sir 647  (New England Biolabs)


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    Name:
    SNAP Cell 647 SiR
    Description:
    SNAP Cell 647 SiR 30 nmol
    Catalog Number:
    S9102S
    Price:
    344
    Category:
    Fluorochromes
    Size:
    30 nmol
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    Structured Review

    New England Biolabs sir 647
    SNAP Cell 647 SiR
    SNAP Cell 647 SiR 30 nmol
    https://www.bioz.com/result/sir 647/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sir 647 - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance"

    Article Title: Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22575-5

    The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell SiR-647. The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.
    Figure Legend Snippet: The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell SiR-647. The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Irradiation, Imaging, Staining

    2) Product Images from "Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α‐actinin 4"

    Article Title: Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α‐actinin 4

    Journal: EMBO Reports

    doi: 10.15252/embr.202050758

    ACTN4 clusters dynamically associate with postmitotic nuclear actin filaments Cells in early G1 stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a time series on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2 B. Scale bar overview (nAC) represents 10 μm. Scale bar time series and magnification 5 μm. Linescan of an actin filament with associated ACTN4 from square 3 in (A). Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in (A). Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2 ). Scale bar is 2 μm. The track length, track displacement (distance between starting point and end point), average speed, and maximum speed were quantified from 3 independent experiments. The black line shows the median and gray dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.
    Figure Legend Snippet: ACTN4 clusters dynamically associate with postmitotic nuclear actin filaments Cells in early G1 stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a time series on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2 B. Scale bar overview (nAC) represents 10 μm. Scale bar time series and magnification 5 μm. Linescan of an actin filament with associated ACTN4 from square 3 in (A). Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in (A). Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2 ). Scale bar is 2 μm. The track length, track displacement (distance between starting point and end point), average speed, and maximum speed were quantified from 3 independent experiments. The black line shows the median and gray dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.

    Techniques Used: Stable Transfection, Expressing, Time-lapse Microscopy, Labeling, Software

    ACTN4 associates with postmitotic nuclear actin filaments Cells stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. The cell is an alternative example of Fig 2 A that additionally covers the mitotic phase. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR (green). The magnification changes at time point 0:00. Scale bar 5 μm.
    Figure Legend Snippet: ACTN4 associates with postmitotic nuclear actin filaments Cells stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. The cell is an alternative example of Fig 2 A that additionally covers the mitotic phase. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR (green). The magnification changes at time point 0:00. Scale bar 5 μm.

    Techniques Used: Stable Transfection, Expressing, Time-lapse Microscopy, Labeling

    3) Product Images from "Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context"

    Article Title: Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

    Journal: Genome Research

    doi: 10.1101/gr.257352.119

    Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.
    Figure Legend Snippet: Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.

    Techniques Used: Derivative Assay, Construct, Staining

    4) Product Images from "Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport"

    Article Title: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08192-9

    PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )
    Figure Legend Snippet: PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )

    Techniques Used: Expressing, Staining, Epifluorescence Microscopy, Labeling

    5) Product Images from "Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings"

    Article Title: Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983924

    SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.
    Figure Legend Snippet: SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.

    Techniques Used: In Vivo Imaging, Labeling, Expressing, Imaging, Staining, SDS Page, Fluorescence, Incubation

    6) Product Images from "Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings"

    Article Title: Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

    Journal: bioRxiv

    doi: 10.1101/2020.03.09.983924

    SNAP-tag enabled  in vivo  imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag.  b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps.  c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown.  d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining.  e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed.  f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.
    Figure Legend Snippet: SNAP-tag enabled in vivo imaging of tubulin in BY-2 cells and Arabidopsis a , Labeling mechanism of SNAP-tag. b , Cortical microtubules in SNAP-TUA5 expressing BY-2 cells with SNAP dyes denoted above images. Images show max intensity projection of confocal z-stack slices taken with 0.5 µm steps. c , Time-lapse imaging of mitotic microtubule dynamics TMR-star labeling of TUA5. Images taken every 30 sec, elapsed time (min) is shown. d , pUBQ10:SNAP-TUA5 and Col-0 (wildtype) seedlings were stained with 500 nM SNAP-Cell TMR-Star for 3h, lysed and analyzed by SDS-PAGE. Left panel: fluorescence; right panel Coomassie blue staining. e , Confocal images of mitotic cells in Arabidopsis root epidermis stained with TMR-Star. Spindles and phragmoplasts were observed. f , Root tip of Arabidopsis coexpressing p35S:YFP-LTI6b, p35:H2B-RFP, and pUBQ10:SNAP-TUA5. 3-day-old seedlings were incubated in 1/2MS containing 500 nM SNAP-Cell 647-SiR for 30 min. Scale bars: 10 µm. Experiments were repeated independently 3 times with comparable results.

    Techniques Used: In Vivo Imaging, Labeling, Expressing, Imaging, Staining, SDS Page, Fluorescence, Incubation

    7) Product Images from "Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context"

    Article Title: Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

    Journal: Genome Research

    doi: 10.1101/gr.257352.119

    Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.
    Figure Legend Snippet: Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.

