Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) Western blots of siCTL–, siRTN4–, and siCLIMP-63–transfected HT-1080 cells were probed with anti-RTN4, anti-CLIMP-63, and anti-β-actin as a loading control. (B) Representative images of ER tubules in HT-1080 cells transfected with ERmoxGFP and siCTL, siRTN4, or siCLIMP-63. Arrowheads indicate the tubules with increased blob length. Scale bar, 2 μm. (C) The quantification of maxima length, variation of maxima length (SD), and maxima-minima intensity differentials of ER tubules in HT-1080 cells transfected with siRTN4, siCLIMP-63, or siCTL. Bar graphs show mean ± SEM and scatter dot plots median with interquartile range. Significance assessed by Student t test from three independent experiments (20–40 line scans/each repeat). ** P < 0.01; *** P < 0.001. Numerical values that underlie the graphs and plots are shown in . CLIMP-63, cytoskeleton-linking membrane protein 63; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4, reticulon4; SD, standard deviation; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRTN4, siRNA to RTN4.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Western Blot, Transfection, Standard Deviation

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) STED images of ERmoxGFP in HT-1080 cells transfected with ERmoxGFP or cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1). Peripheral ER regions (white boxes) are shown as zooms; line scans of selected tubules in these regions (yellow boxes) are shown with ERmoxGFP in green and ER-shaping proteins in red. Scale bar, 5 μm; zooms, 2 μm. (B) Peripheral ER tubule maxima length, variation of maxima length (SD), and maxima-to-minima intensity differential are shown for cells transfected with ERmoxGFP alone (CTL) or cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1). Significance assessed by one-way ANOVA from three independent experiments (40 line scans/each repeat). Bar graphs show mean ± SEM and scatter dot plots median with interquartile range. * P < 0.05; ** P < 0.01; *** P < 0.001. Numerical values that underlie the graphs and plots are shown in . (C) Based on line scan analysis of peripheral ER tubules of HT-1080 cells cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1), percent localization of CLIMP-63, RTN4a, and ATL1 puncta to minima or maxima of lumenal ERmoxGFP-labeled tubules was quantified. Significance assessed by one-way ANOVA from four independent experiments (40 line scans/each repeat). Bar graphs show mean ± SEM. * P < 0.05; *** P < 0.001. Numerical values that underlie the graphs are shown in . ATL, atlastin; CLIMP-63, cytoskeleton-linking membrane protein 63; CTL, control ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4a, reticulon4a; SD, standard deviation; STED, stimulated emission depletion.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Transfection, Labeling, Standard Deviation

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) Representative merged images of single peripheral ER tubules expressing ERmoxGFP or Sec61βGFP labeled for calnexin or derlin-1. The dashed line indicates the site of line scan analysis along tubule. Fluorescence intensities of ER reporter (green) and protein (red) from line scans are presented as graphs. Scale bar, 0.5 μm. (B) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin and derlin-1 puncta to ERmoxGFP or Sec61βGFP maxima and minima was quantified. Values plotted are mean ± SEM from three independent experiments (40 tubules per repeat) with one-way ANOVA for significance. *** P < 0.001. Numerical values that underlie the graphs are shown in . (C) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin and derlin-1 puncta to ERmoxGFP maxima and minima was quantified in cells transfected with siCTL, siCLIMP-63, or siRTN4. Significance was assessed by χ 2 test from three independent experiments (20–40 tubules per repeat). *** P < 0.001. Numerical values that underlie the graphs are shown in . (D) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin puncta to ERmoxGFP maxima and minima was quantified in HT-1080 cells cotransfected with mCherry-CLIMP-63, mCherry-RTN4a, or mCherry-ATL1 compared with CTL. Significance assessed by χ 2 test from three independent experiments (40 tubules per repeat). *** P < 0.001. Numerical values that underlie the graphs are shown in . ATL, atlastin; CLIMP-63, cytoskeleton-linking membrane protein 63; CTL, control; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4a, reticulon4a; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRTN4, siRNA to RTN4.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Expressing, Labeling, Fluorescence, Transfection

