Journal: Cell

Article Title: RIPK3 restricts viral pathogenesis via cell death-independent neuroinflammation

doi: 10.1016/j.cell.2017.03.011

Figure Lengend Snippet: Key Resource Table

Article Snippet: Cells were stained using a primary anti-mCherry antibody (Rockland) for 1 hour and goat anti-rabbit AlexaFluor 594 secondary antibody (ThermoFisher) for 15 minutes, both at room temperature.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software

Journal: Heliyon

Article Title: Median raphe glutamatergic neuron-mediated enhancement of GABAergic transmission and suppression of long-term potentiation in the hippocampus

doi: 10.1016/j.heliyon.2024.e38192

Figure Lengend Snippet: MRN VGLUT3 neuron-mediated glutamatergic transmission in SR/SLM 5-HT3aR neurons. A) A schematic of AAV injection into the MRN. The left panel shows an image of the MRN from VGLUT3 Cre/5-HT3aR-EGFP mouse which received an AAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA injection into the MRN. Scale 150 μm. B) An image of the hippocampus from MRN VGLUT3 ChR2/5-HT3aR-EGFP mouse. The inset shows EGFP-expressing SR/SLM 5-HT3aR neurons and mCherry-expressing MRN VGLUT3 axons. Scale 300 μm/25 μm (inset). C) The upper panel shows a schematic for recording MRN VGLUT3 neuron-mediated transmission in SR/SLM 5-HT3aR neurons. The bottom panel shows example traces for MRN VGLUT3 neuron-mediated EPSCs in SR/SLM 5-HT3aR neurons without drug application, in the presence of tetrodotoxin (TTX, 1 μM), TTX+4-aminopyridine (4-AP, 100 μM) and TTX+4-AP + DNQX (10 μM). Scale 50 pA/10 ms. The blue line indicates light application. D) EPSC amplitude in SR/SLM 5-HT3aR neurons before the drug application, in the presence of TTX, TTX+4-AP, and TTX+4-AP + DNQX (6 neurons from 3 female mice and 6 neurons from 3 male mice). F (1,11) = 22.01, P = 0.001. Data are presented as mean ± SEM. E) EPSC amplitude in female (17 neurons/5 mice) and male (16 neurons/5 mice) groups. Female and male groups did not show statistically significant difference in EPSC amplitude (P = 0.8). The horizontal line in each group represents the mean and the vertical line represents SEM. F) The delay between the onset of the light stimulation and the onset of EPSCs in female (17 neurons/5 mice) and male (16 neurons/5 mice) groups. Female and male groups did not show a statistically significant difference in the delay between the onset of light stimulation and the onset of EPSCs (P = 0.77). n.s. not statistically significant.

Article Snippet: The slices were then incubated in mCherry polyclonal antibody (Rockland Immunochemicals, Cat. No. 600401P16) or Anti-GFP antibody (Abcam, Cat. No. ab13970, RRID: AB_300798 ) at a dilution of 1:1000 in 0.3 % Triton-X/TBS for 24 h at 4 °C [ , ].

