Ditrect STORM imaging and image reconstruction of the transcription initiation marker P-S5 and nuclear PI(4,5)P2 indirectly immunolabeled with AF647 (red) and AF555 (green) in THZ1 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 3h with 1 µM THZ1 (MedChem HY80013) added to the 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 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（Fetal Bovine Serum, FBS）的杜氏改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）中，于37℃、5%二氧化碳体积分数的培养箱中培养。染色前一日将细胞以约50%汇合度铺板于经Hellmanex处理、超声清洗、冲洗、烘干并灭菌的高精度12 mm圆形盖玻片（coverslips）上。向培养基中添加1 μM THZ1（MedChem HY80013）处理细胞3小时。 将U2OS细胞用pH 7.4的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤两次，使用含2%多聚甲醛（Paraformaldehyde, PFA）的PBS溶液固定30分钟，随后用PBS洗涤3次，每次5分钟；接着用含0.1%曲拉通X-100（Triton X-100）的PBS溶液透化20分钟，再用PBS洗涤3次，每次5分钟；之后用过滤后的含5%牛血清白蛋白（Bovine Serum Albumin, BSA）的PBS溶液封闭30分钟。将细胞与浓度为3 μg/mL的兔多克隆免疫球蛋白G（Immunoglobulin G, IgG）抗RNAPII CTD P-S5（Abcam ab5131），以及浓度为5 μg/mL的小鼠腹水免疫球蛋白M（Immunoglobulin M, IgM）抗PI(4,5)P2 2C11（Z-A045; Echelon Biosci. Inc., 美国）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，再与在含5% BSA的PBS中稀释的浓度为10 μg/mL的山羊抗小鼠IgM（μ链）AF555（Jackson ImmunoRes. A24126）、浓度为10 μg/mL的山羊抗兔IgG AF647（Invitrogen A21245）共同孵育30分钟。之后用PBS洗涤3次，每次5分钟，使用含2% PFA的PBS溶液后固定15分钟，再用PBS洗涤3次，每次5分钟。所有操作均在室温（Room Temperature, RT）下进行，成像前将细胞保存在4℃冰箱的PBS中过夜。 将带有细胞的盖玻片安装于Chamlide腔室（Live Cell Instrument, 韩国）中，使用成像缓冲液（PBS pH 7.4, 50 mM MEA）覆盖。单分子定位（Single-molecule localizations, SMLs）数据通过搭载HR Diode 642-150与HR DPSS 561-200激光器的Zeiss Elyra PS.1显微镜采集，搭配Alpha Plan-Apochromat 100x/1.46油浸DIC M27 Elyra物镜、Andor电子倍增电荷耦合器件（Electron Multiplying Charge Coupled Device, EM CCD）iXon DU 897相机，以及Zeiss ZEN Black 2.1 SP3软件（Zeiss）。AF647与AF555的光开关通过HiLo照明与TIRF HP FOV实现，使用功率为100%的642 nm或561 nm激光器，信号分别通过MBS 642 + EF LP 655与MBS 561 + EF BP 570-620 / LP 750滤光片采集。两个通道的曝光时间均为40 ms，电子倍增增益（EM gain）均为300。 单分子定位数据通过Zeiss ZEN Black 2.1 SP3软件以二维方式计算，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。单分子定位数据在ZEN软件中以10 nm/像素的分辨率与1倍PSF扩展因子进行渲染。数据在ZEN中完成基于模型的漂移校正。通过四色微球基准标记物（fiducial markers）进行仿射校准，以对齐两个通道。将完成漂移校正并对齐后的定位坐标导出为文本文件，转换为CSV文件后，通过自行编写的宏（Hoboth等，2021a）导入ImageJ2（Rueden等，2017）插件ThunderSTORM，采用归一化高斯法（Ovesny等，2014）进行可视化。