yfp  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    New England Biolabs yfp
    STIM1 is locally activated in the front of migrating cells. (a) HUVEC cells were co-transfected with <t>YFP-STIM1</t> and the ER-PM junction marker <t>CFP-ER-PM.</t> Confocal images show focal planes at the bottom of the cell. The white arrow marks the direction of migration.YFP-STIM1 was enriched at front ER-PM junctions in migrating cells. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER-PM from front to back in migrating cells. (n =14 cells) (c,d) Similar analysis as in (b) but for cells coexpressing YFP-S1NN and CFP-ER-PM (c) or coexpressing YFP-ER-PM and CFP-ER-PM. A smaller increase in relative S1NN activity was observed in the front (n = 10 cells for S1NN and n = 12 cells for the control group). (e,f) Decreasing luminal ER Ca 2+ levels towards the front of migrating leader cells. (e) Ratio-imaging of a modified luminal ER Ca 2+ FRET probe T1ER (see Methods ) in migrating HUVECs. Adding the SERCA inhibitor thapsigargin (2 μM) and EGTA (3 mM) decreased ER Ca 2+ levels (lower panel). (f) Gradient in luminal ER Ca 2+ measured using the T1ER probe. Note that the lower Ca 2+ levels in the front are still sensitive to EGTA+thapsigargin treatment (n = 79 cells for the control group; n = 49 cells for the thapsigargin + EGTA group).
    Yfp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yfp/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    yfp - by Bioz Stars, 2022-07
    86/100 stars

    Images

    1) Product Images from "A polarized Ca2+, diacylglycerol, and STIM1 signaling system regulates directed cell migration"

    Article Title: A polarized Ca2+, diacylglycerol, and STIM1 signaling system regulates directed cell migration

    Journal: Nature cell biology

    doi: 10.1038/ncb2906

    STIM1 is locally activated in the front of migrating cells. (a) HUVEC cells were co-transfected with YFP-STIM1 and the ER-PM junction marker CFP-ER-PM. Confocal images show focal planes at the bottom of the cell. The white arrow marks the direction of migration.YFP-STIM1 was enriched at front ER-PM junctions in migrating cells. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER-PM from front to back in migrating cells. (n =14 cells) (c,d) Similar analysis as in (b) but for cells coexpressing YFP-S1NN and CFP-ER-PM (c) or coexpressing YFP-ER-PM and CFP-ER-PM. A smaller increase in relative S1NN activity was observed in the front (n = 10 cells for S1NN and n = 12 cells for the control group). (e,f) Decreasing luminal ER Ca 2+ levels towards the front of migrating leader cells. (e) Ratio-imaging of a modified luminal ER Ca 2+ FRET probe T1ER (see Methods ) in migrating HUVECs. Adding the SERCA inhibitor thapsigargin (2 μM) and EGTA (3 mM) decreased ER Ca 2+ levels (lower panel). (f) Gradient in luminal ER Ca 2+ measured using the T1ER probe. Note that the lower Ca 2+ levels in the front are still sensitive to EGTA+thapsigargin treatment (n = 79 cells for the control group; n = 49 cells for the thapsigargin + EGTA group).
    Figure Legend Snippet: STIM1 is locally activated in the front of migrating cells. (a) HUVEC cells were co-transfected with YFP-STIM1 and the ER-PM junction marker CFP-ER-PM. Confocal images show focal planes at the bottom of the cell. The white arrow marks the direction of migration.YFP-STIM1 was enriched at front ER-PM junctions in migrating cells. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER-PM from front to back in migrating cells. (n =14 cells) (c,d) Similar analysis as in (b) but for cells coexpressing YFP-S1NN and CFP-ER-PM (c) or coexpressing YFP-ER-PM and CFP-ER-PM. A smaller increase in relative S1NN activity was observed in the front (n = 10 cells for S1NN and n = 12 cells for the control group). (e,f) Decreasing luminal ER Ca 2+ levels towards the front of migrating leader cells. (e) Ratio-imaging of a modified luminal ER Ca 2+ FRET probe T1ER (see Methods ) in migrating HUVECs. Adding the SERCA inhibitor thapsigargin (2 μM) and EGTA (3 mM) decreased ER Ca 2+ levels (lower panel). (f) Gradient in luminal ER Ca 2+ measured using the T1ER probe. Note that the lower Ca 2+ levels in the front are still sensitive to EGTA+thapsigargin treatment (n = 79 cells for the control group; n = 49 cells for the thapsigargin + EGTA group).

    Techniques Used: Transfection, Marker, Migration, Activity Assay, Imaging, Modification

    Store-operated Ca 2+ (SOC) influx controls cell migration by regulating cell-matrix adhesion in the front of migrating cells. (a) HUVEC migration into open space was monitored by staining cells with CellMask (see Methods ). Accelerated sheet migration was observed in STIM1-depleted compared to control cells. (b) Comparing changes in the rate of sheet migration and cytosolic Ca 2+ levels in HUVECs treated with siRNAs targeting different Ca 2+ signaling regulators. Average cytosolic [Ca 2+ ] was normalized to the level of cells treated with siCntrl (n = 4 experiments for each siRNA). (c) Reduced single cell migration speed in cells over-expressing YFP-STIM1. Cells expressing YFP-ER were used as control (n ~ 10,000 cells per condition). (d,e) Effects of the ER Ca 2+ pump blocker thapsigargin (d) and the SOC inhibitor BTP2 (e) on cytosolic Ca 2+ levels and on sheet migration speed. Notice that increasing cytosolic Ca 2+ levels by thapsigargin decreased migration speed, and lowering Ca 2+ levels by BTP2 increased migration speed (n = 4 experiments per condition). (f,g) Migrating HUVECs were treated with different concentrations of BTP2 or thapsigargin to reduce or elevate cytosolic Ca 2+ levels. Cells were then fixed and stained with anti-phospho-myosin light chain (pMLC) antibody. (f) pMLC signals were lower when SOC was blocked by BTP2 but higher when ER Ca 2+ pumps were blocked by thapsigargin. CAAX: plasma membrane marker. (g) pMLC levels increased with increasing cytosolic [Ca 2+ ] (n = 123, 134, 127, 126, 123, 117 and 142 cells per condition from left to right). (h,i) Effect of BTP2 treatment on cell-matrix adhesion. Focal adhesion formation was monitored by expressing GFP-paxillin. BTP2 treatment rapidly decreased the intensities of GFP-paxillin puncta in the front of migrating cells, consistent with SOC influx promoting cell-matrix adhesion. Bars are mean ± SEM in Fig. 2b,d,e,g.
    Figure Legend Snippet: Store-operated Ca 2+ (SOC) influx controls cell migration by regulating cell-matrix adhesion in the front of migrating cells. (a) HUVEC migration into open space was monitored by staining cells with CellMask (see Methods ). Accelerated sheet migration was observed in STIM1-depleted compared to control cells. (b) Comparing changes in the rate of sheet migration and cytosolic Ca 2+ levels in HUVECs treated with siRNAs targeting different Ca 2+ signaling regulators. Average cytosolic [Ca 2+ ] was normalized to the level of cells treated with siCntrl (n = 4 experiments for each siRNA). (c) Reduced single cell migration speed in cells over-expressing YFP-STIM1. Cells expressing YFP-ER were used as control (n ~ 10,000 cells per condition). (d,e) Effects of the ER Ca 2+ pump blocker thapsigargin (d) and the SOC inhibitor BTP2 (e) on cytosolic Ca 2+ levels and on sheet migration speed. Notice that increasing cytosolic Ca 2+ levels by thapsigargin decreased migration speed, and lowering Ca 2+ levels by BTP2 increased migration speed (n = 4 experiments per condition). (f,g) Migrating HUVECs were treated with different concentrations of BTP2 or thapsigargin to reduce or elevate cytosolic Ca 2+ levels. Cells were then fixed and stained with anti-phospho-myosin light chain (pMLC) antibody. (f) pMLC signals were lower when SOC was blocked by BTP2 but higher when ER Ca 2+ pumps were blocked by thapsigargin. CAAX: plasma membrane marker. (g) pMLC levels increased with increasing cytosolic [Ca 2+ ] (n = 123, 134, 127, 126, 123, 117 and 142 cells per condition from left to right). (h,i) Effect of BTP2 treatment on cell-matrix adhesion. Focal adhesion formation was monitored by expressing GFP-paxillin. BTP2 treatment rapidly decreased the intensities of GFP-paxillin puncta in the front of migrating cells, consistent with SOC influx promoting cell-matrix adhesion. Bars are mean ± SEM in Fig. 2b,d,e,g.

    Techniques Used: Migration, Staining, Expressing, Marker

    STIM1 is enriched in the front of migrating cells. (a,b) Migrating HUVEC expressed YFP-STIM1 (STIM1) and a CFP-tagged ER marker (ER). Merged and ratio images are shown here and in Supplementary Fig. 4a to show the relative enrichment of STIM1 compared to an ER marker towards the front. White arrow indicates the direction of cell migration. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER marker from front to back (see Methods ). YFP-STIM1 was enriched in the front, whereas (c) the control YFP-ER was not (n = 36 cells per condition). (d–f) Enrichment of STIM1 in the front of migrating cells is mediated by binding to the microtubule plus-end binding protein EB1. (d) Domain structure of STIM1 and mutations preventing binding to EB1. (e) Unlike wild-type STIM1 (S1wt) protein (Fig. 4a,b and Supplementary Fig. 4a ), the S1NN mutant was not enriched in the front of migrating cells. (n = 27 cells) (f) Over-expression of the S1NN mutant suppressed cell migration less than overexpression of S1wt. Bars are mean ± SEM (n ~ 10,000 cells per condition).
    Figure Legend Snippet: STIM1 is enriched in the front of migrating cells. (a,b) Migrating HUVEC expressed YFP-STIM1 (STIM1) and a CFP-tagged ER marker (ER). Merged and ratio images are shown here and in Supplementary Fig. 4a to show the relative enrichment of STIM1 compared to an ER marker towards the front. White arrow indicates the direction of cell migration. (b) Quantification of the ratio of YFP-STIM1 / CFP-ER marker from front to back (see Methods ). YFP-STIM1 was enriched in the front, whereas (c) the control YFP-ER was not (n = 36 cells per condition). (d–f) Enrichment of STIM1 in the front of migrating cells is mediated by binding to the microtubule plus-end binding protein EB1. (d) Domain structure of STIM1 and mutations preventing binding to EB1. (e) Unlike wild-type STIM1 (S1wt) protein (Fig. 4a,b and Supplementary Fig. 4a ), the S1NN mutant was not enriched in the front of migrating cells. (n = 27 cells) (f) Over-expression of the S1NN mutant suppressed cell migration less than overexpression of S1wt. Bars are mean ± SEM (n ~ 10,000 cells per condition).