    Techniques Used: Derivative Assay, Construct, Staining

    8) Product Images from "Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication"

    Article Title: Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201906031

    Centrosomes are populated by newly synthesized Nup188, not exchange from NPCs. (A) Like endogenous Nup188, SNAP-Nup188 undergoes mitotic oscillation. Western blots showing the levels of SNAP-Nup188 in total protein samples derived from synchronized cells. Numbers at left are positions of molecular weight standards (in kilodaltons). Approximate cell cycle stage is indicated at the top, referencing Cyclin B synthesis and degradation; α-β-Actin is used to assess total protein loads. (B) As in A, but SNAP-Nup188 protein is visualized by fluorescence. SNAP-Nup188 is first pulse labeled with a 647-SiR dye (red/old) and then chase labeled at the indicated time points with a TMR-Star dye (yellow/new). (C) Plot of fluorescence from B with two additional experimental replicates. The mean fluorescence from these three experiments is represented ±SD. (D) Fluorescence images of HeLa cells expressing SNAP-Nup188 were synchronized with thymidine at S-phase and labeled with 647-SiR dye (red). Subsequently, these cells were released from thymidine block and then allowed to undergo mitosis. The newly synthesized SNAP-Nup188 was labeled with TMR-star dye (yellow) at each time point shown. α-Pericentrin labeling the centrosome is shown in magenta. DNA was visualized by Hoechst staining (blue). Scale bar is 5 µm. Magnifications of boxed regions encompassing centrosomes in red, yellow, and magenta are shown on the right. Scale bar is 0.5 µm. Bottom panels show background fluorescence in cells not expressing SNAP-Nup188 (-dox). n.a., not applicable.
    Figure Legend Snippet: Centrosomes are populated by newly synthesized Nup188, not exchange from NPCs. (A) Like endogenous Nup188, SNAP-Nup188 undergoes mitotic oscillation. Western blots showing the levels of SNAP-Nup188 in total protein samples derived from synchronized cells. Numbers at left are positions of molecular weight standards (in kilodaltons). Approximate cell cycle stage is indicated at the top, referencing Cyclin B synthesis and degradation; α-β-Actin is used to assess total protein loads. (B) As in A, but SNAP-Nup188 protein is visualized by fluorescence. SNAP-Nup188 is first pulse labeled with a 647-SiR dye (red/old) and then chase labeled at the indicated time points with a TMR-Star dye (yellow/new). (C) Plot of fluorescence from B with two additional experimental replicates. The mean fluorescence from these three experiments is represented ±SD. (D) Fluorescence images of HeLa cells expressing SNAP-Nup188 were synchronized with thymidine at S-phase and labeled with 647-SiR dye (red). Subsequently, these cells were released from thymidine block and then allowed to undergo mitosis. The newly synthesized SNAP-Nup188 was labeled with TMR-star dye (yellow) at each time point shown. α-Pericentrin labeling the centrosome is shown in magenta. DNA was visualized by Hoechst staining (blue). Scale bar is 5 µm. Magnifications of boxed regions encompassing centrosomes in red, yellow, and magenta are shown on the right. Scale bar is 0.5 µm. Bottom panels show background fluorescence in cells not expressing SNAP-Nup188 (-dox). n.a., not applicable.

    Techniques Used: Synthesized, Western Blot, Derivative Assay, Molecular Weight, Fluorescence, Labeling, Expressing, Blocking Assay, Staining

    Centrosome associations of Nup188 and PCM proteins are independent of each other. (A) Plot of the mean fluorescence at centrosomes of SNAP-Nup188 labeled with 647-SiR dye in cells treated with indicated siRNAs. More than 30 cells were quantified with mean ± SD shown. (B) Western blots (or fluorescence image/top panel) of total protein from SNAP-NUP188–expressing HeLa cell extracts prepared from cells treated with the indicated siRNAs. α-β-Actin is a protein load reference. Numbers at left are positions of molecular weight standards (in kilodaltons). (C) . Representative plot of α-Cep192 mean fluorescence ± SD at centrosomes ( n > 30). (D) Representative plot of α-Cep152 mean fluorescence ± SD at centrosomes ( n > 30). (E) Western blots of total protein from HeLa cell extracts prepared from cells treated with NUP188 siRNA. α-β-Actin is a protein load reference. n.s., not significant as calculated from two-way ANOVA. Numbers at left are positions of molecular weight standards (in kilodaltons).
    Figure Legend Snippet: Centrosome associations of Nup188 and PCM proteins are independent of each other. (A) Plot of the mean fluorescence at centrosomes of SNAP-Nup188 labeled with 647-SiR dye in cells treated with indicated siRNAs. More than 30 cells were quantified with mean ± SD shown. (B) Western blots (or fluorescence image/top panel) of total protein from SNAP-NUP188–expressing HeLa cell extracts prepared from cells treated with the indicated siRNAs. α-β-Actin is a protein load reference. Numbers at left are positions of molecular weight standards (in kilodaltons). (C) . Representative plot of α-Cep192 mean fluorescence ± SD at centrosomes ( n > 30). (D) Representative plot of α-Cep152 mean fluorescence ± SD at centrosomes ( n > 30). (E) Western blots of total protein from HeLa cell extracts prepared from cells treated with NUP188 siRNA. α-β-Actin is a protein load reference. n.s., not significant as calculated from two-way ANOVA. Numbers at left are positions of molecular weight standards (in kilodaltons).

