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.hTERT永生化BJ1细胞（ATCC CRL-2522）经慢病毒转导，携带基于Linders等人2022年发表于《ACS Chem. Biol.》的设计构建的靶向GALT的比例型报告基因，该报告基因融合了超折叠pHluorin（superecliptic pHluorin）与mRuby3。转导后的细胞通过Sony MA900细胞分选仪基于mRuby3的表达水平进行分选。将BJ1 SEP-mRuby3细胞以40000个/孔的密度接种于24孔玻璃底培养板（Greiner Bio-One）中。接种48小时后，使用0.5 μg/mL的Hoechst 34580（赛默飞世尔科技，货号#H21486）于37℃染色45分钟。随后弃去染色液，洗涤细胞并将其置于添加了10%胎牛血清、1%双抗（Pen-strep）及1× GlutaMAX（赛默飞世尔科技，货号#35050061）的Fluorobrite DMEM培养基（赛默飞世尔科技，货号#A1896701）中进行孵育。针对时间进程实验，采用1 μM巴弗洛霉素A1（Bafilomycin A1，圣克鲁斯生物技术，货号#sc-201550）、1 μM diABZI（Invivogen，货号#tlrl-diabzi）、20 μg/mL cGAMP（Invivogen，货号#tlrl-nacga23-1）配合5 ng/μL洋地黄皂苷（digitonin，普洛麦格，货号#G9441）对细胞进行刺激，刺激时长为1小时，同时设置添加10 μM C53（Cayman，货号#37354）与不添加的平行对照组。所有图像均通过配备自动化XYZ载物台控制及硬件自动对焦功能的Ti2-E倒置落射荧光显微镜（尼康）采集，该系统搭配Yokogawa CSU-W1共聚焦转盘单元与Zyla 4.2 PLUS sCMOS相机。实验采用Okolab笼式培养箱维持37℃、5% CO₂的培养环境。使用405、488、561及640 nm激光线进行荧光激发，所有硬件设备均通过NIS Elements软件控制。成像使用40× 0.95 NA CFI Plan Apo λ物镜（尼康MRD70470），对应激光器与滤光套装如下：Hoechst 34580（405 nm激光，Chroma Multi LED套装#89401）、超折叠pHluorin（488 nm激光，Chroma Multi LED套装#89401）及mRuby3（561 nm激光，Chroma Multi LED套装#89401）。每个视场采集3层Z轴切片，层间距为1.25 μm。视场通过NIS Elements软件的坐标自动选取，无需手动预筛选。图像为多张Z轴堆叠图像的最大强度投影，每个帧对应一个时间点：处理后0、10、20、30、40、50、60分钟。成像通道包括：Hoechst 34580、SEP（超折叠pHluorin，superecliptic pHluorin）、mRuby3及SEP/mRuby3（比例通道）。裁剪区域为论文中展示的视场裁剪片段。