Techniques: Transmission Assay, Injection, Expressing

Journal: Life Science Alliance

Article Title: Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies

doi: 10.26508/lsa.202101269

Figure Lengend Snippet: (A) Schematic representation of the functionalized nanobodies. The standard nanobody (VHH-std) consists of the GFP-specific VHH domain, T7 and HA epitope tags, a biotin acceptor peptide (BAP), and a hexahistidine (His6) purification tag. Other nanobodies in addition contain two tyrosine sulfation sequences (VHH-2xTS) or mCherry (VHH-mCherry). Scale bar in aa. (B) Bacterially expressed and purified nanobodies (30 μg) were analyzed by SDS-gel electrophoresis and Coomassie staining (left). Immunoblot analysis of nanobodies (10 ng) with antibodies against the HA, His6, T7, or mCherry epitopes, or with streptavidin-HRP (SA-HRP). Marker proteins with molecular weights in kilodalton are shown on the left. As previously reported ( ; ), mCherry-containing nanobodies are slightly susceptible to clipping between the VHH and mCherry domains. (C) HeLa cells were transfected with non-targeting siRNA or siRNAs targeting μ1A-adaptin, Vps26, TIP47, or Rab9a. 3 d after transfection, the cells were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) To determine the knockdown (kd) efficiency, the residual protein was quantified in percent of the value after control-kd (mean and SD of three independent experiments). (E) HeLa cells stably expressing EGFP-CDMPR were depleted of μ1A-adaptin, Vps26, TIP47, or Rab9a as in (C). Cells were incubated for 1 h at 37°C with full medium containing 5 μg/ml VHH-mCherry (∼0.1 μM), fixed, stained for EEA1 and nuclei (DAPI, blue), and imaged by fluorescence microscopy. Bar: 10 μm. (F) Quantitation of the percentage of cells displaying the CDMPR localization phenotypes “mainly TGN,” “mainly peripheral,” or “fully peripheral” as in and . For each condition, random frames with a total of 136–140 cells were scored from three independent experiments. (G, H) Total and surface EGFP-CDMPR levels in RNAi-silenced cells were quantified by flow cytometry. Cells were incubated for 30 min at 4°C with VHH-mCherry for exclusive binding to EGFP-CDMPR at the cell surface, washed, dissociated and analyzed for GFP and mCherry fluorescence to determine the levels of total (G) and surface EGFP-CDMPR (H), respectively. Median fluorescence intensities above background of parental HeLa cells without EGFP-CDMPR of each condition were normalized to the average of cells treated with non-targeting control siRNA. For each condition, 50,000 cells were analyzed in each experiment (mean and SD of three independent experiments).

Article Snippet: The anti-mCherry nanobody sequence (LaM4) based on Rout and colleagues ( ) was a kind gift from Kazuhisa Nakayama (#70696; Addgene).

Techniques: Purification, SDS-Gel, Electrophoresis, Staining, Western Blot, Marker, Transfection, Knockdown, Control, Stable Transfection, Expressing, Incubation, Fluorescence, Microscopy, Quantitation Assay, Flow Cytometry, Binding Assay

Journal: Life Science Alliance

Article Title: Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies

doi: 10.26508/lsa.202101269

Figure Lengend Snippet: HeLa cells stably expressing EGFP-CDMPR were transiently transfected with non-targeting siRNA (Control-kd) or siRNAs targeting TIP47, Rab9a, Vps26, μ1A, epsinR, GGA1–3, AP-2α, or clathrin CHC17. 3 d after transfection, cells were incubated with 2 μg/ml VHH-2xTS in PBS at 4°C for 1 h to label the surface fraction of EGFP-CDMPR. Subsequently, cells were washed and lysed, and endogenous protein levels were analyzed by immunoblotting with antibodies against the indicated proteins (VHH-2xTS was detected with anti-His6). Only knockdown of CHC17 influenced surface levels of EGFP-CDMPR (monitored by bound nanobody). Total EGFP-CDMPR remained unaffected in all knockdowns.

Article Snippet: The anti-mCherry nanobody sequence (LaM4) based on Rout and colleagues ( ) was a kind gift from Kazuhisa Nakayama (#70696; Addgene).

Techniques: Stable Transfection, Expressing, Transfection, Control, Incubation, Western Blot, Knockdown

Journal: Life Science Alliance

Article Title: Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies

doi: 10.26508/lsa.202101269

Figure Lengend Snippet: (A) Schematic representation of the functionalized anti-mCherry nanobodies. The standard nanobody (LaM4-std) consists of the mCherry-specific LaM4 domain, T7 and HA epitope tags, a biotin acceptor peptide (BAP), and a hexahistidine (His6) purification tag. Other nanobodies in addition contain one to three tyrosine sulfation sequences (TS). Scale bar in aa. (B) Bacterially expressed and purified nanobodies (30 μg) were analyzed by SDS-gel electrophoresis and Coomassie staining (left). Immunoblot analysis of nanobodies (10 ng) with antibodies against the HA, His6, or T7, or with streptavidin-HRP (SA-HRP). Marker proteins with molecular weights in kilodalton are shown on the left. (C) Parental HeLaM cells and HeLaM-GGA2ks cells stably expressing mCherry-CDMPR were transfected with non-targeting siRNA (−) or siRNAs silencing endogenous GGA1–3 (+). Cell lysates were subjected to immunoblot analysis with antibodies against the indicated proteins. (D) HeLaM-GGA2ks cells stably expressing mCherry-CDMPR were transfected with siRNAs targeting endogenous GGA1–3. These cells were transfected with a plasmid expressing His6/myc-tagged procathepsin D 24–36 h before analysis. Media of cells incubated for 2 h in serum-free medium supplemented with 5 mM mannose-6-phosphate to prevent cathepsin D binding to surface MPRs, and with or without rapamycin (+ or − Rapa, respectively) were analyzed by collecting procathepsin D (pCatD) with Ni/NTA beads and immunoblotting with anti-myc antibodies. Cell lysates were immunoblotted for actin as a control. (E) Procathepsin D missorted upon knocksideways of GGA2 (+Rapa) was quantified from immunoblots as shown in panel (D), normalized to the DMSO-treated (−Rapa) control (mean and SD of four independent experiments). (F) HeLa-GGA2ks cells stably expressing mCherry-CDMPR after silencing endogenous GGA1–3 were treated with or without 500 nM rapamycin for 1 h and processed for fluorescence microscopy to detect mCherry-CDMPR and recombinant GGA2-FKBP. Bar: 10 μm.