    Techniques Used: Fluorescence, Labeling, Western Blot, Expressing, Molecular Weight

    Nup188 turns over rapidly at centrosomes but is stable at NPCs. (A) NUP188 mRNA levels are unchanged between S- and M-phase. Plot shows transcript levels at M-phase relative to S-phase levels as determined by RT-qPCR of the indicated nup messages. Shown are mean transcript levels (± SD) from three independent experiments. Dotted line reflects a ratio of 1, which indicates no change in relative levels between these cell cycle stages. Red, blue, and orange are inner ring, outer ring, and a mixture of cytoplasmic filament/nuclear basket nup genes, respectively. (B) Asynchronous HeLa cells producing SNAP-Nup188 were treated with DMSO (carrier-only control) and then labeled with 647-SiR dye (red) at indicated time points after drug addition, before imaging by fluorescence microscopy. Centrosomes were labeled with α-Pericentrin (green). Bar is 5 µm. Magnifications of boxed regions encompassing centrosomes in red, green, and merge are shown on the right. Scale bar is 0.5 µm. (C) A representative plot (one of three independent replicates) of the mean intensity (± SD) of SNAP-Nup188 fluorescence at centrosomes in individual cells from the experiment in B over time. (D) As in C but plotting nuclear envelope fluorescence. (E–G) As in B–D but cells were treated with cycloheximide. (H and I) As in B and C but cells were treated with MG132. (J) Sample fluorescence images of cells expressing SNAP-Nup188 treated with MG132 for 0 or 4 h. Line profiles of the fluorescence along the white lines shown in images bisecting the nucleus are shown in right panels. Note the y axis scale and intranuclear fluorescence in bottom panels. Arrows denote location of nuclear rim along line profile plot.
    Figure Legend Snippet: Nup188 turns over rapidly at centrosomes but is stable at NPCs. (A) NUP188 mRNA levels are unchanged between S- and M-phase. Plot shows transcript levels at M-phase relative to S-phase levels as determined by RT-qPCR of the indicated nup messages. Shown are mean transcript levels (± SD) from three independent experiments. Dotted line reflects a ratio of 1, which indicates no change in relative levels between these cell cycle stages. Red, blue, and orange are inner ring, outer ring, and a mixture of cytoplasmic filament/nuclear basket nup genes, respectively. (B) Asynchronous HeLa cells producing SNAP-Nup188 were treated with DMSO (carrier-only control) and then labeled with 647-SiR dye (red) at indicated time points after drug addition, before imaging by fluorescence microscopy. Centrosomes were labeled with α-Pericentrin (green). Bar is 5 µm. Magnifications of boxed regions encompassing centrosomes in red, green, and merge are shown on the right. Scale bar is 0.5 µm. (C) A representative plot (one of three independent replicates) of the mean intensity (± SD) of SNAP-Nup188 fluorescence at centrosomes in individual cells from the experiment in B over time. (D) As in C but plotting nuclear envelope fluorescence. (E–G) As in B–D but cells were treated with cycloheximide. (H and I) As in B and C but cells were treated with MG132. (J) Sample fluorescence images of cells expressing SNAP-Nup188 treated with MG132 for 0 or 4 h. Line profiles of the fluorescence along the white lines shown in images bisecting the nucleus are shown in right panels. Note the y axis scale and intranuclear fluorescence in bottom panels. Arrows denote location of nuclear rim along line profile plot.

    Techniques Used: Quantitative RT-PCR, Labeling, Imaging, Fluorescence, Microscopy, Expressing