    Techniques Used: Marker, Migration, Binding Assay, Mutagenesis, Over Expression

    Receptor tyrosine kinase (RTK) signaling is restricted to the front of migrating leader cells. (a,b) bFGF-induced tyrosine phosphorylation was higher in the front of migrating cells (white arrows). Addition of the pan-RTK inhibitor Ponatinib blocked tyrosine kinase signaling in the front, but not in the back of leader cells. Follower cells did not respond to bFGF. HUVECs were fixed and stained with pY20 anti-phospho-tyrosine antibody (n = 107, 105, 115, 110 and 107 cells for SFM, follower cells, and Ponatinib 0 nM, 25 nM and 100 nM, respectively). SFM: serum-free medium. (c,e) Fluorescence ratio images of leader cells co-expressing YFP-Akt-PH (PIP 3 sensor) or YFP-C1AC1A (DAG sensor) and a plasma membrane marker (CFP-mCD4). PIP 3 (c) and DAG (e) were enriched in the front of migrating cells. (d,f) Front-to-back gradients of PIP 3 and DAG were present in leader, but not follower cells (24 leader and 42 follower cells in (d) , 28 and 62 cells in (f) ). (g) Ca 2+ pulses in migrating HUVECs were measured as relative increases in local PM targeted GCaMP6s fluorescence intensity. Higher activities were observed in the front (#1) compared to the middle (#2) or back (#3) of migrating cells. (h) Relative mean amplitudes of local Ca 2+ fluctuations measured over 3 minutes in the front of migrating cells in response to serum or serum plus Ponatinib (see also Supplementary Fig. 1e,f ). Amplitudes of Ca 2+ fluctuations were normalized to basal cytosolic levels (0.3 R.U. means the fluctuation is 30% of the average cytosolic [Ca 2+ ] level; n = 24 cells). (i,j) Migrating HUVECs expressing GCaMP6s-CAAX and the reference membrane marker mCD4 were used to measure Ca 2+ gradients in leader and follower cells (n = 83 leader and n = 86 follower cells). Bars denote mean ± SEM in Fig. 1b,h. Student t test was used for Fig. 1b,d,f,h,j. In Fig. 1d,f,j, p values were calculated by comparing the ratio of the sensor / PM intensity ratios in the front and back (both regions were 10% of cell length).
    Figure Legend Snippet: Receptor tyrosine kinase (RTK) signaling is restricted to the front of migrating leader cells. (a,b) bFGF-induced tyrosine phosphorylation was higher in the front of migrating cells (white arrows). Addition of the pan-RTK inhibitor Ponatinib blocked tyrosine kinase signaling in the front, but not in the back of leader cells. Follower cells did not respond to bFGF. HUVECs were fixed and stained with pY20 anti-phospho-tyrosine antibody (n = 107, 105, 115, 110 and 107 cells for SFM, follower cells, and Ponatinib 0 nM, 25 nM and 100 nM, respectively). SFM: serum-free medium. (c,e) Fluorescence ratio images of leader cells co-expressing YFP-Akt-PH (PIP 3 sensor) or YFP-C1AC1A (DAG sensor) and a plasma membrane marker (CFP-mCD4). PIP 3 (c) and DAG (e) were enriched in the front of migrating cells. (d,f) Front-to-back gradients of PIP 3 and DAG were present in leader, but not follower cells (24 leader and 42 follower cells in (d) , 28 and 62 cells in (f) ). (g) Ca 2+ pulses in migrating HUVECs were measured as relative increases in local PM targeted GCaMP6s fluorescence intensity. Higher activities were observed in the front (#1) compared to the middle (#2) or back (#3) of migrating cells. (h) Relative mean amplitudes of local Ca 2+ fluctuations measured over 3 minutes in the front of migrating cells in response to serum or serum plus Ponatinib (see also Supplementary Fig. 1e,f ). Amplitudes of Ca 2+ fluctuations were normalized to basal cytosolic levels (0.3 R.U. means the fluctuation is 30% of the average cytosolic [Ca 2+ ] level; n = 24 cells). (i,j) Migrating HUVECs expressing GCaMP6s-CAAX and the reference membrane marker mCD4 were used to measure Ca 2+ gradients in leader and follower cells (n = 83 leader and n = 86 follower cells). Bars denote mean ± SEM in Fig. 1b,h. Student t test was used for Fig. 1b,d,f,h,j. In Fig. 1d,f,j, p values were calculated by comparing the ratio of the sensor / PM intensity ratios in the front and back (both regions were 10% of cell length).

    Techniques Used: Staining, Fluorescence, Expressing, Marker

    2) Product Images from "DNA origami signposts for identifying proteins on cell membranes by electron cryotomography"

    Article Title: DNA origami signposts for identifying proteins on cell membranes by electron cryotomography

    Journal: Cell

    doi: 10.1016/j.cell.2021.01.033

    Targeting of fluorescent protein tags by an RNA aptamer The raw data for each experiment can be found in Figure S3 . (A) Isothermal titration calorimetry (ITC) of the RNA aptamer alone binding to different fluorescent proteins. (B) Acrylamide gel-shift assay to confirm aptamer binding to fluorescent proteins. The dotted lines indicate bands corresponding to each fluorescent protein without (top line, left column) and with (bottom line, right column) aptamer. (C) Agarose gel-shift assay to confirm binding to fluorescent proteins of the aptamer incorporated into the signpost DNA origami structure. YFP fluorescence was used for detection. Shown at the top is YFP alone; at the bottom is a fusion protein of YFP and Env, the major membrane glycoprotein of murine leukemia virus. YFP and YFP-Env run as sharper bands (black arrow heads) when bound to SPOTs (right lane) than the proteins alone (left lane), as expected from decreased diffusion with SPOTs bound. YFP-Env is expected to run as two species because of the partial isomerization of a disulphide bond, which produces species with both single and triplicate copies of YFP. Some of the DNA nanostructures and associated proteins aggregate in the well (top of image). These samples were run on a single gel; the image is split to better fit figure spacing. (D) Bio-layer interferometry (BLI) was used to confirm the binding of the aptamer-functionalized origami (SPOT) to isolated fluorescent proteins (top) and to a fluorescent protein fusion to glycoprotein B (gB), a surface glycoprotein from herpes simplex virus 1 in native membrane vesicles (bottom). As observed in the other assays, SPOTs bound to the sfGFP or sfGFP-gB much more than signpost origami alone, and SPOTs specifically bound sfGFP or sfGFP-gB, not mCherry or mCherry-gB. Each experiment was run in triplicate, and the lighter region behind each measurement indicates the standard deviation across the three replicates. (E) Dynamic light scattering was used to check for aggregation in concentrated solutions of SPOTs in buffer (left) and cell culture medium (center). A small amount of aggregate was only observed in culture medium at SPOT concentrations of 200 nM, ∼20x higher than needed for cellular experiments ( Figure S6 ). The 50 nM and 10 nM samples were too dilute to observe in culture medium. The aggregation-induced control sample (gray dotted lines) was stained for electron microscopy (right) to monitor the amount of aggregation (scale bar, 200 nm). Such aggregation was not seen in normal preparations of SPOTs ( Figures 2 B and 2C).
    Figure Legend Snippet: Targeting of fluorescent protein tags by an RNA aptamer The raw data for each experiment can be found in Figure S3 . (A) Isothermal titration calorimetry (ITC) of the RNA aptamer alone binding to different fluorescent proteins. (B) Acrylamide gel-shift assay to confirm aptamer binding to fluorescent proteins. The dotted lines indicate bands corresponding to each fluorescent protein without (top line, left column) and with (bottom line, right column) aptamer. (C) Agarose gel-shift assay to confirm binding to fluorescent proteins of the aptamer incorporated into the signpost DNA origami structure. YFP fluorescence was used for detection. Shown at the top is YFP alone; at the bottom is a fusion protein of YFP and Env, the major membrane glycoprotein of murine leukemia virus. YFP and YFP-Env run as sharper bands (black arrow heads) when bound to SPOTs (right lane) than the proteins alone (left lane), as expected from decreased diffusion with SPOTs bound. YFP-Env is expected to run as two species because of the partial isomerization of a disulphide bond, which produces species with both single and triplicate copies of YFP. Some of the DNA nanostructures and associated proteins aggregate in the well (top of image). These samples were run on a single gel; the image is split to better fit figure spacing. (D) Bio-layer interferometry (BLI) was used to confirm the binding of the aptamer-functionalized origami (SPOT) to isolated fluorescent proteins (top) and to a fluorescent protein fusion to glycoprotein B (gB), a surface glycoprotein from herpes simplex virus 1 in native membrane vesicles (bottom). As observed in the other assays, SPOTs bound to the sfGFP or sfGFP-gB much more than signpost origami alone, and SPOTs specifically bound sfGFP or sfGFP-gB, not mCherry or mCherry-gB. Each experiment was run in triplicate, and the lighter region behind each measurement indicates the standard deviation across the three replicates. (E) Dynamic light scattering was used to check for aggregation in concentrated solutions of SPOTs in buffer (left) and cell culture medium (center). A small amount of aggregate was only observed in culture medium at SPOT concentrations of 200 nM, ∼20x higher than needed for cellular experiments ( Figure S6 ). The 50 nM and 10 nM samples were too dilute to observe in culture medium. The aggregation-induced control sample (gray dotted lines) was stained for electron microscopy (right) to monitor the amount of aggregation (scale bar, 200 nm). Such aggregation was not seen in normal preparations of SPOTs ( Figures 2 B and 2C).

