293 T cells were transfected with WT and indicated TMPRSS2 mutants. 24 h post transfection, the catalytic activity was assessed using BoC-QAR-AMC fluorogenic substrate. Data are mean ± SD of 4 independent experiments. Statistical analysis: a: one-way ANOVA with Dunnett’s multiple comparisons compared to Ctrl cells (transfected with pQCXIP-Empty). **p < 0.01. b. Effect of TMPRSS2 on HKU1 spike expressed on adjacent cells. 293 T cells were transfected either with HKU1A spike or with the different TMPRSS2. 20 h post-transfection, cells were mixed at a 1:1 ratio, and let to settle. 3 or 6 h post-mixing, cells were harvested and lysed for WB. One membrane was probed for S1, TMPRSS2 and actin, another membrane was probed for S2 and actin. For gel source data, see SI 1c, d. Representative blots of 2. Molecular weights: kDa. The arrows and # denote the uncleaved and cleaved protein products, respectively. c. Surface levels of WT and mutant TMPRSS2. 293 T cells were transfected with the indicated doses of TMPRSS2 plasmid. 18 h post-transfection they were stained intracellularly for TMPRSS2 using the commercial antibody. Left: % of TMP positive cells. Right: Median fluorescent intensity (MFI). #: indicates the chosen plasmid ratios to achieve similar TMPRSS2 levels with WT and indicated mutants. Data are mean ± SD of 3 independent experiments. d. Comparison of the anti-TMPRSS2 commercial antibody and VHH staining in 293 T cells. 293 T cells were transfected with WT, R255Q and S441A TMPRSS2 plasmids. Cells were stained 24 h later with the indicated antibodies and analysed by flow cytometry. One experiment representative of 3 is shown. Light grey curves correspond to staining with control antibodies or VHH. Parental: untransfected cells. e. Effect of WT and mutant TMPRSS2 on SARS-CoV-2 pseudovirus infection. 293 T cells expressing ACE2 were transfected with WT and indicated TMPRSS2 mutants. 24 h later, 细胞通过质量编码的SARS-COV-2假病毒感染。48小时后测量发光。数据是4个独立实验的平均±SD。统计分析：对数转换数据的Geisser-Greenouse校正的RM单向方差分析，与CTRL细胞相比，Dunnett的多重比较（用PQCXIP-Empty转染）。** p <0.01。f，g。SB412515，E64D或羟基氯喹（HCQ）对f的影响。hku1a或g。HKU1B假病毒感染。在感染HKU1A或HKU1B假病毒之前，将表达WT或S441A TMPRSS2的293个T细胞与指定药物孵育2小时。感染后48小时阅读发光。左：SB412515。中间：E64D。右：HCQ。数据的平均±SD为3（E64D，HKU1B），4（E64D HKU1A，SB142515 HKU1B），5（HCQ HKU1A）或6（HCQ HKU1B，SB412515 HKU1A）独立实验。统计分析：在非归一化的对数转换数据上进行Geisser-Greenouse校正的RM双向方差分析，与未经处理的条件相比，Dunnett的多重比较。你好。稳定表达WT或S441 TMPRSS2的不同细胞系中TMPRSS2的表面水平。使用VHH-A01-FC对细胞进行TMPRSS2染色，并通过流式细胞仪进行分析。显示了代表性直方图。h。左：U2OS。右：A549。浅灰色：用非目标对照VHH-FC染色的细胞。深灰色：未修饰的父母细胞系。深蓝色：用TMPRSS2或TMPRSS2 S441A突变体转导的细胞。我。caco2。浅灰色：用非目标对照VHH-FC染色的细胞。蓝色：未修饰的父母细胞系。深灰色：TMPRSS2 KO CACO2。深蓝色：TMPRSS2 KO CACO2稳定地通过TMPRSS2 WT或S441A突变体表达向量转导。j。Camostat对CACO2细胞内源性TMPRSS2的影响。在使用HKU1A，HKU1B或SARS-COV-2（D614G或DELTA）pSeudovirus感染之前，将CACO2细胞在100μM凸轮上孵育2小时。在没有药物的情况下，将数据标准化为感染。数据的平均±SD为3（SARS-COV-2） 或4（HKU1）独立实验。统计分析：RM双向方差分析在非归一化对数转换数据上，与未经处理的条件相比，Sidak的多重比较。k。Vero E6和Vero E6-TMPRSS2细胞对HKU1伪病毒感染的敏感性。左：假病毒感染。右：TMPRSS2表面水平。深灰色：父母细胞。深蓝色：用TMPRSS2转导的细胞。数据是4个独立实验的平均±SD。