    Nup188 is stabilized upon proteasome inhibition. (A) SNAP-Nup188 levels are below endogenous Nup188 levels. Western blot (and fluorescence labeling of SNAP-Nup188 with a 647-SiR dye, top panel) of total protein extracts derived from SNAP-Nup188–expressing HeLa cells collected after 48 h in the presence (+) or absence (−) of dox. Note that the putative position of SNAP-Nup188 is indicated, as it is expressed at levels that cannot be detected with the α-Nup188 antibody. α-β-Actin is a protein load reference. (B) Inhibition of the proteasome stabilizes Nup188 levels leaving mitosis. Nocodazole-arrested HeLa-M cells were released in the presence of MG132 or carrier alone (DMSO). Western blots of total protein extracts using the indicated primary antibodies were performed at the indicated time points. α-β-Actin is a protein load reference. (C) Nup188 is ubiquitylated. Western blots of input (5%) and immunoprecipitated (IP) fractions (100%) of FLAG-Nup188 or FLAG alone from cell extracts coexpressing HA-Ubiquitin (HA-Ub) treated with MG132 or DMSO. Use legend at top to interpret whether a given plasmid or treatment has been added (+) or not (−). FLAG-Nup188 was detected with an α-Flag antibody and HA-Ub–conjugates using α-HA antibodies. (D) Western blots examining the levels of BirA*-Nup188 in total protein samples derived from BirA*-Nup188–expressing HeLa cells treated with increasing amounts of dox. Expected position of BirA*-Nup188 is indicated relative to the observed endogenous Nup188; α-α-Tubulin is a reference for total protein loads. Numbers at left of all panels are positions of molecular weight standards (in kilodaltons).
    Figure Legend Snippet: Nup188 is stabilized upon proteasome inhibition. (A) SNAP-Nup188 levels are below endogenous Nup188 levels. Western blot (and fluorescence labeling of SNAP-Nup188 with a 647-SiR dye, top panel) of total protein extracts derived from SNAP-Nup188–expressing HeLa cells collected after 48 h in the presence (+) or absence (−) of dox. Note that the putative position of SNAP-Nup188 is indicated, as it is expressed at levels that cannot be detected with the α-Nup188 antibody. α-β-Actin is a protein load reference. (B) Inhibition of the proteasome stabilizes Nup188 levels leaving mitosis. Nocodazole-arrested HeLa-M cells were released in the presence of MG132 or carrier alone (DMSO). Western blots of total protein extracts using the indicated primary antibodies were performed at the indicated time points. α-β-Actin is a protein load reference. (C) Nup188 is ubiquitylated. Western blots of input (5%) and immunoprecipitated (IP) fractions (100%) of FLAG-Nup188 or FLAG alone from cell extracts coexpressing HA-Ubiquitin (HA-Ub) treated with MG132 or DMSO. Use legend at top to interpret whether a given plasmid or treatment has been added (+) or not (−). FLAG-Nup188 was detected with an α-Flag antibody and HA-Ub–conjugates using α-HA antibodies. (D) Western blots examining the levels of BirA*-Nup188 in total protein samples derived from BirA*-Nup188–expressing HeLa cells treated with increasing amounts of dox. Expected position of BirA*-Nup188 is indicated relative to the observed endogenous Nup188; α-α-Tubulin is a reference for total protein loads. Numbers at left of all panels are positions of molecular weight standards (in kilodaltons).

    Techniques Used: Inhibition, Western Blot, Fluorescence, Labeling, Derivative Assay, Expressing, Immunoprecipitation, Plasmid Preparation, Molecular Weight

    The centrosomal pool of Nup188 is sensitive to siRNA knockdown. (A) Western blots (or fluorescence image/top panel) of total protein from SNAP-NUP188–expressing HeLa cell extracts prepared from cells treated with the indicated siRNAs for 12 or 24 h. α-β-Actin is a protein load reference. Numbers at left are positions of molecular weight standards (in kilodaltons). (B) Fluorescence micrographs of SNAP-Nup188 (labeled with 647-SiR, red) in HeLa cells treated with NUP188 or scrambled siRNA for 12 or 24 h. Centrosomes are labeled with α-Pericentrin (green). Scale bar is 5 µm. Magnifications of boxed region are shown at right. Scale bar is 0.5 µm. (C and D) Plots of mean SNAP-Nup188 fluorescence (647-SiR) ± SD at centrosomes and the nuclear envelope in the indicated conditions where > 30 cells were quantified.
    Figure Legend Snippet: The centrosomal pool of Nup188 is sensitive to siRNA knockdown. (A) Western blots (or fluorescence image/top panel) of total protein from SNAP-NUP188–expressing HeLa cell extracts prepared from cells treated with the indicated siRNAs for 12 or 24 h. α-β-Actin is a protein load reference. Numbers at left are positions of molecular weight standards (in kilodaltons). (B) Fluorescence micrographs of SNAP-Nup188 (labeled with 647-SiR, red) in HeLa cells treated with NUP188 or scrambled siRNA for 12 or 24 h. Centrosomes are labeled with α-Pericentrin (green). Scale bar is 5 µm. Magnifications of boxed region are shown at right. Scale bar is 0.5 µm. (C and D) Plots of mean SNAP-Nup188 fluorescence (647-SiR) ± SD at centrosomes and the nuclear envelope in the indicated conditions where > 30 cells were quantified.

    Techniques Used: Western Blot, Fluorescence, Expressing, Molecular Weight, Labeling

    9) Product Images from "A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression"

    Article Title: A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0791-1

    Subcellular localization of FLAG-SNAP-TERT. IF analysis of fixed HeLa cells expressing FLAG-SNAP-TERT. The SNAP-tag was labeled with SNAP-Cell® 647-SiR dye (scale bar = 5 μm). Telomeres and Cajal bodies were stained with antibodies against TRF2 and coilin, respectively. Edited cells but not parental cells showed FLAG-SNAP-TERT foci that co-localized with telomeres and Cajal bodies. Two independent clones expressing FLAG-SNAP-TERT were used to generate the images shown
    Figure Legend Snippet: Subcellular localization of FLAG-SNAP-TERT. IF analysis of fixed HeLa cells expressing FLAG-SNAP-TERT. The SNAP-tag was labeled with SNAP-Cell® 647-SiR dye (scale bar = 5 μm). Telomeres and Cajal bodies were stained with antibodies against TRF2 and coilin, respectively. Edited cells but not parental cells showed FLAG-SNAP-TERT foci that co-localized with telomeres and Cajal bodies. Two independent clones expressing FLAG-SNAP-TERT were used to generate the images shown