    Techniques Used: Isothermal Titration Calorimetry, Binding Assay, Acrylamide Gel Assay, Shift Assay, Agarose Gel Electrophoresis, Fluorescence, Diffusion-based Assay, Isolation, Standard Deviation, Cell Culture, Staining, Electron Microscopy

    Biophysical data, related to Figure 3 (A) Raw isothermal titration calorimetry data for fluorescent proteins titrated into aptamer (not linked to origami signpost). The fitted parameters are shown in the table for each binding curve. (B) We were unable to confirm binding of other aptamers using acrylamide gel shift assays. Left, an aptamer reported against hexahistidine tags ( Tsuji et al., 2009 ); Right, a reported aptamer against glycoprotein D from Herpes simplex virus I ( Yadavalli et al., 2017 ) Ci-iii. Unprocessed gel images with fluorescent detection in the GFP (i), RFP (ii), and YFP (iii) channels for gel-shift assays in Figures 3 B and 3C. iii Lane 3 - YFP alone. Because of its low molecular weight (∼26 kDa), we expect YFP to diffuse in the agarose gel, resulting in a band that thus appears faint and with a lower apparent motility. Lane 4 - upon addition of SPOTs to YFP, the molecular weight of the fluorescent structure would increase dramatically when they bind (∼5 MDa) so it diffuses much less, and a sharper and therefore brighter band would be seen. Lane 1 - YFP-Env has a much higher molecular weight than YFP (∼125 kDa monomer, ∼375 kDa trimer). As both forms of YFP-Env are much larger than YFP, the molecules should diffuse less in the agarose and thus appear as a more concentrated band. Additionally, there are 3 YFP on each Env trimer such that we would expect 3x the signal of YFP alone. Lane 2 - upon addition of SPOTs to YFP-Env, the trimer population of YFP-Env could bind 3 SPOTs (∼15 MDa) and the monomer population could bind one SPOT (∼5 MDa). As YFP-Env is a membrane protein produced in mammalian cells, it is glycosylated and detergent-solubilized here, both of which may impact the appearance of bands by electrophoresis. D. Example replicate buffer-subtracted and loading-normalized traces for BLI binding of unfunctionalized sign post origami (‘SPO’) or SPOTs to immobilized sfGFP-gB vesicles (i) or mCherry-gB vesicles (ii) from Figure 3 D (bottom panel). The buffer replicates (unloaded tips dipped in analyte nanostructure solutions) and average (used for subtraction) are show in iii, and the loading curves (normalized in the same way as the data) in iv .
    Figure Legend Snippet: Biophysical data, related to Figure 3 (A) Raw isothermal titration calorimetry data for fluorescent proteins titrated into aptamer (not linked to origami signpost). The fitted parameters are shown in the table for each binding curve. (B) We were unable to confirm binding of other aptamers using acrylamide gel shift assays. Left, an aptamer reported against hexahistidine tags ( Tsuji et al., 2009 ); Right, a reported aptamer against glycoprotein D from Herpes simplex virus I ( Yadavalli et al., 2017 ) Ci-iii. Unprocessed gel images with fluorescent detection in the GFP (i), RFP (ii), and YFP (iii) channels for gel-shift assays in Figures 3 B and 3C. iii Lane 3 - YFP alone. Because of its low molecular weight (∼26 kDa), we expect YFP to diffuse in the agarose gel, resulting in a band that thus appears faint and with a lower apparent motility. Lane 4 - upon addition of SPOTs to YFP, the molecular weight of the fluorescent structure would increase dramatically when they bind (∼5 MDa) so it diffuses much less, and a sharper and therefore brighter band would be seen. Lane 1 - YFP-Env has a much higher molecular weight than YFP (∼125 kDa monomer, ∼375 kDa trimer). As both forms of YFP-Env are much larger than YFP, the molecules should diffuse less in the agarose and thus appear as a more concentrated band. Additionally, there are 3 YFP on each Env trimer such that we would expect 3x the signal of YFP alone. Lane 2 - upon addition of SPOTs to YFP-Env, the trimer population of YFP-Env could bind 3 SPOTs (∼15 MDa) and the monomer population could bind one SPOT (∼5 MDa). As YFP-Env is a membrane protein produced in mammalian cells, it is glycosylated and detergent-solubilized here, both of which may impact the appearance of bands by electrophoresis. D. Example replicate buffer-subtracted and loading-normalized traces for BLI binding of unfunctionalized sign post origami (‘SPO’) or SPOTs to immobilized sfGFP-gB vesicles (i) or mCherry-gB vesicles (ii) from Figure 3 D (bottom panel). The buffer replicates (unloaded tips dipped in analyte nanostructure solutions) and average (used for subtraction) are show in iii, and the loading curves (normalized in the same way as the data) in iv .

    Techniques Used: Isothermal Titration Calorimetry, Binding Assay, Acrylamide Gel Assay, Electrophoretic Mobility Shift Assay, Molecular Weight, Agarose Gel Electrophoresis, Multiple Displacement Amplification, Produced, Electrophoresis

    3) Product Images from "Multiple conserved cell adhesion protein interactions mediate neural wiring of a sensory circuit in C. elegans"

    Article Title: Multiple conserved cell adhesion protein interactions mediate neural wiring of a sensory circuit in C. elegans

    Journal: bioRxiv

    doi: 10.1101/148080

    Model for function of cell adhesion protein interactions. (A) Three pathways additively act to achieve the wild type level of HOA-AVG axon fasciculation. The median percentages of HOA-AVG axon fasciculation in mutant and/or PHC ablation studies were used to estimate contribution of each pathway. (B) Schematic showing axons, cell adhesion protein interactions, and their putative function during the L4-adult transition in males. In L4 stage, the PHC axon contacts the AVG axon through RIG-6-SAX-7S interaction. In adult stage, close apposition of HOA and AVG membranes involves CASY-1-BAM-2 interaction, while unidentified protein interaction may mediate contact between HOA and PHC. These protein interactions regulate the probability of axon fasciculation and vulva location behavior possibly by controlling synapse formation between the neurons.
    Figure Legend Snippet: Model for function of cell adhesion protein interactions. (A) Three pathways additively act to achieve the wild type level of HOA-AVG axon fasciculation. The median percentages of HOA-AVG axon fasciculation in mutant and/or PHC ablation studies were used to estimate contribution of each pathway. (B) Schematic showing axons, cell adhesion protein interactions, and their putative function during the L4-adult transition in males. In L4 stage, the PHC axon contacts the AVG axon through RIG-6-SAX-7S interaction. In adult stage, close apposition of HOA and AVG membranes involves CASY-1-BAM-2 interaction, while unidentified protein interaction may mediate contact between HOA and PHC. These protein interactions regulate the probability of axon fasciculation and vulva location behavior possibly by controlling synapse formation between the neurons.

    Techniques Used: Mutagenesis

    Axon fasciculation defects of casy-1 and rig-6 are distinguishable. (A-D) The axons of the three neurons were individually labeled with wCherry (HOA; red), TagBFP (AVG; blue), and GFP (PHC; green) in wild type (A), casy-1(tm718) (B), rig-6(ok1589) (C), or casy-1; rig-6 double mutants (D). Axon fasciculation between each neuronal pair in the dashed box region and schematic of axon fasciculation are shown on the right. Arrowheads indicate the region where two axons are detached from each other. (E) Percentage of axon-axon contact between each neuronal pair in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (F) Developmental timing of axon extension of the three neurons in males. Each developmental stage was determined by the tail morphology of the male. (G) Axon fasciculation of the three neurons in mock-, PHC-, or HOA-ablated animal. Arrowheads indicate the region where HOA and AVG axons are detached from each other. (H) Percentage of axon-axon contact between each neuronal pair in mock-, PHC-, or HOA-ablated animals. The number of animals analyzed is indicated. Scale bars, 20 μm. *, P
    Figure Legend Snippet: Axon fasciculation defects of casy-1 and rig-6 are distinguishable. (A-D) The axons of the three neurons were individually labeled with wCherry (HOA; red), TagBFP (AVG; blue), and GFP (PHC; green) in wild type (A), casy-1(tm718) (B), rig-6(ok1589) (C), or casy-1; rig-6 double mutants (D). Axon fasciculation between each neuronal pair in the dashed box region and schematic of axon fasciculation are shown on the right. Arrowheads indicate the region where two axons are detached from each other. (E) Percentage of axon-axon contact between each neuronal pair in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (F) Developmental timing of axon extension of the three neurons in males. Each developmental stage was determined by the tail morphology of the male. (G) Axon fasciculation of the three neurons in mock-, PHC-, or HOA-ablated animal. Arrowheads indicate the region where HOA and AVG axons are detached from each other. (H) Percentage of axon-axon contact between each neuronal pair in mock-, PHC-, or HOA-ablated animals. The number of animals analyzed is indicated. Scale bars, 20 μm. *, P

    Techniques Used: Labeling, Mutagenesis

    CASY-1 and RIG-6 act in postsynaptic AVG for axon fasciculation. (A and B) Percentage of axon-axon contact between each neuronal pair in animals expressing a full-length casy-1 (A) or rig-6 cDNA (B) in postsynaptic AVG or presynaptic HOA or PHC in a respective mutant background (n = 30). The data for wild type and casy-1 and rig-6 mutants are identical to those in Figure 2 and are shown for comparison. Each dot represents individual animal. Red bar represents the median. *, P
    Figure Legend Snippet: CASY-1 and RIG-6 act in postsynaptic AVG for axon fasciculation. (A and B) Percentage of axon-axon contact between each neuronal pair in animals expressing a full-length casy-1 (A) or rig-6 cDNA (B) in postsynaptic AVG or presynaptic HOA or PHC in a respective mutant background (n = 30). The data for wild type and casy-1 and rig-6 mutants are identical to those in Figure 2 and are shown for comparison. Each dot represents individual animal. Red bar represents the median. *, P

    Techniques Used: Expressing, Mutagenesis

    casy-1 and rig-6 are expressed in AVG in both sexes. Transcriptional reporter lines carrying casy-1p::gfp and rig-6p::gfp were examined for expression in AVG in adult hermaphrodites or males. Location of AVG cell body was identified by Nomarski optics and is indicated as dashed circle. Scale bar, 20 μm.
    Figure Legend Snippet: casy-1 and rig-6 are expressed in AVG in both sexes. Transcriptional reporter lines carrying casy-1p::gfp and rig-6p::gfp were examined for expression in AVG in adult hermaphrodites or males. Location of AVG cell body was identified by Nomarski optics and is indicated as dashed circle. Scale bar, 20 μm.