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: STED live cell imaging (40 ms/frame over 4 seconds) of isolated ROIs of peripheral ERmoxGFP-labeled tubules (a) was performed for COS-7 cells cotransfected with mCherry-CLIMP-63 or mCherry-RTN4a (A) or transfected with siCLIMP-63, siRTN4, or siCTL (B). Kymograms show the distribution of ERmoxGFP at specific sites along ER tubules over time (b). From plots of normalized average intensity over time (c), we determined the CoV along the tubule length as a measure of localized distribution of ERmoxGFP to distinct domains along peripheral ER tubules (d). Scatter dot plots show median with interquartile range from three independent experiments (20–50 tubules per condition) with one-way ANOVA for significance. * P < 0.05; *** P < 0.001. Numerical values that underlie the plots are shown in . CLIMP-63, cytoskeleton-linking membrane protein 63; CoV, coefficient of variation; CTL, control; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; ROI, region of interest; RTN4a, reticulon4a; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRNA, small interfering RNA; siRTN4, siRNA to RTN4; STED, stimulated emission depletion.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Live Cell Imaging, Isolation, Labeling, Transfection, Small Interfering RNA

Journal: Scientific Reports

Article Title: SARS-CoV-2 envelope protein alters calcium signaling via SERCA interactions

doi: 10.1038/s41598-024-71144-5

Figure Lengend Snippet: Homo-oligomerizations of E protein and regulins. ( a ) Fluorescence images of HeLa cells expressing eGFP- (green) and mCherry-tagged (magenta) E protein. The first and second columns show images before (pre) and after (post) photobleaching, respectively. The FRET image shows FRET values calculated by FRETcalc . The scale bar is 10 μm. ( b ) FRET efficiency values produced by homo-oligomers. ctrl0: eGFP/mCherry pair, ctrl: eGFP-SERCA/mCherry-SERCA pair. Pooled data from 3 to 3 independent experiments are shown. Dots represent individual cells. Median and interquartile ranges are indicated with box plots. Data were analyzed by Kolmogorov–Smirnov test (* p < 0.05, **** p < 0.0001).

Article Snippet: For mCherry labelling we replaced eGFP to mCherry from pTK96_mCherry-MRLC2 (Addgene #46358) vector using AgeI and BsrGI sites.

Techniques: Fluorescence, Expressing, Produced

Journal: Scientific Reports

Article Title: SARS-CoV-2 envelope protein alters calcium signaling via SERCA interactions

doi: 10.1038/s41598-024-71144-5

Figure Lengend Snippet: FRET efficiency for hetero-oligomerization among E protein, regulins ( a ), and SERCA ( b ). Ctrl: eGFP-SERCA/mCherry-SERCA pair. PLN # : FRET values for eGFP-PLN tagged at the luminal end and mCherry-SERCA as a positive control for transmembrane measurements). Pooled data from 3 to 3 independent experiments (for SERCA/E pair: 5 independent experiments) are shown. Dots represent individual cells. Median and interquartile ranges are indicated with box plots. Data were analyzed by Kolmogorov–Smirnov test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant). ( c ) The immunoprecipitation experiment using the anti-SERCA IID8 antibody successfully confirmed an association between the eGFP-E protein and mCherry-SERCA2b. Western blot analysis was employed for both the lysates (INPUT) and the eluted fractions (IP), using anti-SERCA IID8 antibody and anti-GFP antibodies as indicated. Non-immune mouse IgG1 was used for IP as a negative control (lanes 3 and 4). Input is ~ 1% of the sample loaded for the immunoprecipitation (~ 1 µg).

Article Snippet: For mCherry labelling we replaced eGFP to mCherry from pTK96_mCherry-MRLC2 (Addgene #46358) vector using AgeI and BsrGI sites.