    Techniques Used: Expressing, Labeling, Staining, Clone Assay

    10) Product Images from "Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Locally Altering Mechanical Properties of Cell Membranes"

    Article Title: Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Locally Altering Mechanical Properties of Cell Membranes

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.171280

    IFITM3 partitions into liquid-disordered membrane domains that support IAV fusion. (A) A diagram of phase-separated GUV. (B) A representative example of phase-separated GUV containing 33.3 mol % DOPC, 33.3 mol % SM, 32.4 mol % cholesterol, 0.5 mol % TopFluor-cholesterol (marker of Lo domain) and 0.5% Liss-Rho-PE (marker of Ld domain). (C) Phase-separated GUVs (33.3 mol % DOPC, 33.3 mol % SM, 32.4 mol % cholesterol, 0.5 mol % TopFluor-cholesterol and 0.5% Liss-Rho-PE) were incubated with 10 μM of Cy-5-labeled AH (56-71) or Scrambled AH(56-71) for 30 min and imaged. Scale bars 10 μm. (D) Plasma membrane spheres were prepared from IFITM3-iSNAP expressing A549 cells by cell swelling. GM1 was crosslinked with Cholera toxin B-AF488 (green) to mark the Lo phase and SNAP tag was stained with SNAP-cell 647-SIR (red). Scale bar 2 μm. (E) A diagram depicting lipid mixing between DiD-labeled IAV and GUV triggered by low pH leading to DiD dequenching. (F) Ld GUVs (top, 97.5 mol % DOPC, 2% GM1, 0.5% NBD-PE) or Lo GUVs (bottom, 66.6 mol % SM, 30.9 mol % cholesterol, 2% GM1, 0.5 mol % TopFluor-cholesterol) were mixed with DiD labeled IAV, lipids mixing was triggered by addition of citrate buffer to achieve the final pH of 5.0, and samples immediately imaged. Scale bars 5 μm. See also Figure S4.
    Figure Legend Snippet: IFITM3 partitions into liquid-disordered membrane domains that support IAV fusion. (A) A diagram of phase-separated GUV. (B) A representative example of phase-separated GUV containing 33.3 mol % DOPC, 33.3 mol % SM, 32.4 mol % cholesterol, 0.5 mol % TopFluor-cholesterol (marker of Lo domain) and 0.5% Liss-Rho-PE (marker of Ld domain). (C) Phase-separated GUVs (33.3 mol % DOPC, 33.3 mol % SM, 32.4 mol % cholesterol, 0.5 mol % TopFluor-cholesterol and 0.5% Liss-Rho-PE) were incubated with 10 μM of Cy-5-labeled AH (56-71) or Scrambled AH(56-71) for 30 min and imaged. Scale bars 10 μm. (D) Plasma membrane spheres were prepared from IFITM3-iSNAP expressing A549 cells by cell swelling. GM1 was crosslinked with Cholera toxin B-AF488 (green) to mark the Lo phase and SNAP tag was stained with SNAP-cell 647-SIR (red). Scale bar 2 μm. (E) A diagram depicting lipid mixing between DiD-labeled IAV and GUV triggered by low pH leading to DiD dequenching. (F) Ld GUVs (top, 97.5 mol % DOPC, 2% GM1, 0.5% NBD-PE) or Lo GUVs (bottom, 66.6 mol % SM, 30.9 mol % cholesterol, 2% GM1, 0.5 mol % TopFluor-cholesterol) were mixed with DiD labeled IAV, lipids mixing was triggered by addition of citrate buffer to achieve the final pH of 5.0, and samples immediately imaged. Scale bars 5 μm. See also Figure S4.

    Techniques Used: Marker, Incubation, Labeling, Expressing, Staining

    11) Product Images from "Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance"

    Article Title: Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22575-5

    The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell SiR-647. The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.
    Figure Legend Snippet: The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell SiR-647. The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Irradiation, Imaging, Staining

    12) Product Images from "New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics"

    Article Title: New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics

    Journal: bioRxiv

    doi: 10.1101/092536

    Comparison of SNAP labeling and nonspecific binding. (A) Wild-type cells were incubated with SNAP-SiR647, washed, and imaged in TIRF as described in the text. (B and C) Cells expressing Pil1p-SNAP (B) or wild-type cells (C) were incubated with SNAP-Alexa647, washed, and imaged as described. Images shown are inverted contrast, Maximum intensity projections of 20-sec movies with median-filter background subtracted. Cell outlines are drawn in orange dash; all image panels are at same scale with scale bar 5 μm.
    Figure Legend Snippet: Comparison of SNAP labeling and nonspecific binding. (A) Wild-type cells were incubated with SNAP-SiR647, washed, and imaged in TIRF as described in the text. (B and C) Cells expressing Pil1p-SNAP (B) or wild-type cells (C) were incubated with SNAP-Alexa647, washed, and imaged as described. Images shown are inverted contrast, Maximum intensity projections of 20-sec movies with median-filter background subtracted. Cell outlines are drawn in orange dash; all image panels are at same scale with scale bar 5 μm.

    Techniques Used: Labeling, Binding Assay, Incubation, Expressing

    13) Product Images from "Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context"

    Article Title: Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

    Journal: Genome Research

    doi: 10.1101/gr.257352.119

    Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.
    Figure Legend Snippet: Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.