    Techniques Used: Expressing

    Cell adhesion protein genes casy-1 and rig-6 are required for axon fasciculation. (A) Schematic of the position of cell bodies and axons of three neurons HOA (red), AVG (blue) and a pair of PHC (green) in the male (ventral view). The dashed box indicates the axons and HOA synaptic output analyzed in (C and D) and imaged in (F and H). (B) Electron micrograph showing the axons and synapses of HOA, PHC and AVG. Arrowheads indicate presynaptic density of dyadic synapses of HOA and PHC. Asterisk indicates a mitochondrion. In the schematic, axons are colored as in (A), and presynaptic density is indicated as gray. (C) The number of electron micrograph sections for axon-axon contact between neuron pairs. N2Y series were analyzed from section #13906 to #14249, except for the PHC-AVG (from #13836 to #14249) (WormWiring: http://wormwiring.org ). See also Supplementary File 1. (D) Synaptic output of HOA and the connectivity of HOA, AVG and PHC in the boxed region shown in (A). (HOA makes additional connections outside this region.) Synaptic weight determined by electron micrograph section numbers is indicated. The number of sections that contain dyadic synapses (HOA > AVG,PHC or PHC > HOA,AVG) is indicated in parenthesis. (E) Expression of transcriptional reporters for neural cell adhesion genes in the three neurons. One hundred out of 106 neural cell adhesion genes (94%) have been examined and the genes having expression in the three neurons are shown. (F) Distribution of a mCherry-tagged presynaptic marker RAB-3 in HOA axon of wild type or indicated mutants. Arrowheads indicate gaps between the presynaptic puncta. (G) Number of mCherry::RAB-3 puncta in mutants was counted and compared to wild type (n = 30). Error bars are SEM. (H) HOA presynaptic puncta (mCherry::RAB-3; red) were simultaneously visualized with GFP-labeled HOA and AVG axons (green) in wild type, or casy-1 or rig-6 mutant animals. Arrowheads indicate the gap region containing smaller or fewer presynaptic puncta. (I) Percentage of axon-axon contact between HOA and AVG in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (J and K) The mCherry::RAB-3 puncta size (J) or number (K) was measured and compared in the contacting and non-contacting axonal segments between HOA and AVG for the indicated genotypes. The number of the puncta (J) or of axon segments (K) analyzed is indicated below each column. Error bars are SEM. Scale bars, 20 μm. *, P
    Figure Legend Snippet: Cell adhesion protein genes casy-1 and rig-6 are required for axon fasciculation. (A) Schematic of the position of cell bodies and axons of three neurons HOA (red), AVG (blue) and a pair of PHC (green) in the male (ventral view). The dashed box indicates the axons and HOA synaptic output analyzed in (C and D) and imaged in (F and H). (B) Electron micrograph showing the axons and synapses of HOA, PHC and AVG. Arrowheads indicate presynaptic density of dyadic synapses of HOA and PHC. Asterisk indicates a mitochondrion. In the schematic, axons are colored as in (A), and presynaptic density is indicated as gray. (C) The number of electron micrograph sections for axon-axon contact between neuron pairs. N2Y series were analyzed from section #13906 to #14249, except for the PHC-AVG (from #13836 to #14249) (WormWiring: http://wormwiring.org ). See also Supplementary File 1. (D) Synaptic output of HOA and the connectivity of HOA, AVG and PHC in the boxed region shown in (A). (HOA makes additional connections outside this region.) Synaptic weight determined by electron micrograph section numbers is indicated. The number of sections that contain dyadic synapses (HOA > AVG,PHC or PHC > HOA,AVG) is indicated in parenthesis. (E) Expression of transcriptional reporters for neural cell adhesion genes in the three neurons. One hundred out of 106 neural cell adhesion genes (94%) have been examined and the genes having expression in the three neurons are shown. (F) Distribution of a mCherry-tagged presynaptic marker RAB-3 in HOA axon of wild type or indicated mutants. Arrowheads indicate gaps between the presynaptic puncta. (G) Number of mCherry::RAB-3 puncta in mutants was counted and compared to wild type (n = 30). Error bars are SEM. (H) HOA presynaptic puncta (mCherry::RAB-3; red) were simultaneously visualized with GFP-labeled HOA and AVG axons (green) in wild type, or casy-1 or rig-6 mutant animals. Arrowheads indicate the gap region containing smaller or fewer presynaptic puncta. (I) Percentage of axon-axon contact between HOA and AVG in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (J and K) The mCherry::RAB-3 puncta size (J) or number (K) was measured and compared in the contacting and non-contacting axonal segments between HOA and AVG for the indicated genotypes. The number of the puncta (J) or of axon segments (K) analyzed is indicated below each column. Error bars are SEM. Scale bars, 20 μm. *, P

    Techniques Used: Expressing, Marker, Labeling, Mutagenesis

    Structure-function analysis for CASY-1 and RIG-6. (A and C) Domain structure of mRFP::CASY-1 and its deletion constructs (A) or of YFP::RIG-6 and its deletion constructs (C) with the summary of rescue activity when expressed in AVG of casy-1 or rig-6 mutant animals. SP, signal peptide; Cad, cadherin domain; LNS, laminin neurexin sex hormone binding protein domain; TM, transmembrane; Intra, intracellular domain; Ig, immunoglobulin domain; FN[III], fibronectin type III domain. (B and D) Percentage of axon-axon contact between each neuronal pair in animals expressing the indicated deletion constructs for CASY-1 (B) or RIG-6 (D) in AVG of casy-1 or rig-6 mutant animals (n = 30). Note that Full-Ct (CASY-1::YFP) in (B) and Full (YFP::RIG-6) in (D) were the same transgenes used for protein localization in Figure 3 , and rescued axon fasciculation defects in mutants. The data for wild type and casy-1 and rig-6 mutants are the same as shown in Figure 2 . Each dot represents individual animal. Red bar represents the median. *, P
    Figure Legend Snippet: Structure-function analysis for CASY-1 and RIG-6. (A and C) Domain structure of mRFP::CASY-1 and its deletion constructs (A) or of YFP::RIG-6 and its deletion constructs (C) with the summary of rescue activity when expressed in AVG of casy-1 or rig-6 mutant animals. SP, signal peptide; Cad, cadherin domain; LNS, laminin neurexin sex hormone binding protein domain; TM, transmembrane; Intra, intracellular domain; Ig, immunoglobulin domain; FN[III], fibronectin type III domain. (B and D) Percentage of axon-axon contact between each neuronal pair in animals expressing the indicated deletion constructs for CASY-1 (B) or RIG-6 (D) in AVG of casy-1 or rig-6 mutant animals (n = 30). Note that Full-Ct (CASY-1::YFP) in (B) and Full (YFP::RIG-6) in (D) were the same transgenes used for protein localization in Figure 3 , and rescued axon fasciculation defects in mutants. The data for wild type and casy-1 and rig-6 mutants are the same as shown in Figure 2 . Each dot represents individual animal. Red bar represents the median. *, P

    Techniques Used: Construct, Activity Assay, Mutagenesis, Binding Assay, Expressing

    SAX-7 interacts with RIG-6. (A) Distribution of a mCherry-tagged presynaptic marker RAB-3 in HOA axon of wild type or sax-7(nj48) mutants. Arrowheads indicate gaps between the presynaptic puncta. (B) Number of mCherry::RAB-3 puncta in sax-7 mutants was counted and compared to wild type (n = 30). Error bars are SEM. (C) Images of axon placement of HOA, AVG and PHC in sax-7 and rig-6; sax-7 double mutants. Axon fasciculation between each neuronal pair in the dashed box region and schematic of axon fasciculation are shown on the right. Arrowheads indicate the region where two axons are detached from each other. (D) Percentage of axon-axon contact between each neuronal pair in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (E) Percentage of axon-axon contact between each neuronal pair in animals expressing the short isoform sax-7s or long isoform sax-7l cDNA in HOA or PHC in sax-7 mutant background (n = 30). In (C-E), the data for wild type and rig-6 mutants are the same as shown in Figure 2 . (F) Co-immunoprecipitation of RIG-6 and SAX-7S. (G) Images of axon placement of HOA and AVG in PHC-ablated animals expressing sax-7s cDNA in HOA ( Ex[HOAp:: sax-7s]) . Arrowheads indicate the region where the HOA axon is detached from the AVG axon. (H) Percentage of axon-axon contact between HOA and AVG in PHC-ablated animals either expressing or not expressing sax-7s cDNA in HOA. The number of animals analyzed is indicated. The data for PHC-ablated wild type animals are the same as shown in Figure 2H . Scale bars, 20 µm. *, P
    Figure Legend Snippet: SAX-7 interacts with RIG-6. (A) Distribution of a mCherry-tagged presynaptic marker RAB-3 in HOA axon of wild type or sax-7(nj48) mutants. Arrowheads indicate gaps between the presynaptic puncta. (B) Number of mCherry::RAB-3 puncta in sax-7 mutants was counted and compared to wild type (n = 30). Error bars are SEM. (C) Images of axon placement of HOA, AVG and PHC in sax-7 and rig-6; sax-7 double mutants. Axon fasciculation between each neuronal pair in the dashed box region and schematic of axon fasciculation are shown on the right. Arrowheads indicate the region where two axons are detached from each other. (D) Percentage of axon-axon contact between each neuronal pair in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (E) Percentage of axon-axon contact between each neuronal pair in animals expressing the short isoform sax-7s or long isoform sax-7l cDNA in HOA or PHC in sax-7 mutant background (n = 30). In (C-E), the data for wild type and rig-6 mutants are the same as shown in Figure 2 . (F) Co-immunoprecipitation of RIG-6 and SAX-7S. (G) Images of axon placement of HOA and AVG in PHC-ablated animals expressing sax-7s cDNA in HOA ( Ex[HOAp:: sax-7s]) . Arrowheads indicate the region where the HOA axon is detached from the AVG axon. (H) Percentage of axon-axon contact between HOA and AVG in PHC-ablated animals either expressing or not expressing sax-7s cDNA in HOA. The number of animals analyzed is indicated. The data for PHC-ablated wild type animals are the same as shown in Figure 2H . Scale bars, 20 µm. *, P

    Techniques Used: Marker, Mutagenesis, Expressing, Immunoprecipitation

    4) Product Images from "Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina"

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    Journal: bioRxiv

    doi: 10.1101/243683

    Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 sgRNA/YFP sgRNA2 (n= 4, red traces; A), SpCas9 sgRNA/LacZ sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p
    Figure Legend Snippet: Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 sgRNA/YFP sgRNA2 (n= 4, red traces; A), SpCas9 sgRNA/LacZ sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p

    Techniques Used: CRISPR, Injection, Two Tailed Test

    In vitro validation of kamikaze CRISPR/Cas construct. (A) Schematic of plasmid constructs for in vitro validation. (B) Representative Western blots of SpCas9 protein expression in cells co-transfected with SpCas9 and kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. (C) Representative images of YFP expression in cells co-transfected with kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. Percentage YFP disruption was assessed by FACS at 10 day after transfection. scale bar: 100 μm. Mean ± SEM for 2 independent replicates.
    Figure Legend Snippet: In vitro validation of kamikaze CRISPR/Cas construct. (A) Schematic of plasmid constructs for in vitro validation. (B) Representative Western blots of SpCas9 protein expression in cells co-transfected with SpCas9 and kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. (C) Representative images of YFP expression in cells co-transfected with kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. Percentage YFP disruption was assessed by FACS at 10 day after transfection. scale bar: 100 μm. Mean ± SEM for 2 independent replicates.