Techniques: Positive Control, Immunoprecipitation, Western Blot, Negative Control

Journal: Aging Cell

Article Title: Astrocyte‐ and NMDA receptor‐dependent slow inward currents differently contribute to synaptic plasticity in an age‐dependent manner in mouse and human neocortex

doi: 10.1111/acel.13939

Figure Lengend Snippet: Slow inward currents (SICs) of murine neocortical pyramidal neurons are slow events mediated by activation of NMDA receptors and astrocytes. (a) The electrophysiological parameters of SICs unambiguously distinguish them from EPSCs. Representative records of a SIC (left) and two EPSCs (right). (b) Statistical comparison of SIC and EPSC amplitudes, rise and decay times and charge transfer data (“area”) of the individual events (hollow columns: average ± SEM, gray dots: individual data). (c) SICs are mediated by NMDA receptors with GluN2B subunits. Representative recordings under control conditions (0 Mg 2+ naCSF), with 500 nM PPPA (GluN2A subunit specific NMDA receptor inhibitor), with additional ifenprodil (5 μM, GluN2B‐specific NMDA receptor blocker) and with 10 μM D‐AP5 (nonspecific NMDA receptor inhibitor). (d) Statistical comparison of charge transfer by SICs in a minute (“SIC activity”) in the presence of the drugs shown in panel ‘c’ (hollow columns: average ± SEM, gray dots: individual data). (e) A neocortical pyramidal cell labeled with biocytin during the patch clamp experiment (green) and astrocytes expressing mCherry tag (red) on two confocal z‐stack images (scale bar: 50 μm). (f) Representative traces under control conditions, with 0.1% DMSO and with 10 μM CNO from a sample expressing hM3D(Gq) chemogenetic actuator under GFAP promoter. (g) Statistical comparison of SIC activity under the conditions shown on panel ‘f’ (hollow columns: average ± SEM, gray dots: individual data). (h) Representative traces recorded in control, with DMSO, and 10 μM CNO from a sample lacking hM3D(Gq) chemogenetic actuator but expressing only mCherry tag under GFAP promoter. (i) Statistical comparison of SIC activity under conditions shown on panel ‘h’ (hollow columns: average ± SEM, gray dots: individual data). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Young adult (9–12 weeks old) lox‐tdTomato mice from both sexes were subjected to stereotaxic injection of ready‐to‐use viruses carrying plasmids encoding hM3D(Gq) chemogenetic actuator and mCherry tag expressed under GFAP promoter (pAAV‐GFAP‐hM3D(Gq)‐mCherry (AAV5); a gift from Bryan Roth; Addgene plasmid # 50478; http://n2t.net/addgene:50478 ; RRID:Addgene_50478, n = 13; titer: 2 × 10 13 GC/mL) or solely mCherry tag as control (pAAV‐GFAP104‐mCherry (AAV5), a gift from Edward Boyden; Addgene plasmid # 58909; http://n2t.net/addgene:58909 ; RRID:Addgene_58909, Perea et al., , n = 10; titer: 1.7 × 10 13 GC/mL).

Techniques: Activation Assay, Comparison, Control, Activity Assay, Labeling, Patch Clamp, Expressing

