ). i , Localization of Ki-67 during spindle-less mitotic exit. Time-lapse microscopy of mitotic HeLa cell as in ( g ), in the presence of nocodazole; flavopiridol was added (t=0 min) to induce mitotic exit. Z-projection. j , Quantification of chromosome convex hull area and EGFP-Ki-67 mean fluorescence on chromosomes of 27 cells as in ( i ). Shaded areas indicate ± SD, scale bars 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Staining, Fluorescence, Time-lapse Microscopy

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-d , Molecular organization of Ki-67 on the surface of mitotic chromosomes before and after flavopiridol addition. a , Ki-67 was tagged on the N-terminus by mCherry, on the C-terminus by EGFP and expressed in HeLa cells. Sister chromatid pairs oriented perpendicular to the imaging plane were imaged in live mitotic cells, lines indicate measurement regions for ( b ). b , Relative positions of mCherry and EGFP along an axis perpendicular to the chromosome surface. Fluorescence densities (dots) were measured along line profiles as in ( a ) and a sum of two Gaussian functions (lines) was separately fitted to the EGFP and mCherry channels, respectively, to determine peak positions (dashed lines). c , The radial displacement of EGFP relative to mCherry peaks was determined based on line profile measurements as in ( a, b ) for mCherry-Ki-67-EGFP (R-Ki-67-G: n = 34 chromosomes pre-flavopiridol, n = 46 chromosomes post-flavopiridol) and a construct where the fluorophores were linked in reverse order, EGFP-Ki-67-mCherry (G-Ki-67-R: n = 40 chromosomes pre-flavopiridol, n = 39 chromosomes post-flavopiridol). Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 2.1 ×10 −8 , ****P = 2.3 × 10 −6 ). d, Model of Ki-67 organization on chromosome surfaces during early mitosis and during mitotic exit. e, Ki-67 KO cell expressing low levels of H2B-mNeonGreen. Representative example of 14 cells. f , Spindle-less mitotic exit in wild-type cells and in Ki-67 knockout (KO) cells overexpressing H2B-mNeonGreen to high levels to suppress the Ki-67 knockout individualization failure phenotype. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Z-projection. g , Normalized chromosome convex hull area quantification of 24 wild-type and 22 Ki-67 KO cells as in ( f ). Lines and shaded areas represent mean ± SD. Scale bars 1 μm ( a ), all others 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Imaging, Fluorescence, Construct, Two Tailed Test, Expressing, Knock-Out

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Time-lapse microscopy of clustering-deficient HeLa Ki-67 KO cell stably expressing H2B-mCherry to high levels during a flavopiridol-induced mitotic exit in the presence of nocodazole. Transient expression of EGFP-Ki-67 to levels at least matching Ki-67 endogenous levels (lower panel). Single Z-slice shown. b , Chromosome convex hull area quantification, normalized to pre-flavopiridol time points, of 29 Ki-67 KO and 28 Ki-67 KO + EGFP-Ki-67 cells as in ( a ). c , Immunoblot analysis of H2B from cell lysates, isolated from wildtype cells (1) or cells transiently expressing H2B-mNeonGreen (2,3). Sample 3 was lysed after 48 h while sample 2 was additionally FACS sorted for the 10% brightest cells which we estimated to represent the population of cells that suppress the Ki-67 knockout individualization failure phenotype. For gel source data, see . n = 2 biological repeats. d, e , Immunofluorescence of acetylated H3 of Ki-67 KO cells arrested in nocodazole with or without trichostatin A. d , Representative examples. Single Z-slices shown. e , Quantification of acetylated H3 mean fluorescence intensity in Ki-67 KO cells arrested in nocodazole with and without trichostatin A. Bars represent mean, significance was tested with a two-tailed Kolmogorov-Smirnov test. (****P = 2.22×10 −16 ). n = 115 cells (Ki-67 KO), n = 111 cells (Ki-67 KO + TSA). f , Time-lapse microscopy of HeLa wild-type or clustering-deficient Ki-67 KO cells with or without transient re-expression of Ki-67 during a flavopiridol-induced mitotic exit in the presence of nocodazole. Cells were treated with trichostatin A 2 h before imaging to rescue the Ki-67 knockout individualization failure phenotype in Ki-67 KO cells. Representative examples stained with SiR-Hoechst, single Z-slices shown. g , Chromosome convex hull area, normalized to pre flavopiridol time points, quantification of 21 Ki-67 KO, 31 wild-type and 32 Ki-67 KO + EGFP-Ki-67 cells as in ( f ). Lines and shaded areas indicate mean ± SD ( b, g ), scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Stable Transfection, Expressing, Western Blot, Isolation, Knock-Out, Immunofluorescence, Fluorescence, Two Tailed Test, Imaging, Staining