    Techniques Used: Derivative Assay, Construct, Staining

    14) Product Images from "Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport"

    Article Title: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08192-9

    PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )
    Figure Legend Snippet: PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )

    Techniques Used: Expressing, Staining, Epifluorescence Microscopy, Labeling

    15) Product Images from "Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products"

    Article Title: Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products

    Journal: Journal of Alzheimer's Disease

    doi: 10.3233/JAD-190898

    A, B) Time series of dual labelled AβPP-SNAP protein over a time frame of two minutes shows vesicular movement. HEK293T cells were transfected with the amber codon machinery and AβPP H13 > amber. Live cells were labeled with SNAP-Cell® 647-SiR (red) and mT-BDP-FL, which reacts with the ncAA (green). Labeled cells were imaged simultaneous by confocal microscopy (63× magnification). Blue arrows indicate nuclei, pink arrows indicate subcellular locations, where most AβPP is located.
    Figure Legend Snippet: A, B) Time series of dual labelled AβPP-SNAP protein over a time frame of two minutes shows vesicular movement. HEK293T cells were transfected with the amber codon machinery and AβPP H13 > amber. Live cells were labeled with SNAP-Cell® 647-SiR (red) and mT-BDP-FL, which reacts with the ncAA (green). Labeled cells were imaged simultaneous by confocal microscopy (63× magnification). Blue arrows indicate nuclei, pink arrows indicate subcellular locations, where most AβPP is located.

    Techniques Used: Transfection, Labeling, Confocal Microscopy

    16) Product Images from "Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport"

    Article Title: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08192-9

    PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )
    Figure Legend Snippet: PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )

    Techniques Used: Expressing, Staining, Epifluorescence Microscopy, Labeling

    17) Product Images from "Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport"

    Article Title: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08192-9

    PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )
    Figure Legend Snippet: PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )

    Techniques Used: Expressing, Staining, Epifluorescence Microscopy, Labeling

    18) Product Images from "Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context"

    Article Title: Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

    Journal: Genome Research

    doi: 10.1101/gr.257352.119

    Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.
    Figure Legend Snippet: Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.

    Techniques Used: Derivative Assay, Construct, Staining

    19) Product Images from "Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane"

    Article Title: Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000998

    The luminal hydrophobic helix is important for TAG clustering within the seipin disk. (A) Snapshots of coarse-grained simulations in the absence of seipin with 2.5 mol% TAG in an ER bilayer. For clarity, PL head groups (yellow) are only shown in the side view, and water is not shown. The acyl chains of TAGs are shown in cyan; glycerol moiety is in light brown. (B) Snapshots of simulations as in (A), but in the presence of seipin. (C) TAG occupancy data of simulations demonstrated in (B). TAG occupancy for each amino acid residue is defined as the probability that the residue has a TAG molecule within 0.5 nm distance from its surface. The TAG occupancies for the initial (0–1 μs) and later (4–5 μs) stages of the simulation are plotted separately. Bars: mean ± SEM, n = 10 replicates. (D) Key residues from (C) are highlighted. (E) A431 SKO cells stably expressing indicated plasmids were delipidated for 3 d and treated with 200 μM OA for 1 h. Cells were fixed, LDs stained, and cells imaged by Airyscan microscopy. Maximum intensity projections of z- stacks. Orange arrowheads: tiny LDs; yellow arrowheads: supersized LDs. (F) Analysis of (E). Bars: mean ± SEM, n ≥ 60 cells/group, 3–4 experiments. Statistics: Kruskal–Wallis test followed by Dunn’s test, comparing against WT-seipin-GFPx7. (G) End-seipin-SNAPf cells and SKO cells stably expressing WT-, S166A-, or S166D-seipin-GFPx7 were co-plated for 2 d in delipidation conditions. Cells were fused with polyethylene glycol, and 12–14 h later 200 μM OA and SNAP-Cell 647-SiR were added to the cells. Four hours after this, LDs were stained with MDH, and fused cells were imaged live. (H) Analysis of (G). The sizes of end-seipin-SNAPf-associated LDs were compared to LDs within the same cell not positive for SNAPf. Bars: mean ± SEM, n = 152–659 LDs/group, 4–20 fused cells/group, 2 experiments. Statistics: Mann–Whitney test. Exemplary micrographs are shown in S1D Fig . Numerical values for the graphs in (C), (F), and (H) can be found in S1 Data . ER, endoplasmic reticulum; KO, knockout; LD, lipid droplet, MDH, monodansylpentane; mol%, mole percent; OA, oleic acid; SKO, seipin knockout; TAG, triacylglycerol; WT, wild-type.
    Figure Legend Snippet: The luminal hydrophobic helix is important for TAG clustering within the seipin disk. (A) Snapshots of coarse-grained simulations in the absence of seipin with 2.5 mol% TAG in an ER bilayer. For clarity, PL head groups (yellow) are only shown in the side view, and water is not shown. The acyl chains of TAGs are shown in cyan; glycerol moiety is in light brown. (B) Snapshots of simulations as in (A), but in the presence of seipin. (C) TAG occupancy data of simulations demonstrated in (B). TAG occupancy for each amino acid residue is defined as the probability that the residue has a TAG molecule within 0.5 nm distance from its surface. The TAG occupancies for the initial (0–1 μs) and later (4–5 μs) stages of the simulation are plotted separately. Bars: mean ± SEM, n = 10 replicates. (D) Key residues from (C) are highlighted. (E) A431 SKO cells stably expressing indicated plasmids were delipidated for 3 d and treated with 200 μM OA for 1 h. Cells were fixed, LDs stained, and cells imaged by Airyscan microscopy. Maximum intensity projections of z- stacks. Orange arrowheads: tiny LDs; yellow arrowheads: supersized LDs. (F) Analysis of (E). Bars: mean ± SEM, n ≥ 60 cells/group, 3–4 experiments. Statistics: Kruskal–Wallis test followed by Dunn’s test, comparing against WT-seipin-GFPx7. (G) End-seipin-SNAPf cells and SKO cells stably expressing WT-, S166A-, or S166D-seipin-GFPx7 were co-plated for 2 d in delipidation conditions. Cells were fused with polyethylene glycol, and 12–14 h later 200 μM OA and SNAP-Cell 647-SiR were added to the cells. Four hours after this, LDs were stained with MDH, and fused cells were imaged live. (H) Analysis of (G). The sizes of end-seipin-SNAPf-associated LDs were compared to LDs within the same cell not positive for SNAPf. Bars: mean ± SEM, n = 152–659 LDs/group, 4–20 fused cells/group, 2 experiments. Statistics: Mann–Whitney test. Exemplary micrographs are shown in S1D Fig . Numerical values for the graphs in (C), (F), and (H) can be found in S1 Data . ER, endoplasmic reticulum; KO, knockout; LD, lipid droplet, MDH, monodansylpentane; mol%, mole percent; OA, oleic acid; SKO, seipin knockout; TAG, triacylglycerol; WT, wild-type.