Journal: bioRxiv

Article Title: Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids

doi: 10.1101/2023.05.25.542343

Figure Lengend Snippet: (A) Schematic showing the derivation of long-term expandable iNPC lines from hPSCs and its versatile applications. (B–D) Immunofluorescence analyses of 11.3-week human fetal kidney sections for SXI2, p-p38 (B), p-SMAD2/3 (C), and p-SMAD1/5/8 (D). Scale bars, 50 μm. (E and F) Immunofluorescence analyses (E) and quantification (F) of YAP expression in iNPCs cultured in hNPSR-v1 medium supplemented with 0, 2, or 4 µM TRULI for 6 days. Scale bars, 50 μm. (G) Bright-field image of iNPCs cultured in hNPSR-v2 medium for 87 days. Scale bar, 100 μm. (H) Growth curve of iNPCs cultured in hNPSR-v2 in a typical 4-day passage cycle starting from 5,000 cells. (I and J) Immunofluorescence analyses (I) and quantification (J) of iNPCs cultured in hNPSR-v2 medium for 21 days for various NPC marker genes as indicated. Scale bars, 100 μm. (K) Time-course bright-field images showing clonal expansion of iNPCs from one single cell in hNPSR-v2 medium. Scale bars, 100 μm. (L) Immunofluorescence analysis of a single cell iNPC clone for SIX2 and PAX2. Scale bars, 50 μm. (M and N) 3D (M) and 2D (N) PCA plots of bulk RNA-seq data. (O) Heatmap showing gene expression of selected marker genes for undifferentiated NPCs and differentiated kidney cell types, in primary and cultured NPCs, as well as FACS-purified SIX2 + or SIX2 + /PAX2 + iNPCs without further culture (D0-iNPC-SIX2 and D0-iNPC-SIX2/PAX2). Primary SIX2-negative non-NPCs (Pri-SIX2-Neg) isolated from human fetal kidneys were used as negative controls. (P and Q) Bright field (BF) and fluorescence images (P) and quantification (Q) of mCherry expression in iNPCs upon lentiviral overexpression of mCherry (lentiviral OE), or targeted CRISPR/Cas9 knock-in of mCherry-expressing cassette into AAVS1 allele (CRISPR KI). Scale bars, 50 μm. (R and S) Whole-mount immunofluorescence analyses of human nephron organoids generated from iNPCs cultured in hNPSR-v2 medium for 42 days for various nephron marker genes as indicated. Scale bars, 200 μm. (T) Whole-mount immunofluorescence analyses of human nephron organoids generated from iNPCs cultured in hNPSR-v2 for 4 days (upper panels) or 32 days (lower panels) for various nephron marker genes as indicated. Scale bars, 200 μm. (U) Whole-mount immunofluorescence analyses of human nephron organoid generated from iNPCs cultured in hNPSR-v2 medium for 42 days for various nephron marker genes as indicated. Scale bar, 200 μm. Data are presented as mean ± SD. Each column represents counts from three biological replicates (n=3). The significance was determined by two-tailed unpaired Student’s t tests; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figures S9, S10, and Tables S12 and 13.

Article Snippet: 24 hours later, iNPCs were transfected with a mixture of two plasmids that provide donor DNA for targeted knockin of CAG promoter-driven mCherry expression cassette at the AAVS1 loci (pAAVS1-P-CAG-mCherry, Addgene, # 80492), and that express Cas9 and sgRNA (pXAT2, Addgene, # 80494), at the ratio of 3:1, using Lipofectamine 3000 transfection reagent.

Techniques: Immunofluorescence, Expressing, Cell Culture, Marker, RNA Sequencing Assay, Purification, Isolation, Fluorescence, Over Expression, CRISPR, Knock-In, Generated, Two Tailed Test

Journal: bioRxiv

Article Title: A chemically inducible muscle ablation system for zebrafish

doi: 10.1101/2022.04.20.488934

Figure Lengend Snippet: ( A, B ) 3 dpf actc1b:epNTR embryos treated with DMSO vehicle (A) or 10 mM MTZ (B) at 1 dpf. ( C, D ) 3 dpf actc1b:epNTR embryos treated with DMSO vehicle (C) or 10 mM MTZ (D) and stained with 10 μM acridine orange. mCherry and Acridine Orange channels are split to observe lack of acridine orange staining in the DMSO treated embryo. Scale Bars=50 μM

Article Snippet: The PCRII-epNTR and actc1b:mcherry-caax plasmids were cut using EcoRI and the respective DNA fragments were purified by gel extraction (NEB).

Techniques: Staining

Journal: bioRxiv

Article Title: A chemically inducible muscle ablation system for zebrafish

doi: 10.1101/2022.04.20.488934

Figure Lengend Snippet: ( A-D ): actc1b:epNTR embryos ablated with 10 mM MTZ at 1 dpf for 30 hours followed by washout. ( A ) actc1b:epNTR embryos imaged for mCherry immediately after washout. ( B ) actc1b:epNTR embryos imaged 1 day after washout. ( C ) actc1b:epNTR embryos imaged 3 days after washout. ( D ) actc1b:epNTR embryos imaged 4 days after washout. ( E-F ): In-situ hybridization for myf5 . ( E ) Non-ablated embryos. ( F ) Ablated embryos. ( G-H ): In-situ hybridization for ttn.2 . ( G ) Non-ablated embryos. ( H ) Ablated embryos.

