Bulk RNA-seq of HFF cell senescence model induced by oncogene activation

To investiage the mechanism of cellular senescence, we established a model of oncogene RAS-induced cellular senescence in human foreskin fibroblasts (HFF cells). We transduced an estrogen receptor fused to the H-RASG12V protein (ER:RAS) in HFF, which can be induced by 4-hydroxytamoxifen (4-OHT). Overall design: Lentiviral packaging of the oncogene-inducible expression vector (pLNC ER:RAS (G12V)) was conducted using HEK293T cells. The collected viral supernatant was then introduced into younger (~P18) HFF cells. Following infection, resistant clones were selected using 200 µg/ml of neomycin at 48-72 hours post-infection. After five days, stable transgenic cell lines were obtained. To activate the expression of H-RAS (G12V), 100 nM of 4-hydroxytamoxifen (4-OHT) was added, and cells exhibiting oncogene-induced senescence (OIS) were harvested one week thereafter.

为探究细胞衰老的分子机制，我们在人包皮成纤维细胞（human foreskin fibroblasts, HFF cells）中构建了癌基因RAS诱导的细胞衰老模型。我们将与雌激素受体融合的H-RASG12V蛋白（ER:RAS）转导至HFF细胞中，该系统可通过4-羟基他莫昔芬（4-hydroxytamoxifen, 4-OHT）诱导激活。实验整体设计如下：利用HEK293T细胞包装携带癌基因诱导型表达载体pLNC ER:RAS (G12V)的慢病毒。收集病毒上清液后，将其感染年轻（约P18代）的HFF细胞。感染后48-72小时，使用200 μg/ml新霉素筛选抗性克隆。培养5天后，获得稳定的转基因细胞系。为激活H-RAS (G12V)的表达，我们添加了100 nM的4-羟基他莫昔芬（4-OHT），并于一周后收集发生癌基因诱导衰老（oncogene-induced senescence, OIS）的细胞。