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Schematic of genotyping strategy to endogenously mutate Ki-67’s PP1 binding motif RVSF to RASA. A newly generated SacII restriction site generated by CRISPR/Cas9 nickase as depicted was used to detect correctly mutated alleles. b , SacII restriction fragments were detected by gel electrophoresis following the assay depicted in ( a ), showing successful recombination of all three Ki-67 alleles present in HeLa cells for clone 43 and 96. Showing one example of 2 biological replicates. c-d , Spindle-less mitotic exit in wild-type cells and in homozygous Ki-67 RASA mutant cells. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Quantification of chromosome convex hull area, normalized to pre-flavopiridol time points ( c ) and representative examples stained with SiR-Hoechst ( d ). Z projection. Lines and shaded areas indicate mean ± SD. n = 22 cells (wild-type), n = 23 cells (RASA) . e , Molecular organization of the Ki-67 RASA mutant on the surface of mitotic chromosomes before and after flavopiridol addition to taxol treated cells. Ki-67(RASA) was tagged by mCherry and EGFP on either protein end, respectively, and expressed in HeLa cells bearing the endogenous RASA mutation in all three copies of Ki-67. Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 8.3 ×10 −8 , ***P = 0.00049). Chromosome numbers: n = 32 (R-Ki-67-G, Pre), n = 28 (R-Ki-67-G, Post), n = 37 (G-Ki-67-R, Pre), n = 34 (G-Ki-67-R, Post). f, g , Immunofluorescence of H3-pS10 during spindle-less mitotic exit in wild-type and Ki-67 KO cells. f , Representative examples of wild-type and Ki-67 KO cells before, 10 and 20 min after flavopiridol addition. Single Z-slice is shown. g , Quantification of H3-pS10 mean fluorescence intensity before (Wild-type n = 38 cells, Ki-67 KO n = 23 cells), 5 (Wild-type n = 61 cells, Ki-67 KO n = 61 cells), 10 (Wild-type n = 72 cells, Ki-67 KO n = 65 cells) and 20 min (Wild-type n = 73 cells, Ki-67 KO n = 55 cells) after mitotic exit induction with flavopiridol in wild-type and Ki-67 KO cells. Values normalized to average of wild-type 5 min time point. Showing combined data of two independent biological replicates. Bars represent mean, significance tested with a two-tailed Mann Whitney test (P = 0.72 for pre-flavopiridol time point, P = 0.96 for 5 min time point, P = 0.71 for 10 min time point and P = 0.26 for 20 min time point). Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Binding Assay, Generated, CRISPR, Nucleic Acid Electrophoresis, Mutagenesis, Staining, Two Tailed Test, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, Time-lapse microscopy of clustering-deficient HeLa Ki-67 KO cell transiently expressing H2B-mCherry to high levels, and stably expressing IBB-EGFP, during a flavopiridol-induced mitotic exit in the presence of nocodazole. b , Quantification of IBB-EGFP mean fluorescence in chromosomal area (green) and quantification of chromosome convex hull area (magenta) in clustering-deficient cells as in ( a ), normalized to pre-flavopiridol. n = 15 cells. c, d , H2B-mCherry expression levels and chromosome area measurements for cells shown in – . , Quantification of H2B-mCherry total fluorescence for cells analyzed in (all data points normalized to mean of wild-type dataset). d , Chromosome convex hull area quantification (normalized to pre-flavopiridol area) for cells quantified in . n = 46 cells (wild-type), n = 29 cells (Ki-67 KO). e , Clustering-proficient HeLa cell imaged as in , but treated with leptomycin B. f , Quantification of total L10 fluorescence in nuclear regions as shown in ( e ), normalized to average of pre-flavopiridol addition. n = 22. g, h , Quantification of H2B-mCherry total fluorescence and chromosome convex hull area for cells analyzed in and in ( f ). Normalizations as in ( c, d ), cell numbers: n = 23 wild-type, n = 8 Ki-67 KO, n = 22 leptomycin B. Individual Ki-67 KO curves shown in . Significance tested with two-sided unpaired t-test (P = 0.28 in GEM cell lines wild-type vs Ki-67 KO, P = 0.12 in L10 cell lines wild-type vs LMB treated and P = 0.13 in L10 cell lines wild-type vs Ki-67 KO). Lines and shaded areas indicate mean ± SD, dashed vertical lines indicate flavopiridol addition. Scale bars 10 μm, showing single Z-slices.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Expressing, Stable Transfection, Fluorescence