Article Snippet: The PCRII-epNTR and actc1b:mcherry-caax plasmids were cut using EcoRI and the respective DNA fragments were purified by gel extraction (NEB).

Techniques: In Situ Hybridization

Journal: bioRxiv

Article Title: A chemically inducible muscle ablation system for zebrafish

doi: 10.1101/2022.04.20.488934

Figure Lengend Snippet: ( A ) A mpeg1:GFP host embryo containing transplanted wild-type cells that lack the actc1b:epNTR transgene shows macrophages (green) that do not linger within muscle tissue. ( B ) A mpeg1:GFP host embryo with transplanted actc1b:epNTR muscle cells (magenta) treated with MTZ and imaged 1 day after washout. Embryos show increased residence of macrophage cells in ablated area. ( C ) A mpeg1:GFP macrophage in an actc1b:epNTR embryo. mCherry puncta indicates uptake of ablated muscle debris from injured cells (white arrows). Scale Bars=50 μM

Article Snippet: The PCRII-epNTR and actc1b:mcherry-caax plasmids were cut using EcoRI and the respective DNA fragments were purified by gel extraction (NEB).

Techniques:

Journal: bioRxiv

Article Title: A chemically inducible muscle ablation system for zebrafish

doi: 10.1101/2022.04.20.488934

Figure Lengend Snippet: Cascade-blue dextran was injected into wild-type donor embryos and transplanted into tg(mpeg1:EGFP) embryos. ( A ) An image showing donor cells in a tg(mpeg1:EGFP) embryo. Green indicates macrophages. Blue indicates transplanted cells in the somites. Magenta (absent) indicates lack of expression of a tg(actc1b:epNTR-mcherry-caax) transgene. ( B ) Single blue channel from merged image in A. ( C ) Single green channel from merged image in A.

Article Snippet: The PCRII-epNTR and actc1b:mcherry-caax plasmids were cut using EcoRI and the respective DNA fragments were purified by gel extraction (NEB).

Techniques: Injection, Expressing

Journal: Nature Communications

Article Title: Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

doi: 10.1038/ncomms12277

Figure Lengend Snippet: ( a ) Schematics of DNA constructs used for the production of EXPLOR. ( b ) Schematic showing fusion proteins and their proposed action. ( c ) HEK293T cells were transiently transfected with CIBN-EGFP-CD9 and mCherry-CRY2 expression vectors. The mCherry fluorescence was imaged before and after 488-nm laser stimulation (15 s in duration, 350 μW cm −2 ). Scale bars, 20 μm (5 μm for inset images). A representative result from at least 10 experiments. ( d ) HEK293T cells transiently transfected with CIBN-EGFP-CD9 and mCherry-CRY2 were imaged for time-lapse imaging of mCherry fluorescence for varying time periods (0–12 min) after a stimulation (black arrow) of 488-nm light (15 s in duration, 350 μW cm −2 ). Scale bars, 5 μm. A representative result of at least 10 experiments. ( e ) Quantification of mCherry fluorescence in the cytoplasm and at the plasma membrane. Data are presented as the mean±s.e.m. ( n =3).

Article Snippet: Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Construct, Transfection, Expressing, Fluorescence, Imaging, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

doi: 10.1038/ncomms12277

Figure Lengend Snippet: ( a ) Cells transiently transfected with CIBN-EGFP-CD9 and mCherry-CRY2 expression vectors were maintained under blue light illumination of varying powers for 48 h. Cell-derived exosomes were subject to immunoblot analysis using antibodies against mCherry, EGFP and CD63, an exosome marker. A representative result from three independent experiments. ( b ) The graph presents densitometry analysis for normalized amount of mCherry-CRY2 protein over CIBN-EGFP-CD9 protein from three independent experiments. Data are presented as the mean±s.e.m. ( n =3), and Tukey's post hoc test was applied to significant group effects (** P <0.01, *** P <0.001) identified by analysis of variance.