    Techniques Used: Stable Transfection, Expressing, Staining, Microscopy, MANN-WHITNEY, Knock-Out

    Related Articles

    Labeling:

    Article Title: Differential turnover of Nup188 controls its levels at centrosomes and role in centriole duplication
    Article Snippet: .. SNAP-Nup188 labeling To label SNAP-Nup188 with fluorophores, we used either a 647-SiR dye (SNAP-Cell 647-SiR; NEB) or TMR-Star dye (SNAP-Cell TMR-Star; NEB). .. Briefly, expression of SNAP-NUP188 was induced by adding doxycyclin (Millipore Sigma) to the culture medium at a final concentration of 100 ng/ml for 48 h. Labeling was performed in living cells by addition of SNAP-Cell 647-SiR or SNAP-Cell TMR-Star directly to the culture medium at a final concentration of 2 µM.

    Article Title: Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α‐actinin 4
    Article Snippet: 24 h before imaging, ACTN4‐SNAP expression was induced by 1 μg/ml doxycycline. .. SNAP‐tag was labeled prior each experiment with SNAP‐Cell 647SiR (NEB). .. Cells at mitotic exit (early G1) were identified by following live cells through mitotic cell division under the microscope until cytokinesis when new daughter cell nuclei are formed.

    Incubation:

    Article Title: Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings
    Article Snippet: For SNAP-Cell SiR647 (#S9102S, NEB) was also prepared from 1 mM dye stock solution in DMSO to a final concentration 1 µM, but to solve the dye into the media the final DMSO concentration was adjusted to 1% (v/v). .. Incubation times varied between 5-60 min depending on the dye and experiments; 5 or 10 mins for SNAP-Cell TMR-star, 30 mins for SNAP-Cell 430, and 60 mins for SNAP-Cell 647-SiR. .. After incubation with SNAP-dyes, cells were washed with 3% (w/v) sucrose several times, then resuspended in fresh LS medium.

    Synthesized:

    Article Title: Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance
    Article Snippet: SNAP-tag labelling of histones For specific labelling of newly synthesized histones , , cells were grown on glass coverslips and pre-existing SNAP-tagged histones were first quenched by incubating cells with 10 μM of the non-fluorescent substrate SNAP-cell Block (New England Biolabs) for 30 min followed by a 30-min wash in fresh medium and a 2-h chase. .. The new SNAP-tagged histones synthesized during the chase were fluorescently labelled with 2 μM of the red-fluorescent reagent SNAP-cell TMR star or SiR-647 (New England Biolabs) during a 15-min pulse step followed by 30-min wash in fresh medium. .. Cells were subsequently permeabilized with Triton X-100, fixed and processed for immunostaining.

    other:

    Article Title: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport
    Article Snippet: SNAP-Cell647-SiR reagent was purchased from New England Biolabs.