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, b, Time-lapse microscopy of spindle-less mitotic exit in clustering-proficient wild-type HeLa cell ( a ) and clustering-deficient Ki-67 KO HeLa cell ( b ). Cells stably expressed GEMs and transiently expressed H2B-mCherry; images show time point 1 min before and 20 min after flavopiridol-induced mitotic exit in presence of nocodazole. c, Quantification of GEM localization in 46 clustering-proficient wild-type and 29 clustering-deficient Ki-67 KO cells as in ( a, b ). Particle numbers were quantified in chromosomal area as in , and normalized to average of particle number pre flavopiridol. t = 0 min refers to addition of flavopiridol. Analyzed cells expressed H2B-mCherry at similar levels in both genetic backgrounds . d, e, Time-lapse microscopy of clustering-proficient wild-type cells and clustering-deficient Ki-67 KO cells ( e ) stably expressing ribosomal protein L10-EGFP and transiently expressing H2B-mCherry, before and after mitotic exit ind-ction in presence of nocodazole. f, Quantification of L10-EGFP total fluorescence within chromosomal area for conditions shown in ( d, e ). t = 0 min refers to flavopiridol addition, data normalized to average values of pre-flavopiridol addition. Analyzed cells expressed H2B-mCherry at similar levels in both conditions . n = 23 cells (wild-type), n = 8 cells (Ki-67 KO). Individual Ki-67 KO curves shown in . g, Model of nuclear assembly during mitotic exit. Lines and shaded areas represent mean ± SD, dashed vertical lines indicate flavopiridol addition. Single Z-slices shown; scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Stable Transfection, Expressing, Fluorescence

Journal: iScience

Article Title: Metabolic nuclear receptors coordinate energy metabolism to regulate Sox9 + hepatocyte fate

doi: 10.1016/j.isci.2021.103003

Figure Lengend Snippet: PPARα activation promotes proliferation of Sox9 + hepatocytes, and FXR suppresses proliferation of Sox9 + hepatocytes after CCl 4 -induced chronic liver injury in Sox9-Cre ERT2 ; Rosa26-mTmG mice (A) Schematic diagram showing mTom/mGFP reporter gene expression in the absence and presence of tamoxifen (TAM)-inducible Cre-mediated recombination. (B) Sox9-Cre ERT2 ; Rosa26-mTmG mice were intraperitoneally injected with a single dose of tamoxifen once per day for three days before treatment. The Sox9-Cre ERT2 ; Rosa26-mTmG mice were received intraperitoneal paraffin oil injection (control group) or CCl 4 injection twice per week for four weeks and these mice were orally gavaged with either Veh, GW7647, or GW4064 four times a week for four weeks. BrdU was injected twice per day for two days before sacrifice. (C) GFP(Sox9)/Hnf4α double staining was performed. Graphs show percentages of GFP + Hnf4α + cell (n = 5). Scale bar represents 20μm. (D) GFP(Sox9)/BrdU double staining was performed. Arrowheads depict the asymmetric division. Graphs show percentages of GFP + BrdU + cell (n = 5). Scale bar represents 20μm. Data are expressed as means ± SD. Comparisons between multiple groups were performed using ordinary one-way ANOVA with the Dunnett's multiple comparison test. Significant difference is presented at the levels of ∗p < 0.05 and ∗∗p < 0.01.