    Techniques Used: In Vitro, CRISPR, Construct, Plasmid Preparation, Western Blot, Expressing, Transfection, FACS

    Kamikaze CRISPR/Cas-mediated genome editing of retinal cells in vivo . (A) High magnification of retinal flat-mount images, showing differences in YFP expression following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA (n= 5), YFP sgRNA2 (n= 5) or SpCas9 sgRNA/LacZ sgRNA (n= 3). scale bar: 20 μm. (B) Percentage YFP disruption was assessed by manual cell counting. Mean ± SEM for 3-5 independent replicates. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (**p
    Figure Legend Snippet: Kamikaze CRISPR/Cas-mediated genome editing of retinal cells in vivo . (A) High magnification of retinal flat-mount images, showing differences in YFP expression following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA (n= 5), YFP sgRNA2 (n= 5) or SpCas9 sgRNA/LacZ sgRNA (n= 3). scale bar: 20 μm. (B) Percentage YFP disruption was assessed by manual cell counting. Mean ± SEM for 3-5 independent replicates. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (**p

    Techniques Used: CRISPR, In Vivo, Expressing, Cell Counting

    Uncropped agarose gel and western blot images. (A) In situ test of designed SpCas9 sgRNAs. (B) in vitro validation of designed SpCas9 sgRNAs. (C) Time course of SpCas9 expression. (D) in vitro validation of YFP targeting kamikaze CRISPR constructs. The β-actin membrane was reprobing without stripping.
    Figure Legend Snippet: Uncropped agarose gel and western blot images. (A) In situ test of designed SpCas9 sgRNAs. (B) in vitro validation of designed SpCas9 sgRNAs. (C) Time course of SpCas9 expression. (D) in vitro validation of YFP targeting kamikaze CRISPR constructs. The β-actin membrane was reprobing without stripping.

    Techniques Used: Agarose Gel Electrophoresis, Western Blot, In Situ, In Vitro, Expressing, CRISPR, Construct, Stripping Membranes

    Quantification of YFP disruption in the retina. The percentage of YFP disruption following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA6, YFP sgRNA6 or SpCas9 sgRNA/LacZ sgRNA was assessed by manual cell counting. Representative data are shown for 3-5 retinas and expressed as the Mean ± SEM. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (*p
    Figure Legend Snippet: Quantification of YFP disruption in the retina. The percentage of YFP disruption following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA6, YFP sgRNA6 or SpCas9 sgRNA/LacZ sgRNA was assessed by manual cell counting. Representative data are shown for 3-5 retinas and expressed as the Mean ± SEM. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (*p

    Techniques Used: Cell Counting

    Schematics of Kamikaze CRISPR/Cas system. A dual AAV vector system was used. One viral vector was used to deliver SpCas9 and the other delivered sgRNAs against SpCas9 and the target locus (YFP), in the presence of mCherry.
    Figure Legend Snippet: Schematics of Kamikaze CRISPR/Cas system. A dual AAV vector system was used. One viral vector was used to deliver SpCas9 and the other delivered sgRNAs against SpCas9 and the target locus (YFP), in the presence of mCherry.

    Techniques Used: CRISPR, Plasmid Preparation

    5) Product Images from "The Dietary Restriction-Like Gene drl-1, Which Encodes a Putative Serine/Threonine Kinase, Is Essential for Orsay Virus Infection in Caenorhabditis elegans"

    Article Title: The Dietary Restriction-Like Gene drl-1, Which Encodes a Putative Serine/Threonine Kinase, Is Essential for Orsay Virus Infection in Caenorhabditis elegans

    Journal: Journal of Virology

    doi: 10.1128/JVI.01400-18

    Orsay RNA replication and GFP expression patterns in Viro-4 mutants carrying a fosmid containing the endogenous drl-1 locus or overexpressing DRL-1. (A) GFP response at 3 days postinfection of Viro-4 mutants expressing the drl-1 locus or ectopically expressing DRL-1 infected with Orsay virus. The Pmyo2 :: YFP plasmid was coinjected as a transgenic marker. (B) Orsay virus RNA2 levels at 3 days postinfection of Viro-4 mutants expressing the drl-1 locus or overexpressing DRL-1 infected with Orsay virus. ***, P
    Figure Legend Snippet: Orsay RNA replication and GFP expression patterns in Viro-4 mutants carrying a fosmid containing the endogenous drl-1 locus or overexpressing DRL-1. (A) GFP response at 3 days postinfection of Viro-4 mutants expressing the drl-1 locus or ectopically expressing DRL-1 infected with Orsay virus. The Pmyo2 :: YFP plasmid was coinjected as a transgenic marker. (B) Orsay virus RNA2 levels at 3 days postinfection of Viro-4 mutants expressing the drl-1 locus or overexpressing DRL-1 infected with Orsay virus. ***, P

    Techniques Used: Expressing, Infection, Plasmid Preparation, Transgenic Assay, Marker

    6) Product Images from "Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD"

    Article Title: Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

    Journal: bioRxiv

    doi: 10.1101/2021.11.29.470318

    Erythromycin-dependent transcriptional termination regulates msrD expression. (A) ERY-dependent induction of msrD (1-3) : yfp . Fluorescent reporters shown on schematics have been introduced in E. coli DB10 and grew in presence of 1 mM IPTG and increasing sublethal ERY concentrations during 17 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. (B) Northern analysis of msrD transcript. RNAs were extracted before adding or not 1 mM IPTG (T 0’ ), 10 min after adding IPTG (T 10’ ), and 5, 15, 30, 60, 120 min after adding or not 100 nM ERY. Location of probe in msrD (1-3) : yfp 3’ UTR is shown on Figure 2A . The presence of a second band was also observed but we hypothesized that it was abortive transcript resulting from the construct insofar as its presence correlates with induction by IPTG and ERY, and is no longer detectable using other probes (data not shown). (C) Deletion of the intrinsic terminator between msrDL and msrD (1-3) : yfp leads to a constitutive induction in absence of ERY. Error bars for both axes represent mean ± s.d. for triplicate experiments.
    Figure Legend Snippet: Erythromycin-dependent transcriptional termination regulates msrD expression. (A) ERY-dependent induction of msrD (1-3) : yfp . Fluorescent reporters shown on schematics have been introduced in E. coli DB10 and grew in presence of 1 mM IPTG and increasing sublethal ERY concentrations during 17 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. (B) Northern analysis of msrD transcript. RNAs were extracted before adding or not 1 mM IPTG (T 0’ ), 10 min after adding IPTG (T 10’ ), and 5, 15, 30, 60, 120 min after adding or not 100 nM ERY. Location of probe in msrD (1-3) : yfp 3’ UTR is shown on Figure 2A . The presence of a second band was also observed but we hypothesized that it was abortive transcript resulting from the construct insofar as its presence correlates with induction by IPTG and ERY, and is no longer detectable using other probes (data not shown). (C) Deletion of the intrinsic terminator between msrDL and msrD (1-3) : yfp leads to a constitutive induction in absence of ERY. Error bars for both axes represent mean ± s.d. for triplicate experiments.

    Techniques Used: Expressing, Fluorescence, Northern Blot, Construct

    MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.
    Figure Legend Snippet: MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.

    Techniques Used: Toeprinting Assay, In Vivo, Expressing

    MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.
    Figure Legend Snippet: MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.

    Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Activity Assay

    MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.
    Figure Legend Snippet: MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.

    Techniques Used: Toeprinting Assay, In Vivo, Expressing

    MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.
    Figure Legend Snippet: MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.

    Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Activity Assay

    7) Product Images from "Endosidin20 targets cellulose synthase catalytic domain to inhibit cellulose biosynthesis"

    Article Title: Endosidin20 targets cellulose synthase catalytic domain to inhibit cellulose biosynthesis

    Journal: bioRxiv

    doi: 10.1101/2020.02.16.946244

    Mutations in amino acids at the catalytic site of CESA6 reduce CSC motility at the PM and reduce CSC delivery to the PM in etiolated hypocotyl cells. A to C , Mutations in amino acids at the catalytic site of CESA6 reduced the velocity of CSCs at the PM. A , Representative time projections of average intensity images from a time-lapse series of CSC particles from YFP-CESA6 lines carrying different mutations. For each time projection, 61 frames collected at 5-s intervals were used. B , Kymographs of trajectories marked in ( A ) showed the movement of CSCs over 5 min. C , Quantification of the velocities of CSCs at the PM in YFP-CESA6 lines carrying different mutations. Data represent mean ± SE ( n > 300 CSC trajectories from 5 seedlings for each mutated line). ***, p
    Figure Legend Snippet: Mutations in amino acids at the catalytic site of CESA6 reduce CSC motility at the PM and reduce CSC delivery to the PM in etiolated hypocotyl cells. A to C , Mutations in amino acids at the catalytic site of CESA6 reduced the velocity of CSCs at the PM. A , Representative time projections of average intensity images from a time-lapse series of CSC particles from YFP-CESA6 lines carrying different mutations. For each time projection, 61 frames collected at 5-s intervals were used. B , Kymographs of trajectories marked in ( A ) showed the movement of CSCs over 5 min. C , Quantification of the velocities of CSCs at the PM in YFP-CESA6 lines carrying different mutations. Data represent mean ± SE ( n > 300 CSC trajectories from 5 seedlings for each mutated line). ***, p

