Ditrect STORM imaging and image reconstruction of the transcription elongation marker P-S2 and nuclear PI(4,5)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in DRB treated cells.

U-2 OS cells were grown in DMEM with 10% FBS at 37°C and 5% CO2. Cells were plated one day before staining in ~50% confluence on the high-precision 12 mm round coverslips treated with Hellmanex, sonicated, washed, dried and sterilized. Cells were treated for 2h with 100 µM DRB (Sigma D1916) added to the cell culture media. U2OS cells were washed twice with PBS (pH 7.4) and fixed for 30 min in 2% PFA in PBS, washed 3-times for 5 min with PBS, then permeabilized in 0.1% Triton X-100 in PBS for 20 min, washed 3-times for 5 min by PBS and blocked in filtered 5% BSA in PBS for 30 min. Cells were incubated for 45 min with rabbit polyclonal IgG anti-RNAPII CTD P-S2 (Abcam ab5095) 3 µg/mL and mouse ascites IgM anti-PI(4,5)P2 2C11 (Z-A045; Echelon Biosci. Inc., USA) 5 µg/mL in 5% BSA in PBS, washed 3-times for 5 min in PBS and incubated for 30 min with goat anti-mouse IgM (µ-chain) AF555 (Jackson ImmunoRes. A24126) 10 µg/mL; goat anti-rabbit IgG AF647 (Invitrogen A21245) 10 µg/mL diluted in 5% BSA in PBS. Then the cells were washed 3-times for 5 min in PBS, post-fixed for 15 min in 2% PFA in PBS and washed 3-times for 5 min in PBS. All procedures were performed at RT and the cells were stored in PBS in the fridge overnight prior imaging. Coverslips with cells were mounted in the Chamlide chamber (Live Cell Instrument, Korea) and covered with imaging buffer (PBS pH 7.4, 50 mM MEA). Single-molecule localizations (SMLs) data were acquired by Zeiss Elyra PS.1 equipped with HR Diode 642-150 and HR DPSS 561-200 lasers, Alpha Plan-Apochromat 100x/1.46 oil DIC M27 Elyra objective and Andor EM CCD iXon DU 897 camera and Zeiss ZEN Black 2.1 SP3 software (Zeiss). AF647 and AF555 photo-switching was achieved by HiLo illumination and TIRF HP FOV with 100% power of 642nm or 561nm laser, and the signal was acquired via MBS 642 + EF LP 655 and MBS 561 + EF BP 570-620 / LP 750 filters, respectively. Exposure time was 40 ms and EM gain was 300 for both channels. SMLs were calculated in 2D by Zeiss ZEN Black 2.1 SP3 software using x,y 2D Gauss fit with point spread function (PSF) half width 177.9 nm, peak mask size 9 pixels and peak intensity to noise 6 and accounted for overlap in 2D with max cluster size 10. SMLs were rendered in ZEN software with 10 nm/px resolution and 1x PSF expansion factor. The data were model-based drift corrected in ZEN. Two channels were aligned using tetraspec beads fiducial markers for affine calibration. Drift-corrected and aligned localization coordinates were exported as text files. Text files were converted into csv files and imported using self-written macro (Hoboth et al., 2021a) into the ImageJ2 (Rueden et al., 2017) plug-in ThunderSTORM, visualized by normalized Gaussian method (Ovesny et al., 2014).将U-2 OS细胞（U-2 OS cells）置于添加有10%胎牛血清（FBS）的DMEM培养基中，于37℃、5% CO₂条件下培养。染色前一日将细胞以约50%汇合度接种于经Hellmanex处理、超声清洗、漂洗、干燥并灭菌的高精度12 mm圆形盖玻片上。向细胞培养基中加入100 μM DRB（Sigma D1916）处理细胞2小时。 用pH 7.4的磷酸盐缓冲液（PBS）漂洗U-2 OS细胞两次，使用含2%多聚甲醛（PFA）的PBS溶液固定30分钟；随后用PBS漂洗3次，每次5分钟。将细胞置于含0.1% Triton X-100的PBS溶液中透化20分钟，再用PBS漂洗3次，每次5分钟。使用经过滤的含5%牛血清白蛋白（BSA）的PBS溶液封闭细胞30分钟。将细胞与含3 μg/mL兔源多克隆IgG抗RNA聚合酶II C端结构域磷酸化丝氨酸2（RNAPII CTD P-S2，Abcam ab5095）及5 μg/mL小鼠腹水IgM抗磷脂酰肌醇4,5-二磷酸（PI(4,5)P₂）2C11（Z-A045；美国Echelon Biosci.公司）的5% BSA-PBS溶液共孵育45分钟；随后用PBS漂洗3次，每次5分钟，再与含10 μg/mL山羊抗小鼠IgM（μ链）AF555（Jackson ImmunoRes. A24126）及10 μg/mL山羊抗兔IgG AF647（Invitrogen A21245）的5% BSA-PBS溶液孵育30分钟。之后用PBS漂洗3次，每次5分钟，使用含2% PFA的PBS溶液进行后固定15分钟，再用PBS漂洗3次，每次5分钟。所有操作均在室温（RT）下完成，成像前将细胞保存在PBS中并置于冰箱过夜。 将载有细胞的盖玻片安装于Chamlide腔室（Live Cell Instrument，韩国）中，加入成像缓冲液（PBS pH 7.4，50 mM MEA）。单分子定位（single-molecule localizations, SMLs）数据通过配备HR Diode 642-150与HR DPSS 561-200激光器、Alpha Plan-Apochromat 100x/1.46油浸DIC M27 Elyra物镜以及Andor EM CCD iXon DU 897相机的Zeiss Elyra PS.1系统采集，并使用Zeiss ZEN Black 2.1 SP3软件（Zeiss）进行控制。通过HiLo照明与TIRF HP FOV实现AF647与AF555的光开关切换，分别采用功率为100%的642nm或561nm激光器，信号分别通过MBS 642 + EF LP 655滤光片组与MBS 561 + EF BP 570-620 / LP 750滤光片组采集。两个通道的曝光时间均为40 ms，电子倍增增益（EM gain）均设置为300。 使用Zeiss ZEN Black 2.1 SP3软件以二维方式计算SMLs，采用x、y二维高斯拟合，点扩散函数（point spread function, PSF）半高宽为177.9 nm，峰值掩膜尺寸为9像素，峰值强度与噪声比为6，并考虑二维重叠情况，最大簇尺寸为10。使用ZEN软件以10 nm/像素的分辨率与1倍PSF扩展因子渲染SMLs数据，并在ZEN中基于模型进行漂移校正。使用四色微球基准标记物进行仿射校准以配准两个成像通道。将经漂移校正与配准后的定位坐标导出为文本文件，将文本文件转换为逗号分隔值（csv）文件，通过自行编写的宏程序（Hoboth等，2021a）导入ImageJ2（Rueden等，2017）的ThunderSTORM插件，并使用归一化高斯法（Ovesny等，2014）进行可视化。