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. 2021 Jul 16;15(11):2989–3002. doi: 10.1002/1878-0261.13050

Fig. 4.

Fig. 4

CDKN1A is a direct target of EHMT1 in lung cancer. (A) Cell growth assay after transfection of the cells with siEHMT1, siCont, and siEHMT1/siCDKN1A for 48 h. A549 and H1299 cells were fixed in 100% methanol and stained with CV solution. Scale bar, 500 μm (upper). Quantification of cell numbers in CV staining assay. Mean ± SD of three independent experiments. The P values were calculated using Student’s t‐tests (***P < 0.001) (below). (B) Western blot analysis after siEHMT1, siCont, and siEHMT1/siCDKN1A transfection using anti‐EHMT1, anti‐PARP, and anti‐ACTB antibodies. ACTB was used as the internal control in A549 and H1299 cells. (Arrowhead: cleaved PARP) (C) FACS analysis using Muse Caspase‐3/7 working solution was performed after transfection of siEHMT1, siCont, and siEHMT1/siCDKN1A. The upper right panel indicates the apoptotic and dead cell proportions (left). Quantification of caspase‐3/7 activity. Mean ± SD of three independent experiments. The P values were calculated using Student’s t‐tests (***P < 0.001) (right). (D) FACS analysis of Annexin V staining was carried out after transfection of the cells with siEHMT1, siCont, and siEHMT1/siCDKN1A. The lower right and upper right quadrants indicate early apoptosis and late apoptosis, respectively (left). Quantification of apoptosis. Mean ± SD of three independent experiments. The P values were calculated using Student’s t‐tests (***P < 0.001 and **P < 0.01) (right). (E) FACS analysis using PI staining was performed after transfection of A549 and H1299 cells with siEHMT1, siCont, and siEHMT1/siCDKN1A (upper). Quantification of cell cycle analysis. Mean ± SD of three independent experiments. The P values were calculated using Student’s t‐tests (***P < 0.001) (below). (F) Cell growth assay after transfection with siCDKN1A and siCont for 48 h. A549 and H1299 cells were fixed in 100% methanol and stained with CV solution. Scale bar, 500 μm. (G) Graphical abstract for ChIP primer design on the CDKN1A promoter region (upper). The ChIP assay was performed with anti‐H3K9me2 antibody. The result is shown as a relative enrichment compared to the control in A549 cells after siEHMT1 transfection. Mean ± SD of three independent experiments. P values were calculated using Student’s t‐test (***P < 0.001).