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453 results for “sting”
Data from: Ethnobotany of stinging nettle (Urtica simensis Hochst. ex. A. Rich.) in the Oromia region of central and southeastern highlands of Ethiopia
<p>The data was generated to document ethnobotanical uses of <em>U. simensis</em> and the associated traditional knowledge of the indigenous people and to identify the factors limiting harvesting and utilization of stinging nettle in North Shewa (R4), Bale and Arsi zones of the Oromia region, central and southeastern highlands of Ethiopia. Thirteen districts were purposively selected from the three zones and a total of 130 respondents were sampled, with consideration of gender, age, occupation, and wealth status. Data were collected using semistructured interviews, tour-guided field observations, and focus group discussions.</p>
STING OPS: BJ1 Secondary Screen Single-Cell Features
<p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for BJ1 fibroblast secondary screen.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/SEC_BJ1.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p>
STING OPS: HeLa cGAMP (6 hours) Secondary Screen Single-Cell Features
<div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa secondary screen, 6 hours post-cGAMP, with or without rolling ball background subtraction.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/SEC_HeLa.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> <p> </p> </div>
STING OPS: HeLa Unstimulated Secondary Screen Single-Cell Features
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa secondary screen, unstimulated, without rolling ball background subtraction.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/SEC_HeLa.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> <p> </p> </div> <p> </p> </div>
STING OPS: HeLa Unstimulated Secondary Screen Single-Cell Features (Background-Subtracted)
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa secondary screen, unstimulated, with rolling ball background subtraction.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/SEC_HeLa.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> <p> </p> </div> <p> </p> </div>
STING OPS: HeLa Genome-wide Screen Single-Cell Features (Part 2/5)
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa genome-wide screen, Zenodo dataset part 2/5.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/GW*.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> </div> </div>
STING OPS: HeLa Genome-wide Screen Single-Cell Features (Part 5/5)
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa genome-wide screen, Zenodo dataset part 5/5.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/GW*.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> </div> </div>
STING OPS: HeLa cGAMP (4 hours) Secondary Screen Single-Cell Features
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa secondary screen, 4 hours post-cGAMP, with or without rolling ball background subtraction.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/SEC_HeLa.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> </div> </div>
STING OPS: HeLa Genome-wide Screen Single-Cell Features (Part 1/5)
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa genome-wide screen, Zenodo dataset part 1/5.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/GW*.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> </div> </div>
STING OPS: HeLa Genome-wide Screen Single-Cell Features (Part 3/5)
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa genome-wide screen, Zenodo dataset part 3/5.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/GW*.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> </div> </div>
STING OPS: HeLa Genome-wide Screen Single-Cell Features (Part 4/5)
<div> <div> <p><strong>Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening</strong></p> <p>Single-cell features and coordinates for HeLa genome-wide screen, Zenodo dataset part 4/5.</p> <p>Images available at gs://opspublic-east1/STINGOpticalPooledScreen/Images/GW*.</p> <p>README for additional image information is at gs://opspublic-east1/STINGOpticalPooledScreen/STING_README.</p> </div> </div>
Supplementary Data for "Molecular dynamics simulations provide structural insight into binding of cyclic dinucleotides to human STING protein"
<p>Supplementary Data for "Molecular dynamics simulations provide structural insight into binding of cyclic dinucleotides to human STING protein", Journal of Biomolecular Structure and Dynamics, 2021, 10.1080/07391102.2021.1942213</p> <p>A random selection of 10 representative structures from each MSM state of STING/CDN complexes is provided in .pdb file format. The selected MSM representatives are aligned and available as PyMOL session files.</p>
