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. <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. 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.转hTERT永生化的BJ1细胞（ATCC CRL-2522）采用基于Linders等人2022年发表于《ACS Chem. Biol.》的设计构建的靶向GALT的慢病毒比例报告基因（ratiometric reporter）进行转导，该报告基因融合了超荧光pHluorin（superecliptic pHluorin）与mRuby3（mRuby3）荧光蛋白。转导后的细胞基于mRuby3的表达水平，使用索尼MA900分选仪完成分选。将BJ1 SEP-mRuby3细胞以每孔40,000个的密度接种于24孔玻璃底培养板（Greiner Bio-One）中。接种48小时后，使用浓度为0.5 µg/mL的Hoechst 34580（赛默飞世尔科技，货号#H21486）于37℃染色45分钟。随后洗涤细胞，并将其置于添加了10%胎牛血清（FBS）、1%青霉素-链霉素双抗（Pen-strep）以及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。在时间进程实验中，分别使用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）搭配5 ng/µL洋地黄皂苷（digitonin，普洛麦格，货号#G9441）对细胞刺激1小时，同时设置添加或不添加10 µM C53（Cayman，货号#37354）的对照组别。所有图像均通过尼康Ti2-E倒置落射荧光显微镜采集，该系统配备自动化XYZ载物台控制模块、硬件自动对焦功能，以及搭载Zyla 4.2 PLUS sCMOS相机的横河Yokogawa CSU-W1共聚焦转盘扫描单元。使用Okolab箱式培养箱维持37℃、5% CO₂的培养环境。采用405 nm、488 nm、561 nm及640 nm激光线进行荧光激发，所有硬件设备均通过NIS Elements软件进行控制。图像采集采用40×0.95 NA CFI Plan Apo λ物镜（尼康MRD70470），搭配对应激光与滤光套装：Hoechst 34580对应405 nm激光（Chroma Multi LED套装#89401）、超荧光pHluorin对应488 nm激光（Chroma Multi LED套装#89401）、mRuby3对应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，无需人工预先选定。图像为多张Z轴堆栈的最大强度投影，每个帧对应处理后的一个时间点：0分钟、10分钟、20分钟、30分钟、40分钟、50分钟及60分钟。成像通道包括：Hoechst 34580通道、SEP（超荧光pHluorin）通道、mRuby3通道以及SEP/mRuby3比例通道。裁剪区域标注了论文中展示的裁剪后视场。