    Techniques Used:

    Mutations in amino acids at the catalytic site cause reduced sensitivity to ES20 treatment at the cellular level in root epidermal cells. A to C , Mutations in amino acids at the catalytic site of CESA6 reduced the sensitivity to ES20 inhibition of CSC velocity. A , Representative time projections of average intensity images from a time-lapse series of YFP-CESA6 particles from YFP-CESA6 lines carrying different mutations after DMSO or ES20 treatment. For each time projection, 61 frames collected at 5-s intervals were used. B , Kymographs of trajectories marked in ( A ) showed the movement of CSCs over 5 min. C , Quantification of the velocities of CSCs at the PM in YFP-CESA6 lines carrying different mutations after ES20 treatment. Data represent mean ± SE ( n > 300 CSC trajectories from 6 seedlings for each mutated line). D and E , Mutations in amino acids at the catalytic site of CESA6 cause reduced sensitivity to ES20 inhibition of CSC density at the PM. Representative images ( D ) and quantification of ( E ) of PM-localized YFP-CESA6 carrying different mutations in root epidermal cells after 0.1% DMSO or 6 μM ES20 treatment. Data represent mean ± SE ( n = 24 cells from 12 seedlings). F and G , Some mutations in amino acids at the catalytic site of CESA6 cause reduced sensitivity to ES20 induction of cortical SmaCCs. The density of cortical SmaCCs was increased by ES20 treatment (30 min). Data represent mean ± SE ( n = 24 cells from 12 seedlings). * represents p
    Figure Legend Snippet: Mutations in amino acids at the catalytic site cause reduced sensitivity to ES20 treatment at the cellular level in root epidermal cells. A to C , Mutations in amino acids at the catalytic site of CESA6 reduced the sensitivity to ES20 inhibition of CSC velocity. A , Representative time projections of average intensity images from a time-lapse series of YFP-CESA6 particles from YFP-CESA6 lines carrying different mutations after DMSO or ES20 treatment. For each time projection, 61 frames collected at 5-s intervals were used. B , Kymographs of trajectories marked in ( A ) showed the movement of CSCs over 5 min. C , Quantification of the velocities of CSCs at the PM in YFP-CESA6 lines carrying different mutations after ES20 treatment. Data represent mean ± SE ( n > 300 CSC trajectories from 6 seedlings for each mutated line). D and E , Mutations in amino acids at the catalytic site of CESA6 cause reduced sensitivity to ES20 inhibition of CSC density at the PM. Representative images ( D ) and quantification of ( E ) of PM-localized YFP-CESA6 carrying different mutations in root epidermal cells after 0.1% DMSO or 6 μM ES20 treatment. Data represent mean ± SE ( n = 24 cells from 12 seedlings). F and G , Some mutations in amino acids at the catalytic site of CESA6 cause reduced sensitivity to ES20 induction of cortical SmaCCs. The density of cortical SmaCCs was increased by ES20 treatment (30 min). Data represent mean ± SE ( n = 24 cells from 12 seedlings). * represents p

    Techniques Used: Inhibition

    ES20 treatment inhibits CSC delivery to the PM but does not affect CSC trafficking from ER to the Golgi. A to C , ES20 reduced the delivery rate of CSCs to PM in root epidermal cells. A , Representative images of CSCs at the PM during FRAP analysis. B, Representative kymographs of trajectories of newly delivered CSCs after photobleaching. C . Quantification of CSC delivery rates based on FRAP assays described in ( A ). Data represent mean ± SE ( n = 18 ROI from 15 seedlings). D and E , ES20 did not affect the delivery of CSCs from ER to the Golgi in root epidermal cells. D . Representative images of Golgi-localized YFP-CESA6 during a FRAP assay. E . Quantification of the relative recovery of CSCs at Golgi at different time points during FRAP assay. Data represent mean ± SE ( n = 12 from 12 seedlings per treatment). Scale bars: 5 μm. *** indicates p
    Figure Legend Snippet: ES20 treatment inhibits CSC delivery to the PM but does not affect CSC trafficking from ER to the Golgi. A to C , ES20 reduced the delivery rate of CSCs to PM in root epidermal cells. A , Representative images of CSCs at the PM during FRAP analysis. B, Representative kymographs of trajectories of newly delivered CSCs after photobleaching. C . Quantification of CSC delivery rates based on FRAP assays described in ( A ). Data represent mean ± SE ( n = 18 ROI from 15 seedlings). D and E , ES20 did not affect the delivery of CSCs from ER to the Golgi in root epidermal cells. D . Representative images of Golgi-localized YFP-CESA6 during a FRAP assay. E . Quantification of the relative recovery of CSCs at Golgi at different time points during FRAP assay. Data represent mean ± SE ( n = 12 from 12 seedlings per treatment). Scale bars: 5 μm. *** indicates p

    Techniques Used: FRAP Assay

    ES20 directly interacts with CESA6. A to F , ES20 interacts with CESA6 in DARTS assay. A , C , and E , Representative western blots of DARTS assays for YFP-CESA6 with ES20 ( A ), YFP-CESA6 with Ampicillin ( C ), and YFP-CESA6 P595S with ES20 ( E ), respectively. B , D , and F , Quantitative analysis of DARTS assays for YFP-CESA6 with ES20 ( B ), YFP-CESA6 with Ampicillin ( D ), and YFP-CESA6 P595S with ES20 ( F ), respectively. G to I , the central cytoplasmic domain of CESA6 interacts with ES20 and UDP-glucose in MST assay. G . Purified GFP-CESA6c with a His-SUMO tag (lane 2). H , Thermophoresis binding curve shows direct interaction between GFP-CESA6c and ES20. I , Thermophoresis binding curve shows direct interaction between GFP-CESA6c and UDP-glucose. J to L , the central cytoplasmic domain of CESA6 P595S interacts with ES20 and UDP-glucose in MST assay. J . Purified GFP-CESA6 P595S c with a His-SUMO tag (lane 2). K , Thermophoresis binding curve shows direct interaction between GFP-CESA6 P595S c and ES20. L , Thermophoresis binding curve shows direct interaction between GFP-CESA6 P595S c and UDP-glucose. M and N , UDP-glucose could partially complement the root swollen caused by ES20. M , Representative images of seedlings treated with DMSO (0.1%), ES20 (0.8 μM), UDP-glucose (1 mM) and ES20 (0.8 μM) + UDP-glucose (1 mM). N , Quantification on the root width at the elongation zone of seedlings with different treatments as shown in M . The letters in N indicate the statistically significant differences determined by one-way ANOVA tests followed by Tukey’s multiple comparison tests in different samples. Different letters indicate significant differences between groups (p
    Figure Legend Snippet: ES20 directly interacts with CESA6. A to F , ES20 interacts with CESA6 in DARTS assay. A , C , and E , Representative western blots of DARTS assays for YFP-CESA6 with ES20 ( A ), YFP-CESA6 with Ampicillin ( C ), and YFP-CESA6 P595S with ES20 ( E ), respectively. B , D , and F , Quantitative analysis of DARTS assays for YFP-CESA6 with ES20 ( B ), YFP-CESA6 with Ampicillin ( D ), and YFP-CESA6 P595S with ES20 ( F ), respectively. G to I , the central cytoplasmic domain of CESA6 interacts with ES20 and UDP-glucose in MST assay. G . Purified GFP-CESA6c with a His-SUMO tag (lane 2). H , Thermophoresis binding curve shows direct interaction between GFP-CESA6c and ES20. I , Thermophoresis binding curve shows direct interaction between GFP-CESA6c and UDP-glucose. J to L , the central cytoplasmic domain of CESA6 P595S interacts with ES20 and UDP-glucose in MST assay. J . Purified GFP-CESA6 P595S c with a His-SUMO tag (lane 2). K , Thermophoresis binding curve shows direct interaction between GFP-CESA6 P595S c and ES20. L , Thermophoresis binding curve shows direct interaction between GFP-CESA6 P595S c and UDP-glucose. M and N , UDP-glucose could partially complement the root swollen caused by ES20. M , Representative images of seedlings treated with DMSO (0.1%), ES20 (0.8 μM), UDP-glucose (1 mM) and ES20 (0.8 μM) + UDP-glucose (1 mM). N , Quantification on the root width at the elongation zone of seedlings with different treatments as shown in M . The letters in N indicate the statistically significant differences determined by one-way ANOVA tests followed by Tukey’s multiple comparison tests in different samples. Different letters indicate significant differences between groups (p