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    New England Biolabs sir 647
    The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell <t>SiR-647.</t> The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.
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    The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell SiR-647. The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

    doi: 10.1038/s41467-021-22575-5

    Figure Lengend Snippet: The UV damage sensor DDB2 promotes linker histone displacement from damaged chromatin. a Scheme of the experiment for simultaneous detection of H1 and H3.3 in live cells exposed to UVC laser damage. H1 variants are transiently expressed as mCherry-tagged fusions in NIH/3T3 GFP-DDB2 cells stably expressing H3.3-SNAP, which is labelled with SNAP-cell SiR-647. The levels of H1 variants and H3.3 are measured in UVC-damaged regions, identified by GFP-DDB2 accumulation (white arrowheads), relative to the whole nucleus at the indicated time points after laser damage. Results normalized to before laser damage are presented on the graphs. b mCherry-H1.4 signal in damaged heterochromatin domains (white arrowheads) 30 min after UVC laser micro-irradiation analyzed by live imaging in the indicated cell lines. CPD staining in fixed cells highlights the damaged chromocenter. The scatter plot represents the mCherry-H1.4 signal loss in UVC-damaged chromatin regions in both cell lines. Data are presented as mean values ± SD from n cells scored in at least three independent experiments. Comparisons of histone signal loss are based on non-linear regression with a polynomial quadratic model ( a ). Statistical significance in ( b ) is calculated via two-sided Student’s t test. Scale bars, 10 μm. Source data are provided as a Source Data file.

    Article Snippet: The new SNAP-tagged histones synthesized during the chase were fluorescently labelled with 2 μM of the red-fluorescent reagent SNAP-cell TMR star or SiR-647 (New England Biolabs) during a 15-min pulse step followed by 30-min wash in fresh medium.

    Techniques: Stable Transfection, Expressing, Irradiation, Imaging, Staining

    ACTN4 clusters dynamically associate with postmitotic nuclear actin filaments Cells in early G1 stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a time series on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2 B. Scale bar overview (nAC) represents 10 μm. Scale bar time series and magnification 5 μm. Linescan of an actin filament with associated ACTN4 from square 3 in (A). Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in (A). Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2 ). Scale bar is 2 μm. The track length, track displacement (distance between starting point and end point), average speed, and maximum speed were quantified from 3 independent experiments. The black line shows the median and gray dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.

    Journal: EMBO Reports

    Article Title: Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α‐actinin 4

    doi: 10.15252/embr.202050758

    Figure Lengend Snippet: ACTN4 clusters dynamically associate with postmitotic nuclear actin filaments Cells in early G1 stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a time series on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2 B. Scale bar overview (nAC) represents 10 μm. Scale bar time series and magnification 5 μm. Linescan of an actin filament with associated ACTN4 from square 3 in (A). Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in (A). Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2 ). Scale bar is 2 μm. The track length, track displacement (distance between starting point and end point), average speed, and maximum speed were quantified from 3 independent experiments. The black line shows the median and gray dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.

    Article Snippet: SNAP‐tag was labeled prior each experiment with SNAP‐Cell 647SiR (NEB).

    Techniques: Stable Transfection, Expressing, Time-lapse Microscopy, Labeling, Software

    ACTN4 associates with postmitotic nuclear actin filaments Cells stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. The cell is an alternative example of Fig 2 A that additionally covers the mitotic phase. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR (green). The magnification changes at time point 0:00. Scale bar 5 μm.

    Journal: EMBO Reports

    Article Title: Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α‐actinin 4

    doi: 10.15252/embr.202050758

    Figure Lengend Snippet: ACTN4 associates with postmitotic nuclear actin filaments Cells stably expressing nAC‐mCherry and doxycycline‐inducible ACTN4‐SNAP were analyzed by time‐lapse microscopy. The cell is an alternative example of Fig 2 A that additionally covers the mitotic phase. ACTN4‐SNAP was labeled by SNAP‐Cell 647SiR (green). The magnification changes at time point 0:00. Scale bar 5 μm.

    Article Snippet: SNAP‐tag was labeled prior each experiment with SNAP‐Cell 647SiR (NEB).

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

    Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.

    Journal: Genome Research

    Article Title: Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

    doi: 10.1101/gr.257352.119

    Figure Lengend Snippet: Localization of LIMK1-SNAP -derived products in cells. ( A , B ) Products from sP and wt constructs, respectively (red, stained with SNAP-Cell 647-SiR), counterstained with α-tubulin/Alexa Fluor 488 antibodies (cyan). ( Bottom ) Line profile analysis across three contacting cells shows the predominantly peripheral distribution of sP products, in contrast to more uniform cytosolic distribution of the wt products. ( C ) Colocalization of sP products (red) and F-actin (green, stained with phalloidin-Alexa 546). Images represent stacks of four ( A , B ) or three ( C ) focal planes taken with a 5 µm step.

    Article Snippet: Protein isolation and electrophoresisCells were washed with 1× PBS, and whole-cell lysates were prepared in a standard RIPA buffer supplemented with 0.01 μM SNAP-Cell 647-SiR fluorescent substrate (NEB).

    Techniques: Derivative Assay, Construct, Staining

    PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )

    Journal: Nature Communications

    Article Title: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport

    doi: 10.1038/s41467-018-08192-9

    Figure Lengend Snippet: PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Ac tub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in ( c ) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Ac tub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g , h HPNE ( g ) and NIH3T3 ( h ) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Ac tub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 μm for ( c , f – i )

    Article Snippet: SNAP-Cell647-SiR reagent was purchased from New England Biolabs.

    Techniques: Expressing, Staining, Epifluorescence Microscopy, Labeling