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opencc-by-4.0Apr 2024View details →

Datasets for the manuscript "Metabolomic and Sphingolipidomic Profiling of Human Hepatoma Cells Exposed to Widely Used Pharmaceuticals"

See experimental details on the main text of the manuscript "Metabolomic and Sphingolipidomic Profiling of Human Hepatoma Cells Exposed to Widely Used Pharmaceuticals" <a title="Persistent link using digital object identifier" href="https://doi.org/10.1016/j.jpba.2024.116378" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.jpba.2024.116378</a> <h2>Data description</h2> This study investigates the impact of three commonly used pharmaceuticals (amoxicillin, carbamazepine, and trazodone) on human liver cells. To mimic real-world conditions, liver cells were encapsulated in spheroids and exposed to various concentrations of these drugs for 24 hours. The study employs metabolomic and sphingolipid analyses to identify metabolic changes induced by drug exposure. <div></div> <div> <div> <div> <h3>LC-MS/MS Method for Sphingolipid Analysis</h3> Targeted sphingolipid analysis was conducted using a Waters ACQUITY UPLC System coupled to a Waters Xevo TQ-S system equipped with an Electrospray Ion Source (ESI) and ScanWave™ collision cell technology, operating in positive mode [16]. Sphingolipids were quantified using a Zorbax Rapid Resolution RRHD C18 Column (80 Å, 1.8 µm, 2.1 mm × 100 mm). <h3>LC-HRMS Method for Semi-Targeted Metabolomic Analysis</h3> LC-HRMS analysis was performed on an Agilent 1290 Affinity II HPLC system coupled to an Agilent 6550 iFunnel QTOF mass spectrometer equipped with a dual AJS electrospray ionization source operating in both positive and negative modes. Polar metabolite screening was conducted using a SeQuant® ZIC®-pHILIC 5 µm polymer 100 × 2.1 mm column. </div> </div> </div>   <h2>Funding</h2> The research leading to these results has received funding from the Spanish Ministry of Science and Innovation MCIN/AEI/ 10.13039/501100011033, Grants CTQ2017-82598-P and CEX2018-000794-S. The authors also want to grant support from the Catalan Agency for Management of University and Research Grants (AGAUR, Grant 2017SGR753). Miriam Pérez-Cova acknowledges a predoctoral FPU 16/02640 scholarship from the Spanish Ministry of Education and Vocational Training (MEFP), and Post-graduate department from CSIC for the funding of the research stay in Karolinska Institute, via the award to best outreach video in the YoInvestigoYosoyCsic contest, 2019 edition.

opencc-zeroApr 2023View details →

Cell tracking data from: Automated timelapse data segmentation reveals in vivo cell state dynamics

Embryonic development proceeds as a series of orderly cell state transitions built upon noisy molecular processes. Here, we defined gene expression and cell motion states using single cell RNA sequencing data and in vivo timelapse cell tracking data of the zebrafish tailbud. We performed a parallel identification of these states using dimensional reduction methods and a change point detection algorithm. Both types of cell states were quantitatively mapped onto embryos, and we utilized the cell motion states to study the dynamics of biological state transitions over time. The time average pattern of cell motion states is reproducible among embryos. However, individual embryos exhibit transient deviations from the time average forming left-right asymmetries in collective cell motion. Thus, the reproducible pattern of cell states and bilateral symmetry arises from temporal averaging. In addition, collective cell behavior can be a source of asymmetry rather than a buffer against noisy individual cell behavior.

Prevalence Of Sickle Cell Trait and Needs Assessment Forfor Uptake of Sickle Cell Screening Among Secondary School Students in Kampala City, Uganda

This study aimed to determine the burden of SCT and factors influencing uptake of screening services among secondary school students in Uganda. Data was collected using semi-structured questionnaires and analyzed using descriptive statistics and logistic regression.

Cultured renal proximal tubular epithelial cells resemble stressed/damaged kidney while supporting BKV infection

Supplementary tables for the manuscript: Cultured renal proximal tubular epithelial cells resemble stressed/damaged kidney while supporting BKV infection The scRNA-Seq of RPTE and hTERT-RPTE cells infected by BKV (10X Genomics) is available under NCBI Bioproject PRJNA961060 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA961060)

Genetic control of the dynamic transcriptional response to immune stimuli and glucocorticoids at single cell resolution

Supplementary Tables for article "Genetic control of the dynamic transcriptional response to immune stimuli and glucocorticoids at single cell resolution"

opencc-zeroApr 2023View details →

Mathematical model results for: Dynamic fibronectin assembly and remodeling by leader neural crest cells prevents jamming in collective cell migration