    Techniques Used: Western Blot, Purification, Binding Assay

    ES20 reduces CSC localization at the PM and increases CSC at the Golgi. A to C , ES20 reduced the velocity of CSCs at the PM. A , Representative time projections using average intensity images from a time-lapse series of YFP-CESA6 particles in root epidermal cells. B , Kymographs of trajectories marked in ( A ). C , Histogram showed the frequencies of YFP-CESA6 particle velocity after treatment with 0.1% DMSO or 6 μM ES20 for 30 min. Data represent mean ± SD ( n = 320 CSC trajectories from 18 seedlings per treatment). D and E , ES20 reduced PM-localized YFP-CESA6 in root epidermal cells after ES20 treatment. Representative images ( D ) and quantification ( E ) of PM-localized YFP-CESA6 in root epidermal cells after 0.1% DMSO or 6 μM ES20 treatment were shown. Data represent mean ± SE ( n = 20 cells from 10 seedlings). F and G , The density of cortical SmaCCs, as indicated by red circles, was increased by ES20 treatment (30 min). Data represent mean ± SE ( n = 20 cells from 10 seedlings per treatment). H and I , ES20 increased the abundance of CSC at the Golgi. H , Representative images of Golgi-localized YFP-CESA6 and ManI-CFP after 0.1% DMSO (top) or 6 μM ES20 (bottom) treatment for 1 h. I , Quantification of integrated fluorescence intensity of Golgi-localized CSCs and ManI as described in ( H ). Data represent mean ± SE ( n = 60 from 14 seedlings). J , CSCs were depleted from the PM after treatment with 6 μM ES20 for 2 h, whereas microtubule-associated CESA compartments accumulated, as indicated by white arrows. K , Magnified view on the association of CESA compartment (pointed by white arrows) with microtubules in time course image after 6 μM ES20 treatment for 2h. L , Kymograph image to show the association of CESA compartment with the microtubules as shown in K . Scale bars, 5 μm. ** indicates p
    Figure Legend Snippet: ES20 reduces CSC localization at the PM and increases CSC at the Golgi. A to C , ES20 reduced the velocity of CSCs at the PM. A , Representative time projections using average intensity images from a time-lapse series of YFP-CESA6 particles in root epidermal cells. B , Kymographs of trajectories marked in ( A ). C , Histogram showed the frequencies of YFP-CESA6 particle velocity after treatment with 0.1% DMSO or 6 μM ES20 for 30 min. Data represent mean ± SD ( n = 320 CSC trajectories from 18 seedlings per treatment). D and E , ES20 reduced PM-localized YFP-CESA6 in root epidermal cells after ES20 treatment. Representative images ( D ) and quantification ( E ) of PM-localized YFP-CESA6 in root epidermal cells after 0.1% DMSO or 6 μM ES20 treatment were shown. Data represent mean ± SE ( n = 20 cells from 10 seedlings). F and G , The density of cortical SmaCCs, as indicated by red circles, was increased by ES20 treatment (30 min). Data represent mean ± SE ( n = 20 cells from 10 seedlings per treatment). H and I , ES20 increased the abundance of CSC at the Golgi. H , Representative images of Golgi-localized YFP-CESA6 and ManI-CFP after 0.1% DMSO (top) or 6 μM ES20 (bottom) treatment for 1 h. I , Quantification of integrated fluorescence intensity of Golgi-localized CSCs and ManI as described in ( H ). Data represent mean ± SE ( n = 60 from 14 seedlings). J , CSCs were depleted from the PM after treatment with 6 μM ES20 for 2 h, whereas microtubule-associated CESA compartments accumulated, as indicated by white arrows. K , Magnified view on the association of CESA compartment (pointed by white arrows) with microtubules in time course image after 6 μM ES20 treatment for 2h. L , Kymograph image to show the association of CESA compartment with the microtubules as shown in K . Scale bars, 5 μm. ** indicates p

    Techniques Used: Fluorescence

    8) Product Images from "LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring"

    Article Title: LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-12-0874

    Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.
    Figure Legend Snippet: Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.

    Techniques Used:

    LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.
    Figure Legend Snippet: LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.

    Techniques Used: Fluorescence, Microscopy

    Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.
    Figure Legend Snippet: Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.

    Techniques Used: Imaging, Transgenic Assay, Labeling

    9) Product Images from "Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD"

    Article Title: Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

    Journal: bioRxiv

    doi: 10.1101/2021.11.29.470318

    Erythromycin-dependent transcriptional termination regulates msrD expression. (A) ERY-dependent induction of msrD (1-3) : yfp . Fluorescent reporters shown on schematics have been introduced in E. coli DB10 and grew in presence of 1 mM IPTG and increasing sublethal ERY concentrations during 17 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. (B) Northern analysis of msrD transcript. RNAs were extracted before adding or not 1 mM IPTG (T 0’ ), 10 min after adding IPTG (T 10’ ), and 5, 15, 30, 60, 120 min after adding or not 100 nM ERY. Location of probe in msrD (1-3) : yfp 3’ UTR is shown on Figure 2A . The presence of a second band was also observed but we hypothesized that it was abortive transcript resulting from the construct insofar as its presence correlates with induction by IPTG and ERY, and is no longer detectable using other probes (data not shown). (C) Deletion of the intrinsic terminator between msrDL and msrD (1-3) : yfp leads to a constitutive induction in absence of ERY. Error bars for both axes represent mean ± s.d. for triplicate experiments.
    Figure Legend Snippet: Erythromycin-dependent transcriptional termination regulates msrD expression. (A) ERY-dependent induction of msrD (1-3) : yfp . Fluorescent reporters shown on schematics have been introduced in E. coli DB10 and grew in presence of 1 mM IPTG and increasing sublethal ERY concentrations during 17 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. (B) Northern analysis of msrD transcript. RNAs were extracted before adding or not 1 mM IPTG (T 0’ ), 10 min after adding IPTG (T 10’ ), and 5, 15, 30, 60, 120 min after adding or not 100 nM ERY. Location of probe in msrD (1-3) : yfp 3’ UTR is shown on Figure 2A . The presence of a second band was also observed but we hypothesized that it was abortive transcript resulting from the construct insofar as its presence correlates with induction by IPTG and ERY, and is no longer detectable using other probes (data not shown). (C) Deletion of the intrinsic terminator between msrDL and msrD (1-3) : yfp leads to a constitutive induction in absence of ERY. Error bars for both axes represent mean ± s.d. for triplicate experiments.

    Techniques Used: Expressing, Fluorescence, Northern Blot, Construct

    MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.
    Figure Legend Snippet: MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.

    Techniques Used: Toeprinting Assay, In Vivo, Expressing

    MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.
    Figure Legend Snippet: MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.

    Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Activity Assay

    MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.
    Figure Legend Snippet: MsrDL is a macrolide-sensing nascent chain inhibiting its translation termination. (A) Toeprinting assay of msrDL in the absence (-) or in the presence of 50 µM of various macrolide antibiotics. Empty arrow indicates initiation codon. Plain arrows indicate ribosome stalling. Chemical structure of antibiotics is shown, C3 cladinose sugar of ERY and AZI being highlighted. See also Figure S3 . (B) In vivo induction of msrD (1:3) : yfp by various PTC/NPET targeting antibiotics. Bacteria containing pMMB- msrDL - msrD (1:3): yfp were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 50 nM antibiotics (red histograms). Error bars represent mean ± s.d. for triplicate experiments. (C) Toeprinting assay depleted of release factors was performed in the absence of ribosome (line 1), in presence of 50 µM RTP to assess start codon (line 2), without or with 50 µM ERY (line 3 and 4), without or with 50 µM ERY then supplemented with 100 µM puromycin (line 5 and 6), without or with 50 µM ERY in presence of RF1/RF2/RF3 (line 7 and 8). The schematic indicates position of toe-printing signal on the synthetic mRNA, P site codon of MsrDL-SRC is underlined. See also Figure S3 . (D and E) Effects of msrDL variants on the expression of msrD (1:3): yfp . Bacteria were grown during 17 h in presence of 1 mM IPTG, in the absence (grey histograms) or in the presence of 100 nM ERY (red histograms). Error bars represent mean ± s.d. for triplicate experiments.

    Techniques Used: Toeprinting Assay, In Vivo, Expressing

    MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.
    Figure Legend Snippet: MsrD negatively regulates its own synthesis upon erythromycin exposure. (A) Effects of MsrD variants on MsrDL. E. coli DB10 containing pMMB- msrDL-msrD (1-3) :yfp and expressing various msrD mutants were grown in presence of 0.2 % L-Arabinose, 1 mM IPTG and 300 nM ERY, both OD 600 and fluorescence being recorded over 24 h. Fluorescence has been plotted against OD 600 , error bars for both axes represent mean ± s.d. for triplicate experiments. Color code is same as Figures 1F to 1H . Light green rectangle indicates bacterial growth over control plasmid. (B) Model of MsrD regulating its own expression and providing antibiotic resistance. In absence of ERY, RNAP drops off at Rho-independent transcription terminator. In presence of ERY, the ribosome following the RNAP stalls and unwinds the terminator leading to msrD transcription. Once translated, MsrD negatively regulates its own expression on one side, and provides antibiotic resistance on the other side. ATP-bound MsrD recognizes ERY-stalled ribosome and may either expel the antibiotic or dissociate the ribosome, ATP site II being active. Activity in ATP site I leads to MsrD dissociation and recycling.

    Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Activity Assay

    10) Product Images from "LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring"

    Article Title: LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-12-0874

    Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.
    Figure Legend Snippet: Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.

    Techniques Used:

    LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.
    Figure Legend Snippet: LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.

    Techniques Used: Fluorescence, Microscopy

    Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.
    Figure Legend Snippet: Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.

    Techniques Used: Imaging, Transgenic Assay, Labeling

    11) Product Images from "LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring"

    Article Title: LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-12-0874

    Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.
    Figure Legend Snippet: Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.

    Techniques Used:

    LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.
    Figure Legend Snippet: LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.

    Techniques Used: Fluorescence, Microscopy

    Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.
    Figure Legend Snippet: Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.

    Techniques Used: Imaging, Transgenic Assay, Labeling

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    New England Biolabs prset yfp
    Gene expression of <t>pRSET-YFP</t> with 100 mg/mL E . coli lysates with different concentration of gene.
    Prset Yfp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prset yfp/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    prset yfp - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    95
    New England Biolabs yfp
    Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 <t>sgRNA/YFP</t> sgRNA2 (n= 4, red traces; A), SpCas9 <t>sgRNA/LacZ</t> sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p
    Yfp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/yfp/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    yfp - by Bioz Stars, 2022-07
    95/100 stars
      Buy from Supplier

    Image Search Results


    Gene expression of pRSET-YFP with 100 mg/mL E . coli lysates with different concentration of gene.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Cell lysates and egg white create homeostatic microenvironment for gene expression in cell-free system

    doi: 10.1016/j.synbio.2018.10.004

    Figure Lengend Snippet: Gene expression of pRSET-YFP with 100 mg/mL E . coli lysates with different concentration of gene.

    Article Snippet: 2.4 Construction of genes All enzymes used for the construction of pRSET-eGFP and pRSET-YFP were purchased from New England Biolabs, USA.

    Techniques: Expressing, Concentration Assay

    Gene expression of pRSET-YFP with 100 mg/mL E . coli lysates with different concentration of gene.

    Journal: Synthetic and Systems Biotechnology

    Article Title: Cell lysates and egg white create homeostatic microenvironment for gene expression in cell-free system

    doi: 10.1016/j.synbio.2018.10.004

    Figure Lengend Snippet: Gene expression of pRSET-YFP with 100 mg/mL E . coli lysates with different concentration of gene.

    Article Snippet: All enzymes used for the construction of pRSET-eGFP and pRSET-YFP were purchased from New England Biolabs, USA.