Article Snippet: The anti-mCherry nanobody sequence (LaM4) based on Rout and colleagues ( ) was a kind gift from Kazuhisa Nakayama (#70696; Addgene).

Techniques: Purification, SDS-Gel, Electrophoresis, Staining, Western Blot, Marker, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Incubation, Binding Assay, Control, Fluorescence, Microscopy, Recombinant

Journal: Life Science Alliance

Article Title: Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies

doi: 10.26508/lsa.202101269

Figure Lengend Snippet: HeLa cells transiently expressing mCherry-CDMPR or TfR-mCherry were incubated for 1 h at 37°C with full medium containing 5 μg/ml LaM4-EGFP (∼0.1 μM), fixed, stained for nuclei (DAPI, blue), and imaged by fluorescence microscopy. Bar: 10 μm.

Article Snippet: The anti-mCherry nanobody sequence (LaM4) based on Rout and colleagues ( ) was a kind gift from Kazuhisa Nakayama (#70696; Addgene).

Techniques: Expressing, Incubation, Staining, Fluorescence, Microscopy

Journal: Life Science Alliance

Article Title: Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies

doi: 10.26508/lsa.202101269

Figure Lengend Snippet: (A, D) Schematic outline of the retrograde (A) anterograde (D) transport sulfation assays, respectively, with HeLa-GGA2ks/mCherry-CDMPR cells. (B) HeLa-GGA2ks cells stably expressing mCherry-CDMPR were siRNA-silenced for endogenous GGA1–3. The cells were starved for sulfate for 1 h, and then labeled for up to 75 min with [ 35 S]sulfate in the presence of LaM4-3xTS nanobodies either in the absence (−) or presence of 500 nM rapamycin to inactivate GGA2-FKBP (+Rapa). The nanobodies were isolated by Ni/NTA beads and subjected to SDS-gel electrophoresis followed by immunoblot analysis (anti-His6) and autoradiography ([ 35 S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. (C) Experiments as shown in panel B were quantified and presented as the percentage of the value in the absence of rapamycin after 75 min (mean and SD of three independent experiments). Without rapamycin is shown as black squares, with rapamycin as gray circles; uptake as open symbols, sulfation as filled symbols. (E) HeLa-GGA2ks cells stably expressing mCherry-CDMPR were siRNA-silenced for endogenous GGA1–3, followed by starvation for sulfate in the presence of LaM4-3xTS to preload mCherry-CDMPR in the surface/endosome/TGN pool. The cells were then labeled with [ 35 S]sulfate for up to 75 min in the continued presence of LaM4-3xTS, without or with addition of 500 nM rapamycin after 15 min (arrow) to inactivate GGA2 (+Rapa). (B) Nanobody analysis was performed as above (B). (F) Experiments as shown in panel E were quantified and presented as the percentage of the value in the absence of rapamycin after 75 min (mean and SD of three independent experiments). Without rapamycin is shown as black squares, with rapamycin as gray circles; uptake as open symbols, sulfation as filled symbols.

Article Snippet: The anti-mCherry nanobody sequence (LaM4) based on Rout and colleagues ( ) was a kind gift from Kazuhisa Nakayama (#70696; Addgene).