Collective cell migration plays an essential role in vertebrate development, yet the extent to which dynamically changing microenvironments influence this phenomenon remains unclear. Observations of the distribution of the extracellular matrix (ECM) component fibronectin during the migration of loosely connected neural crest cells (NCCs) lead us to hypothesize that NCC remodeling of an initially punctate ECM creates a scaffold for trailing cells, enabling them to form robust and coherent stream patterns. We evaluate this idea in a theoretical setting by developing an agent-based model that incorporates reciprocal interactions between NCCs and their ECM. ECM remodeling, haptotaxis, contact guidance, and cell-cell repulsion are sufficient for cells to establish streams in silico, however additional mechanisms, such as chemotaxis, are required to consistently guide cells along the correct target corridor. Further investigations of the model imply that contact guidance and differential cell-cell repulsion between leader and follower cells are key contributors to robust collective cell migration by preventing stream breakage. Global sensitivity analysis and simulated underexpression/overexpression experiments suggest that long-distance migration without jamming is most likely to occur when leading cells specialize in creating ECM fibers, and trailing cells specialize in responding to environmental cues by upregulating mechanisms such as contact guidance. This dataset contains summary statistics, movies, parameter values, and photos obtained from individual realizations of the mathematical model.

opencc-by-4.0Mar 2022View details →

Designing single-cell experiments to harvest fluctuation information while rejecting measurement noise

Numerical simulation outputs and model schematic figures for the Python portion (main text 3.1 and Supporting Information) in the preprint Analysis and design of single-cell experiments to harvest fluctuation information while rejecting measurement noise Huy D. Vo, Linda Forero, Luis Aguilera, Brian Munsky bioRxiv doi: https://doi.org/10.1101/2021.05.11.443611

opencc-zeroApr 2023View details →

Response to primary chemoradiotherapy of locally advanced oropharyngeal carcinoma is determined by the degree of cytotoxic T cell infiltration within tumor cell aggregates

Background: Effective anti-tumor immune responses are mediated by T cells and require organized, spatially coordinated interactions within the tumor microenvironment (TME). Understanding coordinated T-cell behavior and deciphering mechanisms of radiotherapy resistance mediated by tumor stem cells will advance risk stratification of oropharyngeal cancer (OPSCC) patients treated with primary chemoradiotherapy (RCTx). Methods: To determine the role of CD8 T cells (CTL) and tumor stem cells in response to RCTx, we employed multiplex immunofluorescence stains on pre-treatment biopsy specimens from 86 advanced OPSCC patients and correlated these quantitative data with clinical parameters. Multiplex stains were analyzed at the single-cell level using QuPath and spatial coordination of immune cells within the TME was explored using the R-package Spatstat. Results: Our observations demonstrate that a strong CTL-infiltration into the epithelial tumor compartment (HR for overall survival, OS: 0.35; p<0.001) and the expression of PD-L1 on CTL (HR: 0.36; p<0.001) were both associated with a significantly better response and survival upon RCTx. As expected, p16 expression was a strong predictor of improved OS (HR: 0.38; p=0.002) and correlated with overall CTL infiltration (r: 0.358, p<0.001). By contrast, tumor cell proliferative activity, expression of the tumor stem cell marker CD271 and overall CTL infiltration, regardless of the affected compartment, were not associated with response or survival. Conclusion: In this study, we could demonstrate the clinical relevance of the spatial organization and the phenotype of CD8 T cells within the TME. In particular, we found that the infiltration of CD8 T cells specifically into the tumor cell compartment was an independent predictive marker for response to chemoradiotherapy, which was strongly associated with p16 expression. Meanwhile, tumor cell proliferation and the expression of stem cell markers showed no independent predictive effect in response to RCTx and require further study.

Allosteric activation of cell wall synthesis during bacterial growth

This repository contains single-molecule FRET data related to this manuscript organized by figure. Note that data that appear both in the main and in the supplementary figures are provided only once, in the relevant main figure folders. Each figure folder contains all relevant datasets, deposited as zipped folders with pre-processed raw trajectories in the .dat format. These trajectories list donor excitation/donor emission (column 1) and donor excitation/acceptor emission (column 2) values as a function of time, and can be visualized and further processed using either custom code or the publicly available ebFRET software (http://ebfret.github.io/).

opencc-by-4.0Jan 2023View details →

Microglia-mediated T cell Infiltration Drives Neurodegeneration in Tauopathy

Extracellular amyloid-β (Aβ) deposition as neuritic plaques and intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles (NFT) are two of the characteristic hallmarks in Alzheimer’s disease (AD). The regional progression of brain atrophy in AD highly correlates with tau accumulation but not amyloid deposition  and the mechanisms of tau-mediated neurodegeneration remain elusive. Innate immune responses represent a common pathway for the initiation and progression of some neurodegenerative diseases. To date, little is known about the extent or role of the adaptive immune response in the presence of Aβ or tau pathology. We systematically compared the immunological milieus in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not amyloid, developed a unique innate and adaptive immune response and that depletion of microglia or T-cells blocked tau-mediated neurodegeneration. T cells, especially cytotoxic T cells, were markedly increased in areas with tau pathology in mice with tauopathy and in the AD brain. T cell numbers correlated with the extent of neuronal loss, and dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of IFN-γ and PD-1signaling both significantly ameliorated brain atrophy. Our results thus reveal a tauopathy and neurodegeneration-related immune hub involving activated microglia and T cell responses, which could serve as therapeutic targets for preventing neurodegeneration in AD and primary tauopathies.