Article Snippet: Samples were fixed and permeabilized, saturated, and processed for immunostaining with primary antibody Sox9(1:100 dilution Millipore, AB5535)/Hnf4α(1:100 dilution Abcam, ab41898), Sox9(1:100 dilution)/BrdU(1:100 dilution), GFP(1:200 dilution Proteintech, 50430-2-AP)/Hnf4α(1:100 dilution), GFP(1:200 dilution)/BrdU(1:200 dilution), GFP(1:200 dilution)/Ck19(1:200 dilution Servicebio, GB12197), Ck19(1:200 dilution Abcam ab52625)/BrdU(1:200 dilution), GFP(1:200 dilution)/Notch1(1:100 dilution Santa Cruz, sc-376403).

Techniques: Activation Assay, Expressing, Injection, Double Staining

Journal: iScience

Article Title: Metabolic nuclear receptors coordinate energy metabolism to regulate Sox9 + hepatocyte fate

doi: 10.1016/j.isci.2021.103003

Figure Lengend Snippet: PPARα activation increases differentiation of Sox9 + hepatocytes, and FXR activation promotes self-renewal of Sox9 + hepatocytes in Sox9-Cre ERT2 ; Rosa26-mTmG mice The model of CCl 4 -induced chronic liver injury in Sox9-Cre ERT2 ; Rosa26-mTmG mice that was described in Figure 6 B. (A) GFP(Sox9)/CK19 staining was performed in the indicated groups. Graphs show percentages of GFP + CK19 + cells (n = 5). Scale bar represents 20μm. (B) GFP(Sox9)/Notch1 staining was performed in the indicated groups. Scale bar represents 20μm. (C) Hepatic expression levels of Cpt1a were determined by QRT-PCR analysis (n = 5). (D) Hepatic expression levels of PDK4 were determined by QRT-PCR analysis (n = 5). (E) ATP concentration measurements of liver samples (n = 5). Data are expressed as means ± SD. Comparisons between multiple groups were performed using ordinary one-way ANOVA with the Dunnett's multiple comparison test. Significant difference is presented at the levels of ∗p < 0.05 and ∗∗p < 0.01.

Article Snippet: Samples were fixed and permeabilized, saturated, and processed for immunostaining with primary antibody Sox9(1:100 dilution Millipore, AB5535)/Hnf4α(1:100 dilution Abcam, ab41898), Sox9(1:100 dilution)/BrdU(1:100 dilution), GFP(1:200 dilution Proteintech, 50430-2-AP)/Hnf4α(1:100 dilution), GFP(1:200 dilution)/BrdU(1:200 dilution), GFP(1:200 dilution)/Ck19(1:200 dilution Servicebio, GB12197), Ck19(1:200 dilution Abcam ab52625)/BrdU(1:200 dilution), GFP(1:200 dilution)/Notch1(1:100 dilution Santa Cruz, sc-376403).

Techniques: Activation Assay, Staining, Expressing, Quantitative RT-PCR, Concentration Assay

Journal: iScience

Article Title: Metabolic nuclear receptors coordinate energy metabolism to regulate Sox9 + hepatocyte fate

doi: 10.1016/j.isci.2021.103003

Figure Lengend Snippet: PPARα induces proliferation and differentiation of Sox9+ hepatocytes by enhancing OXPHOS, and FXR promote self-renewal of Sox9 + hepatocytes by increasing glycolysis and inhibiting OXPHOS (A) GFP + primary mouse hepatocytes were stained with BrdU. Scale bar represents 20μm. The morphology of mitochondria in GFP + hepatocytes. Scale bar represents 1μm. (B) GFP + primary mouse hepatocytes were stained with Notch1. Scale bar represents 20μm. (C) ATP concentration, O 2 consumption and Glycolysis measurements of primary mouse hepatocytes. Data are expressed as means ± SD. Comparisons between multiple groups were performed using ordinary one-way ANOVA with the Dunnett's multiple comparison test. Significant difference is presented at the levels of ∗p < 0.05 and ∗∗p < 0.01