Movie of the molecular dynamics simulation of a crucial step in the activation of the STING protein
<p>STING (stimulator of interferon genes) is a homodimer protein involved in regulation of the innate immune system and plays a role in antitumor immunity by inducing the production of cytokines. Activation of STING stems from binding of endogenous cyclic dinucleotides (CDNs), which induce a conformational change to initiate signaling. The MP4 movie shows a molecular dynamics simulation of a crucial step of the activation process.</p>
Human STING is a proton channel (Live-cell MGAT Super-Resolution Experiment)
<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) expressing SEP-mRuby3 targeted to cis/medial Golgi (MGAT) were transduced with pXPR023 (lentiCRISPRv2) expressing an sgRNA targeting STING and selected with 0.1 µg/mL puromycin for 5 days. Cells were then transduced with blasticidin-STING-miRFP680 and selected using 10 µg/mL blasticidin HCl for 5 days. Cells were plated in 96-well glass-bottom plates (Greiner Bio-One) at 6,000 cells/well. After 48 hours, cells were incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061) and stimulated with 1 µM diABZI (Invivogen, #tlrl-diabzi). All images were acquired using an LSM980 with Airyscan2 (Zeiss) with 37°C with 5% CO2 incubation. 8 z-stacks were acquired with 0.15 µm z-step. Images were acquired using a 63X 1.40 NA DIC M27 objective with Immersol 518F 37°C oil. Acquired images were Airyscan processed and then analyzed as described in the image analysis section.</p> <p>Each frame represens one timepoint imaged every 5 minutes post diABZI treatment. Channels are: SEP (super-ecliptic pHluorin), mRuby3, and STING-miRFP680.</p> <p> </p>
Human STING is a proton channel (Live-cell GALT pH Measurement upon STING agonist treatment with or without C53)
<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to GALT constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. BJ1 SEP-mRuby3 cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For time-course experiments, cells were stimulated with 1 µM Bafilomycin A1 (Santa Cruz Biotechnology cat. #sc-201550),<strong> </strong>1 µM diABZI (Invivogen, #tlrl-diabzi), 20 µg/mL cGAMP (Invivogen, #tlrl-nacga23-1) with 5 ng/µL digitonin (Promega, #G9441) for 1 hr with or without the addition of 10 µM C53 (Cayman, #37354). All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks with each frame representing one timepoint: 0, 10, 20, 30, 40, 50, 60 minutes post treatment. Channels are: Hoechst 34580, SEP (super-ecliptic pHluorin), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.</p>
Human STING is a proton channel (Live-cell MGAT pH Measurement upon STING agonist treatment with or without C53)
<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to MGAT constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. BJ1 SEP-mRuby3 cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For time-course experiments, cells were stimulated with 1 µM Bafilomycin A1 (Santa Cruz Biotechnology cat. #sc-201550),<strong> </strong>1 µM diABZI (Invivogen, #tlrl-diabzi), 20 µg/mL cGAMP (Invivogen, #tlrl-nacga23-1) with 5 ng/µL digitonin (Promega, #G9441) for 1 hr with or without the addition of 10 µM C53 (Cayman, #37354). All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks with each frame representing one timepoint: 0, 10, 20, 30, 40, 50, 60 minutes post treatment. Channels are: Hoechst 34580, SEP (super-ecliptic pHluorin), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.</p>
Human STING is a proton channel (Live-cell MGAT STING WT or S53L pH Measurement upon STING agonist treatment with or without C53)
<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to MGAT constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. Cells then were transduced with pXPR023 (lentiCRISPRv2) expressing an sgRNA targeting STING and selected with 0.1 µg/mL puromycin for 5 days. Finally, cells were transduced with blasticidin-STING-HA (WT or S53L) and selected using 10 µg/mL blasticidin HCl for 5 days.<strong> </strong> BJ1 SEP-mRuby3 STING-HA (WT or S53L) cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For time-course experiments, cells were stimulated with 1 µM diABZI (Invivogen, #tlrl-diabzi) for 1 hr with or without the addition of 10 µM C53 (Cayman, #37354). All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks with each frame representing one timepoint: 0, 10, 20, 30, 40, 50, 60 minutes post treatment. Channels are: Hoechst 34580, SEP (super-ecliptic pHluorin), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.</p>
Human STING is a proton channel (HEK293T Autophagy Experiment)
