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Fig. 2 | Molecular Cancer

Fig. 2

From: p53-targeted lincRNA-p21 acts as a tumor suppressor by inhibiting JAK2/STAT3 signaling pathways in head and neck squamous cell carcinoma

Fig. 2

NF-YA is essential for lincRNA-p21 regulation by mutant P53 but not wild-type p53. a The expression of lincRNA-p21 was detected by qRT-PCR in cells transfected with si-p53 for 24 h. b The expression of lincRNA-p21 was detected by qRT-PCR in cells treated with 500 nM doxorubicin (DOX) for 24 h. c, d The expression of TP53 mRNA and lincRNA-p21 were detected by qRT-PCR in SCC25 cells transfected with the wild-type 53 or mutant p53 vector for 24 h. e, f Detroit562 and HN30 cells were treated with 500 nM DOX or transfected with si-p53 for 48 h. The binding of p53 at the lincRNA-p21 promoter region was detected by a chromatin immunoprecipitation assay. g A probe targeting the binding sites of p53 in the lincRNA-p21 promoter was designed according to ChIP assays. h HN6, Cal27 and HN30 cells were treated with 500 nM DOX for 48 h. A biotin probe and cold oligo probes were incubated with nuclear extract followed by an EMSA. For the cold competitive EMSA, 150 × nonlabelled probes were used. i SCC25 cells were co-transfected with lincRNA-p21 promoter-luciferase truncations and p53 plasmids, and the luciferase activity was determined using a dual luciferase reporter assay after 24 h. j The correlation between lincRNA-p21 and TP53 mRNA was analyzed in 70 HNSCC patients by qPCR. k The cellular localization of NF-YA and p53 was visualized by immunofluorescence in HN6 cells. Scale bars, 20 μm. l, m NF-YA mRNA and protein levels were detected after transfection with siRNA targeting NF-YA. n The expression of lincRNA-p21 was detected in HN6, Cal27 and HN30 cells after transfection with si-NF-YA for 24 h. o lincRNA-p21 promoter activity was analyzed after co-transfection with p53 R175H or wild-type p53 plasmids and si-NF-YA for 24 h. p The binding of p53 to NF-YA in HN6, Cal27 and HN30 cells was determined by coimmunoprecipitation. *: P < 0.05; **: P < 0.01

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