Article Snippet: Samples were fixed and permeabilized, saturated, and processed for immunostaining with primary antibody Sox9(1:100 dilution Millipore, AB5535)/Hnf4α(1:100 dilution Abcam, ab41898), Sox9(1:100 dilution)/BrdU(1:100 dilution), GFP(1:200 dilution Proteintech, 50430-2-AP)/Hnf4α(1:100 dilution), GFP(1:200 dilution)/BrdU(1:200 dilution), GFP(1:200 dilution)/Ck19(1:200 dilution Servicebio, GB12197), Ck19(1:200 dilution Abcam ab52625)/BrdU(1:200 dilution), GFP(1:200 dilution)/Notch1(1:100 dilution Santa Cruz, sc-376403).

Techniques: Staining, Concentration Assay

Journal: iScience

Article Title: Metabolic nuclear receptors coordinate energy metabolism to regulate Sox9 + hepatocyte fate

doi: 10.1016/j.isci.2021.103003

Figure Lengend Snippet:

Article Snippet: Samples were fixed and permeabilized, saturated, and processed for immunostaining with primary antibody Sox9(1:100 dilution Millipore, AB5535)/Hnf4α(1:100 dilution Abcam, ab41898), Sox9(1:100 dilution)/BrdU(1:100 dilution), GFP(1:200 dilution Proteintech, 50430-2-AP)/Hnf4α(1:100 dilution), GFP(1:200 dilution)/BrdU(1:200 dilution), GFP(1:200 dilution)/Ck19(1:200 dilution Servicebio, GB12197), Ck19(1:200 dilution Abcam ab52625)/BrdU(1:200 dilution), GFP(1:200 dilution)/Notch1(1:100 dilution Santa Cruz, sc-376403).

Techniques: Recombinant, Detection Assay, Cell Based Assay, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Staining, Multiplex Assay, Mutagenesis, Software, Microscopy

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 1. Generation of human Tspan15-expressing MEFs as an immunogen and validation of resulting mouse anti-human Tspan15 mAbs. (A) ADAM10-knockout MEFs (–) and ADAM10- knockout MEFs stably overexpressing FLAG-tagged Tspan15 (+) were lysed in 1% Triton X-100 lysis buffer and subjected to anti-FLAG (top panel) and anti-α-tubulin (bottom panel) western blotting. (B) Wild-type (WT) and Tspan15-knockout (KO) Jurkat human T cells were analysed by flow cytometry with tissue culture supernatant for each of the four mouse anti-human Tspan15 hybridomas (1C12, 4A4, 5D4 or 5F4; solid line), or with mouse IgG1 as a negative control (dotted line). Histograms are representative of two independent experiments. (C) HEK-293T cells were transfected with FLAG-tagged human TspanC8 expression constructs (except for Tspan10, which was of mouse origin) or an empty vector control (–),

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Stable Transfection, Lysis, Western Blot, Flow Cytometry, Negative Control, Transfection, Construct, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 3. Tspan15 mAbs 1C12 and 4A4 partially inhibit ADAM10/Tspan15 activity. (Ai) Wild-type (WT), ADAM10-knockout (A10 KO) and Tspan15-knockout (T15 KO) HEK-293T cells were transfected with a VE-cadherin expression construct. Cells were treated with 10 μM DAPT to prevent post-ADAM10 proteolysis by γ-secretase, followed by 2 mM NEM for 30 minutes to activate ADAM10. Cells were lysed in 1% Triton X-100 lysis buffer and subjected to western blotting with an antibody against the cytoplasmic tail of VE-cadherin. No C-terminal fragment was detected in the absence of NEM (data not shown). (Aii) VE-cadherin cleavage data were quantitated to calculate the percentage cleaved. Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001 compared to WT). Error bars represent standard error of the mean from three independent experiments. (B) Wild-type HEK-293T cells were transfected with VE-cadherin, treated with Tspan15 mAbs or MOPC-21 negative control mAb for 30 minutes, and stimulated with NEM as described for panel

