Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(3,4)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-S5 (Abcam ab5131) 3 µg/mL and mouse monoclonal IgG2 anti-PI(3,4)P2 (Z-P034; 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 donkey anti-mouse IgG AF555 (Invitrogen A31570) 10 µg/mL and 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).U-2 OS细胞（U-2 OS cells）于添加10%胎牛血清(FBS)的DMEM培养基中，在37℃、5% CO₂的培养条件下扩增培养。染色前1天，将细胞以约50%汇合密度接种于经Hellmanex处理、超声清洗、烘干灭菌的高精度12 mm圆形盖玻片上。向培养基中加入100 μM DRB（Sigma D1916），处理细胞2小时。 U2OS细胞用pH 7.4的磷酸盐缓冲液(PBS)洗涤两次，随后用2%多聚甲醛(PFA)的PBS溶液固定30分钟，再用PBS洗涤3次，每次5分钟；之后用含0.1% Triton X-100的PBS透化20分钟，再次用PBS洗涤3次，每次5分钟，最后用过滤后的5%牛血清白蛋白(BSA)的PBS溶液封闭30分钟。将细胞与3 μg/mL兔源多克隆IgG抗RNA聚合酶II C端结构域丝氨酸5磷酸化抗体（RNAPII CTD P-S5，Abcam ab5131）以及5 μg/mL小鼠单克隆IgG2抗磷脂酰肌醇(3,4)二磷酸（PI(3,4)P2，Z-P034；美国Echelon Biosciences公司）在含5% BSA的PBS中共同孵育45分钟；随后用PBS洗涤3次，每次5分钟，再与10 μg/mL驴抗小鼠IgG AF555（Invitrogen A31570）和10 μg/mL山羊抗兔IgG AF647（Invitrogen A21245）在含5% BSA的PBS中稀释后共同孵育30分钟。之后用PBS洗涤3次，每次5分钟，再用2% PFA的PBS溶液后固定15分钟，最后用PBS洗涤3次，每次5分钟。所有操作均在室温(RT)下进行，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样品池（韩国Live Cell Instrument公司）中，加入成像缓冲液（pH 7.4的PBS，含50 mM 巯基乙胺(MEA)）覆盖样品。单分子定位(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、Andor电子倍增电荷耦合器件相机(EM CCD) iXon DU 897以及Zeiss ZEN Black 2.1 SP3软件（Zeiss公司）的Zeiss Elyra PS.1系统采集。AF647与AF555的光开关通过HiLo照明与全内反射荧光(TIRF)高视场(HP FOV)实现，分别使用100%功率的642 nm或561 nm激光器激发；信号分别通过MBS 642 + EF LP 655滤光组与MBS 561 + EF BP 570-620 / LP 750滤光组采集。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。 单分子定位数据通过Zeiss ZEN Black 2.1 SP3软件进行二维计算，采用x、y方向二维高斯拟合，点扩散函数(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。单分子定位结果通过ZEN软件以10 nm/像素的分辨率与1倍PSF扩展因子进行渲染。数据在ZEN软件中基于模型进行漂移校正。两个通道通过四色微球基准标记物（tetraspec beads fiducial markers）进行仿射校准以实现配准。经漂移校正与配准后的定位坐标以文本文件格式导出。将文本文件转换为逗号分隔值(CSV)文件，通过自行编写的宏代码（Hoboth等，2021a）导入ImageJ2（Rueden等，2017）的ThunderSTORM插件，并通过归一化高斯方法（Ovesny等，2014）进行可视化。