opencc-zeroMay 2023View details →

Diet-mediated constitutive induction of novel IL4+ ILC2 cells maintains intestinal homeostasis in mice

Group 2 innate lymphoid cells (ILC2s) expressing IL-5 and IL-13 are localized at various mucosal tissues and play critical roles in the induction of type 2 inflammation, response to helminth infection, and tissue repair. Here we reveal a unique ILC2 subset in the mouse intestine that constitutively expresses IL-4 together with GATA3, ST2, KLRG1, IL-17RB, and IL-5. In this subset, IL-4 expression is regulated by mechanisms similar to but distinct from those observed in T cells and is partly affected by IL-25 signaling. Although the absence of the microbiota had marginal effects, feeding mice with a vitamin B1-deficient diet compromised the number of intestinal IL-4+ ILC2s. The decrease in the number of IL-4+ ILC2s caused by the vitamin B1 deficiency was accompanied by a reduction in IL-25-producing tuft cells. Our findings reveal that dietary vitamin B1 plays a critical role in maintaining interaction between tuft cells and IL-4+ ILC2s, a previously uncharacterized immune cell population that may contribute to maintaining intestinal homeostasis.

opencc-zeroMay 2023View details →

Data from: The fat body cortical actin network regulates Drosophila inter-organ nutrient trafficking, signaling, and adipocyte cell size

Defective nutrient storage and adipocyte enlargement (hypertrophy) are emerging features of metabolic syndrome and type 2 diabetes. How the cytoskeletal network contributes to nutrient uptake, fat storage, and adipocyte size remains poorly understood. Utilizing the Drosophila larval fat body (FB) as a model adipose tissue, we show that a specific actin isoform—Act5C—forms the cortical actin network necessary for inter-organ lipid trafficking. Act5C also promotes FB tissue expansion during larval development so larvae can store sufficient biomass for metamorphosis. We find FB-specific loss of Act5C, but not other Drosophila actins, perturbs FB triglyceride (TG) storage in lipid droplets (LDs), resulting in developmentally delayed larvae that fail to develop into flies. Act5C localizes to the FB cell surface where it intimately contacts peripheral LDs (pLDs), forming a cortical actin network together with spectrins for cell architectural support. While both the cortical actin and spectrin cytoskeletons maintain FB cell surface architecture, we find that only the actin network is required for fat storage. Mechanistically, we show that FBs lacking the Act5C cortical cytoskeleton exhibit a block in lipoprotein (Lpp) secretion from FB cells, and a subsequent disruption of gut:FB inter-organ lipid transport, resulting in mid-gut fat accumulation. Utilizing temporal RNAi-depletion approaches, we also reveal that Act5C is indispensable post-embryogenesis during larval feeding to promote FB cell expansion. Act5C-deficient FBs fail to expand cell sizes, leading to lipodystrophic larvae unable to accrue sufficient biomass for metamorphosis. Collectively, we propose that the Act5C-mediated cortical actin network of Drosophila adipose tissue plays an essential role in post-embryonic inter-organ nutrient transport and FB cell size determination for organismal energy homeostasis and development.

opencc-zeroMay 2023View details →

Lipid analysis of S.c. cells expressing ProtA-ScNdc1 (261-655), ProtA-AHNdc1-ProtA, or ProtA-ScHmg1

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, and few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

opencc-by-4.0May 2023View details →

fluorescence microscopy image containing more than 65536 cells

1. A montage of fluorescence images containing more than 65536 cells. 2. The output label image from Cellpose, as obtained from the BIOP Cellpose wrapper

Human STING is a proton channel (Live-cell MGAT Super-Resolution Experiment)

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. Each frame represens one timepoint imaged every 5 minutes post diABZI treatment. Channels are: SEP (super-ecliptic pHluorin), mRuby3, and STING-miRFP680.

Human STING is a proton channel (Live-cell GALT pH Measurement upon STING agonist treatment with or without C53)

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. ACS Chem. Biol. 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), 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. 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.

Human STING is a proton channel (Live-cell MGAT pH Measurement upon STING agonist treatment with or without C53)

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. ACS Chem. Biol. 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), 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. 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.

Human STING is a proton channel (Live-cell MGAT STING WT or S53L pH Measurement upon STING agonist treatment with or without C53)

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. ACS Chem. Biol. 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. 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. 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.

Human STING is a proton channel (Live-cell pH Calibration Meaurements)

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. ACS Chem. Biol. 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. 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.

Human STING is a proton channel (Live-cell MGAT, GALT, and LAMP1 pH Measurements Upon BafA1 and diABZI treatment)

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. ACS Chem. Biol. 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. 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.