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Activity Assay, Knock-Out, Transfection, Expressing, Construct, Lysis, Western Blot, Transformation Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 4. Tspan15 and ADAM10 co-localise on the cell surface. (Ai) A549 cells were fixed and stained with anti-ADAM10 mAb (red) and either anti-Tspan15 mAb 5D4 (green) or anti-CD9 mAb 1AA2 (green). ADAM10, Tspan15 and CD9 on the basal membrane were imaged using TIRF microscopy. Images shown are representative of 48 fields of view from four independent experiments (scale bar 10 µm). (Aii) The degree of co-localisation between ADAM10 and Tspan15 or CD9 was determined using Manders’ coefficients to measure the proportion of overlapping pixels contained within total ADAM10 signal in the red channel (M1) and total Tspan15 or CD9 signal in the green channel (M2). Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Tukey’s multiple comparisons test to compare M1 and M2, within and between Tspan15 and CD9 (***p<0.001 for all pairwise comparisons). Error bars represent standard error of the mean.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Staining, Membrane, Microscopy, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 5. ADAM10 is the principal Tspan15-interacting protein in HEK-293T cells. Wildtype (WT) and Tspan15-knockout (KO) HEK-293T cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with Tspan15 mAb 1C12 cross-linked to protein G sepharose beads. Proteins were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Proteomic profiles of WT and Tspan15 KO HEK-293T immunoprecipitates are presented in a volcano plot to identify differentially expressed proteins. The minus log10 transformed p-value of each protein was plotted against the log2 transformed protein label free quantification ratio between the Tspan15 co-immunoprecipitation of WT samples and the control co-immunoprecipitation of Tspan15 KO samples. Proteins with significant fold change (p<0.05) are depicted in red; blue dots represent proteins with no significant changes in expression. A permutation-based false discovery rate estimation was applied and visualised as hyperbolic curves in grey.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Knock-Out, Lysis, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Quantitative Proteomics, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 6. Tspan15 protein expression requires ADAM10. (A) Tspan15 surface expression in wildtype (WT), Tspan15-knockout (KO) and ADAM10 KO Jurkat, HEK-293T and A549 cell lines were analysed

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 7. The requirement of Tspan15 for ADAM10 surface expression is cell type dependent. (A) ADAM10 surface expression in WT, ADAM10 KO and Tspan15 KO Jurkat, HEK-293T and A549 cells was measured by flow cytometry and quantitated as described in Figure 4A. (B) HUVECs were transfected with two different Tspan15 siRNAs or negative control siRNA and surface expression of ADAM10 was measured by flow cytometry and analysed as described in Figure 6A.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Flow Cytometry, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 8. ADAM10 and Tspan15 form dynamic bimolecular fluorescence complementation (BiFC) complexes. (A) Schematic representation of ADAM10 tagged with the C-terminal half of superfolder GFP (sfGFP-C), Tspan15 tagged with the N-terminal half of superfolder GFP (sfGFP-N) and the predicted ADAM10/Tspan15 BiFC dimer. Solid ovals represent N-glycosylation. (B) HEK-293T cells were transfected with the ADAM10 and Tspan15 BiFC expression constructs, fixed and stained with Alexa Fluor® 647-conjugated Tspan15 mAb 5D4, and analysed by confocal microscopy. The image shown is representative of middle plane sections taken from two independent experiments (scale bar 10 µm). (C-D) Fluorescence correlation spectroscopy (FCS) measurements from the upper membrane of HEK-293T expressing the ADAM10/Tspan15 BiFC complexes were used to determine the average particle concentration (C) and diffusion co-efficient (D) of the complexes. (E) Fluorescence fluctuations from the FCS reads were also subjected to photon counting histogram (PCH) analysis to obtain the average molecular brightness (ε) of particles within the confocal volume. The FCS data were separated into groups that preferentially fit to a one-component or a two-component PCH model with dimmer and brighter subcomponents. Data were obtained from 43 individual measurements from three independent experiments. Error bars represent standard errors of the mean, N is the number of particles, and cpm is the counts per molecule. Data were log-transformed and statistically analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test (***p<0.001).

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Fluorescence, Glycoproteomics, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Spectroscopy, Membrane, Concentration Assay, Diffusion-based Assay, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 9. A synthetic ADAM10/Tspan15 fusion protein is a functional scissor. (A) Schematic representation of the synthetic ADAM10/Tspan15 fusion protein that has the C-terminus of ADAM10

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Functional Assay