Fig 1: Overexpression of COL22A1 rescues cell proliferation and cycle changes caused by downregulation of EN1. (A) CCK-8 assay detects cell proliferation capacity after simultaneous knockdown of EN1 and overexpression of COL22A1. (B) Single-cell clone formation ability after simultaneous knockdown of EN1 and overexpression of COL22A1 in two NPC cell lines. (C) Flow cytometric cell cycle assay detects cell cycle changes in 5-8F and CNE-2Z cell lines. (D) The SA-ß-gal assay detects proportion of positive cells in 5-8F and CNE-2Z cell lines. The results of CCK-8 assay are analysed with one-way anova. The other results are shown as mean ± SD. All data from triplicate experiments. *p < 0.05, ** p < 0.01, ***p < 0.001
Fig 2: EN1 is highly expressed in NPC tissue cells. (A) Heat map of GEO data differentially expressed genes. (B) EN1 is highly expressed in TCGA‐HNSCC. (C) Expression of EN1 in nasopharyngeal carcinoma microarrays. (D) Sectoral statistics of EN1 expression in NPG and NPC. (E) Cardinality test for statistical EN1 expression in NPG and NPC. (F) Expression of EN1 in various cell lines of NPC. *p < 0.05, **p < 0.01
Fig 3: Downregulation of EN1 inhibited cell proliferation in vivo. (A) Overall view of the tumour after tumour formation in nude mice. (B) The growth curve of the tumour. (C) HE staining of the tumour. (D) Weight of the tumour. The results of the growth curve of the tumour are analysed with one-way anova. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 4: Changes in cell function after downregulating EN1. (A) Validation by qRT-PCR after downregulation of EN1. (B) Verified by immunoblotting after downregulation of EN1 in 5-8F and Cen-2Z cell lines. (C) Heat map of the alteration of some genes after downregulation of EN1. (D) KEGG bubble diagram shows the function of EN1-altered genes. (E) GO maps plotted for changes in genes after downregulation of EN1. (F) TEM showed many secondary lysosomes (including lipofuscin) appear in the shEN1 group. (G) the SA-ß-gal assays showed a higher proportion of positive cells in the shEN1 group. The results show the mean ± SD from triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 5: EN1 regulates cell proliferation and cycle via COL22A1-CDK4/6-Cyclin D1-Rb signalling axis. (A) Dual luciferase reporter shows EN1 transcriptional regulation of COL22A1. (B) Immunoblotting experiments showed the expression of COL22A1, CDK4/6, Cyclin D1 and p-Rb proteins. The results show the mean ± SD from triplicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from OriGene Technologies for EN1 Human qPCR Primer Pair (NM_001426)