    Techniques: Expressing, Concentration Assay

    Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 sgRNA/YFP sgRNA2 (n= 4, red traces; A), SpCas9 sgRNA/LacZ sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p

    Journal: bioRxiv

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    doi: 10.1101/243683

    Figure Lengend Snippet: Long-term effect of AAV2-mediated CRISPR/Cas administration on retinal function. Averaged ERG waveforms at selected intensities for control (black traces) and SpCas9 sgRNA/YFP sgRNA2 (n= 4, red traces; A), SpCas9 sgRNA/LacZ sgRNA (n = 4, blue traces; C) and YFP sgRNA2 (n= 5, green traces; E) injected eyes. Group average (± SEM) photoreceptoral (a-wave), bipolar cell (b-wave), amacrine cell (oscillatory potentials, OPs) and ganglion cell (scotopic threshold response, STR) amplitude relative to contralateral control eyes (%) for each group (B, D and F). Effect of SpCas9 sgRNA/YFP sgRNA2, SpCas9 sgRNA/LacZ sgRNA and YFP sgRNA2 on retinal structure measured with OCT (G). Group average (± SEM) retinal nerve fibre layer thickness (H) for SpCas9 sgRNA/YFP sgRNA2 treated (filled red, n= 4) and their contralateral controls (unfilled red, n= 4), SpCas9 sgRNA/LacZ sgRNA treated (filled blue, n= 4) and their contralateral controls (unfilled blue, n= 4) and YFP sgRNA2 treated (filled green, n= 5) and their contralateral controls (unfilled green, n=5). Total retinal thickness (I). Statistical analysis between injected and control eyes was performed using two-tailed Student t-test (*p

    Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

    Techniques: CRISPR, Injection, Two Tailed Test

    In vitro validation of kamikaze CRISPR/Cas construct. (A) Schematic of plasmid constructs for in vitro validation. (B) Representative Western blots of SpCas9 protein expression in cells co-transfected with SpCas9 and kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. (C) Representative images of YFP expression in cells co-transfected with kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. Percentage YFP disruption was assessed by FACS at 10 day after transfection. scale bar: 100 μm. Mean ± SEM for 2 independent replicates.

    Journal: bioRxiv

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    doi: 10.1101/243683

    Figure Lengend Snippet: In vitro validation of kamikaze CRISPR/Cas construct. (A) Schematic of plasmid constructs for in vitro validation. (B) Representative Western blots of SpCas9 protein expression in cells co-transfected with SpCas9 and kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. (C) Representative images of YFP expression in cells co-transfected with kamikaze (SpCas9 sgRNA/YFP sgRNA and SpCas9 sgRNA/LacZ sgRNA) or non-kamikaze (YFP sgRNA and LacZ sgRNA) constructs. Percentage YFP disruption was assessed by FACS at 10 day after transfection. scale bar: 100 μm. Mean ± SEM for 2 independent replicates.

    Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

    Techniques: In Vitro, CRISPR, Construct, Plasmid Preparation, Western Blot, Expressing, Transfection, FACS

    Kamikaze CRISPR/Cas-mediated genome editing of retinal cells in vivo . (A) High magnification of retinal flat-mount images, showing differences in YFP expression following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA (n= 5), YFP sgRNA2 (n= 5) or SpCas9 sgRNA/LacZ sgRNA (n= 3). scale bar: 20 μm. (B) Percentage YFP disruption was assessed by manual cell counting. Mean ± SEM for 3-5 independent replicates. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (**p

    Journal: bioRxiv

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    doi: 10.1101/243683

    Figure Lengend Snippet: Kamikaze CRISPR/Cas-mediated genome editing of retinal cells in vivo . (A) High magnification of retinal flat-mount images, showing differences in YFP expression following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA (n= 5), YFP sgRNA2 (n= 5) or SpCas9 sgRNA/LacZ sgRNA (n= 3). scale bar: 20 μm. (B) Percentage YFP disruption was assessed by manual cell counting. Mean ± SEM for 3-5 independent replicates. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (**p

    Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

    Techniques: CRISPR, In Vivo, Expressing, Cell Counting

    Uncropped agarose gel and western blot images. (A) In situ test of designed SpCas9 sgRNAs. (B) in vitro validation of designed SpCas9 sgRNAs. (C) Time course of SpCas9 expression. (D) in vitro validation of YFP targeting kamikaze CRISPR constructs. The β-actin membrane was reprobing without stripping.

    Journal: bioRxiv

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    doi: 10.1101/243683

    Figure Lengend Snippet: Uncropped agarose gel and western blot images. (A) In situ test of designed SpCas9 sgRNAs. (B) in vitro validation of designed SpCas9 sgRNAs. (C) Time course of SpCas9 expression. (D) in vitro validation of YFP targeting kamikaze CRISPR constructs. The β-actin membrane was reprobing without stripping.

    Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

    Techniques: Agarose Gel Electrophoresis, Western Blot, In Situ, In Vitro, Expressing, CRISPR, Construct, Stripping Membranes

    Quantification of YFP disruption in the retina. The percentage of YFP disruption following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA6, YFP sgRNA6 or SpCas9 sgRNA/LacZ sgRNA was assessed by manual cell counting. Representative data are shown for 3-5 retinas and expressed as the Mean ± SEM. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (*p

    Journal: bioRxiv

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    doi: 10.1101/243683

    Figure Lengend Snippet: Quantification of YFP disruption in the retina. The percentage of YFP disruption following AAV2-mediated delivery of SpCas9 sgRNA/YFP sgRNA6, YFP sgRNA6 or SpCas9 sgRNA/LacZ sgRNA was assessed by manual cell counting. Representative data are shown for 3-5 retinas and expressed as the Mean ± SEM. Statistical analysis between groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test (*p

    Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

    Techniques: Cell Counting

    Schematics of Kamikaze CRISPR/Cas system. A dual AAV vector system was used. One viral vector was used to deliver SpCas9 and the other delivered sgRNAs against SpCas9 and the target locus (YFP), in the presence of mCherry.

    Journal: bioRxiv

    Article Title: Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

    doi: 10.1101/243683

    Figure Lengend Snippet: Schematics of Kamikaze CRISPR/Cas system. A dual AAV vector system was used. One viral vector was used to deliver SpCas9 and the other delivered sgRNAs against SpCas9 and the target locus (YFP), in the presence of mCherry.

    Article Snippet: Subsequently, the selected SpCas9 sgRNA (SpCas9 sgRNA4) was sub-cloned into a AAV package plasmid (pX552-hsyn-mCherry-YFP sgRNA2, sgRNA6 or pX552-LacZ sgRNA) at the MluI (catalog no. R3198; New England Biolabs) restriction site to generate YFP or LacZ targeting kamikaze CRISPR/Cas construct.

    Techniques: CRISPR, Plasmid Preparation

    Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.

    Journal: Molecular Biology of the Cell

    Article Title: LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

    doi: 10.1091/mbc.E16-12-0874

    Figure Lengend Snippet: Gα is not directly required for furrowing in the AB cell. (A) Spinning-disk confocal images from time-lapse movies of YFP::LET-99 embryos. Representative embryos; both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow) to generate a plot of pixel intensities along the AP axis. (B) Average line scans for all embryos (control n = 11; goa-1/gpa-16(RNAi) n = 9). NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body and a dashed line representing the plane of division. (C) YFP::LET-99 or YFP::LET-99; goa-1/gpa-16(RNAi) cortical images at NEB and furrowing onset. White arrowheads mark the YFP::LET-99 at the furrow in the AB cell, brackets mark the YFP::LET-99 band in the P1 cell. (D) Bright-field images to show the maximum extent of furrow ingression. (E) Furrow ingression in the AB cell is quantified as described in Figure 1 . Images in A and C are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Scale bars, 10 μm. Error bars represent ± SEM.

    Article Snippet: Further, the position of the peak of YFP::LET-99 cortical pixel intensities in goa-1;gpa-16(RNAi) embryos was indistinguishable from wild type at NEB (54.0 ± 1.4% EL; ).

    Techniques:

    LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.

    Journal: Molecular Biology of the Cell

    Article Title: LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

    doi: 10.1091/mbc.E16-12-0874

    Figure Lengend Snippet: LET-99 localization is myosin dependent. ( A) Kymograph of cortical images of YFP::LET-99 and NMY-2::mKate2 in the same embryo. Time is shown on the y -axis with 9 s between frames; M, A, and F represent metaphase, anaphase, and furrowing onset, respectively. Scale bar, 10 μm. Yellow arrows mark concomitant movement of NMY-2::mKate2 and YFP::LET-99 toward the furrow. (B) Stills from time-lapse fluorescence microscopy of YFP::LET-99 in control and strong depletion of NMY-2 by RNAi. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Arrowheads mark the band in control embryos. (C) Cortical kymograph of YFP::LET-99 in either nmy-2(RNAi) or par-3(it71) embryos. Colored lines represent metaphase, anaphase, or furrowing onset in green, magenta, and cyan, respectively.

    Article Snippet: Further, the position of the peak of YFP::LET-99 cortical pixel intensities in goa-1;gpa-16(RNAi) embryos was indistinguishable from wild type at NEB (54.0 ± 1.4% EL; ).

    Techniques: Fluorescence, Microscopy

    Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.

    Journal: Molecular Biology of the Cell

    Article Title: LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

    doi: 10.1091/mbc.E16-12-0874

    Figure Lengend Snippet: Either astral or central spindle microtubules are sufficient to localize LET-99 at anaphase. (A) Spinning-disk confocal images from time-lapse imaging of YFP::LET-99 transgenic embryos in the labeled backgrounds. Images are pseudocolored with Fire LUT such that the highest pixel values are pink/white and the lowest pixel values are blue/purple. Time relative to anaphase onset is shown in seconds. To generate a plot of intensity from anterior to posterior, both sides of the embryo cortex were traced from anterior (white arrow) to posterior (yellow arrow); arrowheads mark the LET-99 band as determined by the width of the peak of intensity at half-maximum. Scale bars represent 10 μm. (B) Average line scans for all embryos of a given genotype at NEB and late anaphase are shown with an asterisk marking peaks in signal from the polar body. Each graph corresponds to the genotype in the same row; n = number of embryos. Error bars represent ± SEM. See the text and Tables S1 and S2 for quantification of peak intensities and band position.

    Article Snippet: Further, the position of the peak of YFP::LET-99 cortical pixel intensities in goa-1;gpa-16(RNAi) embryos was indistinguishable from wild type at NEB (54.0 ± 1.4% EL; ).

    Techniques: Imaging, Transgenic Assay, Labeling