Fig 2: SETD1A knockdown in NSCLC cells decreases the expression of several critical oncogenes in lung cancer. (a),(b) Immunofluorescence of A549 cells (shSETD1A‐1 or control) with antibody to SETD1A or H3K4me3. The scale bar was 50 μm. (c)–(h) ChIP assay to detect location of H3K4me3 methylation in genomics. (i) Western blot for critical oncogenes with H3K4me3 methylation at the transcriptional start site. (j) Relative protein levels of oncogenic proteins in I. ACTB was used as the internal control (n = 4). P1 = 0.0193, P2 = 0.0385, P3 = 0.003, P4 = 0.0238, P5 = 0.0212, P6 = 0.0317, P7 = 0.0047, P8 = 0.0358, P9 = 0.0079, P10 = 0.0799, P11 = 0.0191, P12 = 0.0183. Statistical tests were analyzed by using paired t‐test after log transformation. n means independent biological sample

Fig 3: Upregulated SETD1A is associated with the disease process in lung cancer. (a) The mRNA levels of SETD1A with different SETD1A DNA copy numbers in lung cancer. (Shallow deletion, n = 79; diploid, n = 260; gain, n = 158; amplification, n = 6). Data were from the cBioportal database. (b) Lung adenocarcinoma and adjacent nontumor lung tissue were analyzed using the Illumina human WG‐6 v3.0 expression Beadchip and the expression of SETD1A between the two groups was determined (GEO DataSets, GSE32863, n = 58). (c) SETD1A mRNA levels in different stages of lung adenocarcinoma from a public dataset (GEO DataSets, GSE32863. Normal, n = 58; stage I, n = 34; stage II, n = 11; stage III/IV, n = 13). (d) Fold‐change of SETD1A levels in lung cancer cell lines compared to small airway epithelial cell (SAEC). Data were from previous reports.12 (e) Kaplan–Meier plot of cumulative overall survival of patients with lung carcinoma in the TCGA database. Patients in the 90% (SETD1A higher expression group, blue line, n = 440) were compared with patients in the lower 10% (SETD1A lower expression group, yellow line, n = 48). The p‐value of the log‐rank test comparing the two survival curves was 0.067. Statistical test of (a)–(c) were analyzed by using the Welch's t‐test after log transformation. n means independent biological sample

Fig 4: Downregulation of SETD1A in NSCLC cells suppresses cancer EMT. (a),(b), the images of A549 cells with different expression of SETD1A were captured after wound injury. Scale bar was 200 μm. The motility of lung cancer cells was measured at the indicated time points (n = 5). (c)–(f), the same number of A549 or H1299 cells (shSETD1A‐1 or control) were seeded in Transwell chambers and the cells that passed through the porous membrane were stained and captured (A549, (c) upper row; H1299, (e) upper row). In addition, the cells that passed through the Matrigel and porous membrane were also stained and captured (A549, (c) lower row; H1299, (e) lower row). The number of stained cells in five random fields was counted (A549, D; H1299, F) (n = 3). (g),(h) Western blot to detect EMT‐related proteins in SETD1A silenced A549 (g) or H1299 (h) cell lines (n = 4). Statistical tests were analyzed by using paired t test after log transformation. n means independent biological sample

Fig 5: SETD1A silencing prevents tumorigenesis and EMT of NSCLC cells in nude mice. (a) The image of tumor tissue from nude mice with A549 cell transplantation at the end point (35 days after inoculation). (b) Average tumor weight of A549 xenografts with SETD1A knockdown at the end point (n = 6). (c) Tumor volumes of A549 xenografts with SETD1A knockdown in nude mice (n = 6). (d) The tumor images from nude mice with H1299 cell transplantation at the end point (25 days after inoculation). (e),(f) Tumor weight and volumes of H1299 xenografts with SETD1A knockdown in nude mice (n = 6). (g),(h) qPCR results for mRNA levels of oncogenes and EMT‐related genes in A549 xenografts (n = 6). P1 = 0.0017, P2 = 0.005, P3 = 0.0201, P4 = 0.005, P5 = 0.0015, P6 = 0.0012, P7 = 0.005, P8 = 0.0001. (i),(j) qPCR results for mRNA levels of oncogenes and EMT‐related genes in H1299 xenografts (n = 6). P1 = 0.0089, P2 = 0.0157, P3 = 0.0056, P4 = 0.0138, P5 = 0.0063, P6 = 0.0055, P7 = 0.0158, P8 = 0.0062. Statistical tests were analyzed by using the Welch's t‐test after log transformation (b),(e),(g)–(j) or two‐way ANOVA (c),(f). n means independent biological sample