Techniques: Stable Transfection, Expressing, Labeling, Isolation, SDS-Gel, Electrophoresis, Western Blot, Autoradiography, Control

Journal: Biomedical Optics Express

Article Title: Temporal variance mapping with machine learning for label-free 3D chromatin imaging using optical interferometric microscopy

doi: 10.1364/BOE.583584

Figure Lengend Snippet: Machine learning-based inference of chromatin fluorescence from label-free structure and dynamics maps. (a) Schematic of the training and validation process for a CNN model f S D from paired label-free inputs ( S , D ) to fluorescence outputs f S D ( S , D ) . (b) Quantitative evaluation of prediction accuracy using MS - SSIM ( Y ˆ , Y ) to compare the model-generated fluorescence images Y ˆ , with ground truth confocal images Y . In the box plots, the lower and upper boundaries of the box indicate the 25th and 75th percentiles, respectively. Within the box, a solid line marks the median. [*** P < 0.001 (Student’s t test)] (c–f) Confocal fluorescence image of H2B-mCherry as ground truth Y (c), and CNN predictions obtained with combined inputs Y ˆ S D = f S D ( S , D ) , (d), dynamics map only Y ˆ D = f D ( D ) , (e), and structure map only Y ˆ S = f S ( S ) , (f) as input.

Article Snippet: A stable U2OS cell line expressing H2B-mCherry was established by Omisc Bio (Taiwan) using a knock-in technique with the H2B-mCherry plasmid (pH2B_mCherry_IRES_ neo3, Addgene #21044).

Techniques: Fluorescence, Biomarker Discovery, Generated

Journal: Biomedical Optics Express

Article Title: Temporal variance mapping with machine learning for label-free 3D chromatin imaging using optical interferometric microscopy

doi: 10.1364/BOE.583584

Figure Lengend Snippet: Label-free, high-resolution imaging of nuclear structure and dynamics in live cells. (a) Schematic of the COBRI microscope integrated with a confocal fluorescence channel, enabling simultaneous label-free and fluorescence imaging. (b) COBRI image of a live cell nucleus showing clearly resolved nucleoli (indicated by arrows). (c) Temporal variance map of the same nucleus, calculated from a sequence of COBRI images acquired at 1,000 frames per second. The spatial variation in variance reflects differences in scattering intensity caused by dynamic molecular structures. (d) Confocal fluorescence image of the same nucleus expressing H2B-mCherry, illustrating the spatial distribution of chromatin for reference.

Article Snippet: A stable U2OS cell line expressing H2B-mCherry was established by Omisc Bio (Taiwan) using a knock-in technique with the H2B-mCherry plasmid (pH2B_mCherry_IRES_ neo3, Addgene #21044).

Techniques: Imaging, Microscopy, Fluorescence, Sequencing, Expressing

Journal: Biomedical Optics Express

Article Title: Temporal variance mapping with machine learning for label-free 3D chromatin imaging using optical interferometric microscopy

doi: 10.1364/BOE.583584

Figure Lengend Snippet: Correlation between dynamics maps and confocal fluorescence images of chromatin. (a) Joint intensity distribution of the pixel-wise differential temporal variance values from the dynamics map (computed from 2,000 frames) and fluorescence intensities of H2B-mCherry, showing a modest positive correlation. (b) Correlation between the dynamics maps computed with varying numbers of frames and the reference dynamics map computed with 2,000 frames. All image data are recorded at 1000 Hz with a frame time of 1 ms. (c) Representative dynamics maps of a cell nucleus computed using increasing numbers of frames, ranging from 20 to 2,000. (d) Confocal fluorescence image of H2B-mCherry from the same nucleus shown in (c).

Article Snippet: A stable U2OS cell line expressing H2B-mCherry was established by Omisc Bio (Taiwan) using a knock-in technique with the H2B-mCherry plasmid (pH2B_mCherry_IRES_ neo3, Addgene #21044).

Techniques: Fluorescence

Journal: Biomedical Optics Express

Article Title: Temporal variance mapping with machine learning for label-free 3D chromatin imaging using optical interferometric microscopy

doi: 10.1364/BOE.583584

Figure Lengend Snippet: Predicted confocal fluorescence images of chromatin and their corresponding ground truths. (a) Seven representative paired images of confocal fluorescence signals predicted by our algorithm using structural and dynamical maps as input, alongside their ground truth counterparts. (b) Joint intensity distribution of pixel-wise predicted fluorescence values and H2B-mCherry fluorescence intensities (outliers removed), demonstrating a strong positive correlation with improved linearity.

Article Snippet: A stable U2OS cell line expressing H2B-mCherry was established by Omisc Bio (Taiwan) using a knock-in technique with the H2B-mCherry plasmid (pH2B_mCherry_IRES_ neo3, Addgene #21044).

Techniques: Fluorescence