<p>RFP-LC3 and STING-HA-expressing FIP200 KO 293T cells were seeded on Fibronectin bovine plasma coated 24-well glass-bottom plates (Greiner Bio-One) the night before stimulation. Cells were then stimulated with 20 µg/ml cGAMP (Invivogen, #tlrl-nacga23-1) with 5ug/ml digitonin (Promega, #G9441) or 1 µM DIABZI (Invivogen, #tlrl-diabzi) with or without the addition of 10 µM C53 (Cayman, #37354) for 1 hour. Cells were then fixed with 2% Paraformaldehyde (Electron Microscopy Sciences) in PHEM buffer (Electron Microscopy Sciences) for 30 minutes at 37°C, washed three times with PBS and quenched with freshly prepared 0.1M Glycine for 10 minutes. Cells were permeabilized in 100% methanol for 30 minutes and stained with anti-HA (Millipore, #11867423001) for 1 hour at room temperature in 3% BSA, washed 5 times, and then stained with Alexa 647 anti-rat IgG (H+L) (Thermo, A-21247) in 3% BSA for 1 hour. After five washes, cells were incubated in 2X SSC with 200 ng/mL DAPI (Thermo Fisher) and imaged using the Nikon microscope used for organelle pH images. Images were acquired using a 60X 1.40 NA Plan Apo λ oil immersion objective (Nikon MRD01605) with Nikon type F immersion oil with the following lasers and filters: DAPI (405 nm laser, Chroma <a href="https://www.chroma.com/products/parts/et455-50m">ET455/50</a>), RFP-LC3B (561 nm laser, Chroma ET605/52), and STING-HA (640 nm laser, Chroma ET705/72), assaying five z planes per field of view with 0.625 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks. Channels are: DAPI, LC3B-RFP, and STING.</p>
Human STING is a proton channel (Live-cell pH Calibration Meaurements)
<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to GALT constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. BJ1 SEP-mRuby3 cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For pH calibration experiments, cells were treated with buffers at defined pH values supplemented with nigericin and valinomycin following manufacturer’s instructions (Thermo Fisher Scientific cat. # P35379). Additional buffers at pH 7, 6, and 5 were created by titration with acid. All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks with each frame representing one pH value: 7.5, 7, 6.5, 6, 5.5, 5, and 4.5 (except for replicate 1, which omitted pH 7). Channels are: Hoechst 34580, SEP (super-ecliptic GFP), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.</p>
Human STING is a proton channel (Live-cell MGAT, GALT, and LAMP1 pH Measurements Upon BafA1 and diABZI treatment)
<p>hTERT-immortalized BJ1 cells (ATCC CRL-2522) were transduced with lentiviral ratiometric reporters targeted to MGAT, GALT, or LAMP1 constructed based on designs reported in Linders et al. <em>ACS Chem. Biol. </em>2022, with superecliptic pHluorin and mRuby3. Transduced cells were sorted based on mRuby3 expression using a Sony MA900 sorter. BJ1 SEP-mRuby3 cells were plated in 24-well glass-bottom plates (Greiner Bio-One) at 40,000 cells/well. After 48 hours, cells were stained for 45 minutes at 37°C with 0.5 µg/ml Hoechst 34580 (Thermo Fisher Scientific, cat. #H21486). Cells were then washed and incubated in Fluorobrite DMEM (Thermo Fisher Scientific, cat. #A1896701) medium supplemented with 10% FBS, 1% Pen-strep, and 1x GlutaMAX (Thermo Fisher Scientific, cat. #35050061). For time-course experiments, cells were stimulated with 1 µM Bafilomycin A1 (Santa Cruz Biotechnology cat. #sc-201550), or 1 µM diABZI (Invivogen, #tlrl-diabzi). All images were acquired using a Ti2-E inverted epifluorescence microscope (Nikon) with automated XYZ stage control, hardware autofocus, and a Yokogawa CSU-W1 confocal spinning disk unit with Zyla 4.2 PLUS sCMOS camera. An Okolab cage incubator was set to 37°C with 5% CO2. 405, 488, 561, and 640 nm laser lines were used for fluorescence illumination and all hardware was controlled using NIS elements software. Images were acquired using a 40X 0.95 NA CFI Plan Apo λ objective (Nikon MRD70470) with the following lasers and filters: Hoechst (405 nm laser, Chroma Multi LED set #89401), superecliptic pHluorin (488 nm laser, Chroma Multi LED set #89401), and mRuby3 (561 nm laser, Chroma Multi LED set #89401), assaying three z planes per field of view with 1.25 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection.</p> <p>Images are maximum projections of multiple z-stacks with each frame representing one timepoint: 0, 10, 20, 30, 40, 50, 60 minutes post treatment (except for replicate 1, which omitted the final timepoint). Channels are: Hoechst 34580, SEP (super-ecliptic pHluorin), mRuby3, and SEP/mRuby3 (ratio). Crops indicate cropped fields of view presented in the manuscript.</p>
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