Article Snippet: Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Expressing, Derivative Assay, Western Blot, Marker

Journal: Nature Communications

Article Title: Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

doi: 10.1038/ncomms12277

Figure Lengend Snippet: ( a ) HEK293T cells were transiently transfected with luciferase-mCherry-CRY2 expression vector alone, XPack-luciferase-mCherry expression vector or co-transfected with CIBN-EGFP-CD9 and luciferase-mCherry-CRY2 expression vectors. After 24 h, cells were imaged by fluorescence microscopy for the expression profile of mCherry fusion proteins. A representative result from five independent experiments. Scale bars, 20 μm. ( b ) Quantification of mCherry fluorescence. Data are presented as the mean±s.e.m. ( n =5), and Tukey's post hoc test was applied to significant group effects identified by analysis of variance (ANOVA). Control: untransfected HEK293T cells; OVER: cells transiently transfected with a luciferase-mCherry-CRY2 vector; XP: cells transfected with an XPACK-luciferase-mCherry vector; and EXPLOR: cells transfected with both luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 vectors. ( c ) Cells transiently transfected with various vectors were maintained for 48 h. In the case of EXPLOR-producing cells, cells were maintained in the absence (OFF) or presence (ON) of blue light illumination; 5 × 10 8 particles of the isolated exosomes were analysed for luciferase activity. Data are presented as the mean±s.e.m. ( n =3), and Tukey's post hoc test was applied to significant group effects (** P <0.01, *** P <0.001) identified by ANOVA. ( d ) Loading efficiency of various protein-loaded exosomes was calculated by dividing the number of luciferase molecules in exosomes with the number of luciferase molecules in the exosome-producing cells. Numbers of luciferase molecules were estimated from a standard curve using recombinant luciferase. Data are presented as the mean±s.e.m. ( n =3), and Tukey's post hoc test was applied to significant group effects (*** P <0.001) identified by ANOVA. NS, not significant.

Article Snippet: Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Control, Isolation, Activity Assay, Recombinant

Journal: Nature Communications

Article Title: Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

doi: 10.1038/ncomms12277

Figure Lengend Snippet: ( a , b ) HeLa cells were incubated in the absence or presence of 5 × 10 9 particles of various isolated exosomes for 24 h and imaged by fluorescence microscopy. The fluorescence intensities of mCherry were quantified by two imaging processing tools, ImageJ and Cellprofiler. Data are presented as the mean±s.e.m. ( n =15), and Tukey's post hoc test was applied to significant group effects (*** P <0.001) identified by analysis of variance (ANOVA). Scale bars, 100 μm. ( c , d ) HeLa cells were incubated in the absence or presence of 0.1 mg ml −1 mCherry:EXPLORs or Bax-mCherry:EXPLORs for 12 h, and fixed with 4% paraformaldehyde. Then, cytochrome c was stained with an antibody conjugated with Alexa Fluor 647 and imaged by confocal microscopy. The ratios of cytochrome c localization were analysed by cell counting. Data are presented as the mean±s.e.m. ( n =3), and Tukey's post hoc test was applied to significant group effects (** P <0.01) identified by ANOVA. Scale bars, 20 μm. ( e ) HeLa cells were incubated in the absence or presence of 0.1 mg ml −1 mCherry:EXPLORs or srIκB:EXPLORs for 12 h, treated with 10 ng ml −1 tumour necrosis factor- α (TNF-α) for an additional 30 min, and fixed with 4% paraformaldehyde. NF-κB p65 was stained with an antibody conjugated with Alexa Fluor 488 and imaged by confocal microscopy. ( f ) The nuclear extracts of cells were assayed for the DNA-binding activity of p65/c-Rel (NF-κB). Data are presented as the mean±s.e.m. ( n =3), and Tukey's post hoc test was applied to significant group effects (** P <0.01) identified by ANOVA. Scale bars, 20 μm. NS, not significant.

Article Snippet: Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Isolation, Fluorescence, Microscopy, Imaging, Staining, Confocal Microscopy, Cell Counting, Binding Assay, Activity Assay