Fig 2: ELK1 is highly expressed in patients with CRC especially patients with recurrent disease and positively correlated with CEA expression. A, Predicted transcription factors (TFs) that have the capability to bind to the CEA promoter, using bioinformatics analysis. The maximum matrix dissimilarity is set as zero, and with 18 output TFs. B, ELK1 mRNA levels in CRC tissues (n = 275) were significantly higher compared with normal tissues (n = 349) based on analysis from the GEPIA2 database. C, ELK1 mRNA in the recurrent patients with CRC (n = 272) maintains high compared with patients with no recurrent disease (n = 256) after radical operation as assessed by TCGA database. D, ELK1 mRNA expression in the diagnosed 30 CRC in Beijing Friendship Hospital, Capital Medical University is increased compared with corresponding adjacent normal tissues. E, ELK1 protein as well as its active form, the p-ELK1, is clearly increased in the 30 CRC tissues in comparison with adjacent normal tissues. Left: Representative western blot bands for ELK1 and p-ELK1 with ß-actin as control. Right: Statistical analysis on the 30 samples. F, Representative immunohistochemical staining images of ELK1 in CRC tissues and adjacent normal tissues. ELK1 staining is more intensive in CRC tissues, and was mainly located in the cytoplasm and nucleus. The framed views in the upper panel are magnified in the lower panel. G, ELK1 mRNA expression is positively correlated with the CEA mRNA analyzed on GEPAI2 database. *P < .05, **P < .01, ***P < .001

Fig 3: 17ß-Estradiol mediates Elk-1 phosphorylation in HT1080 cells via ER and activated MAP Kinase-ERK1/2 signalling pathway. HT1080 cells were transiently transfected with hERa-vector and analysed after 24 h of treatment with Dextrin (vehicle), 17ß-estradiol (10-8 M), ICI 182,780 (10-5 M) and PD98059 (10 µM) by immunoblotting using anti p-Elk-1 antibody. (A and B) 17ß-Estradiol significantly increased phosphorylation-status of Elk-1 in HT1080 cells, whereas ICI 182,780 and PD98059 significantly inhibited E2-mediated Elk-1 phosphorylation. Membranes were subsequently re-probed with a specific GAPDH antibody as internal standard. Protein-expression levels of cells treated with 17ß-estradiol, ICI 182,780 and PD98059 are expressed as fold induction of vehicle-treated cells. Bars represent means ± SEM (n = 3). **P = 0.01, ##P = 0.01, #P = 0.05.

Fig 4: RAD51 gene expression is regulated by the MAPK signaling pathway in melanoma cells via Elk1.a The influence of vemurafenib treatment (2 µM, 24 h) on the expression of 84 different DNA repair genes in the A375 cell line was analyzed by PCR array. DMSO (0.02%, 24 h) treated cell samples served as control. log2 fold changes in the expression of the corresponding genes between the vemurafenib treated group and the control group and log10 p-values of the respective gene expression changes (unpaired Student’s t-test) are shown in the volcano plot (n = 3 per group). The data of the HRR genes are marked in blue and named. The data of other DNA repair genes are shown as gray dots. b The log2 of fold change in the expression of HRR genes between the vemurafenib (2 µM, 24 h) treated samples and DMSO (0.02%, 24 h) treated control samples from five melanoma cell lines is plotted (RT-qPCR, n = 3 in triplicates, mean ± SEM, unpaired Student’s t-test *: p < 0.05). c Immunoblot analysis of vemurafenib (vem., 2 or 5 µM, 24 h) treated or untreated (0) melanoma cell line samples. d The binding sites of the critical transcription factors in the indicated HRR gene promoter regions were determined via the ENCODE website (b.r.: binding region). e Immunoblot analysis of vemurafenib (vem., 2 or 5 µM, 24 h) treated or untreated (0) Mel1617 cell line samples. F. Elk1 knockdown was induced by respective siRNA transfection (siElk1A and siElk1B) in A375 cells and its effect on Rad51 expression was analyzed by immunoblot analysis (48 h after transfection). Samples of non-coding siRNA transfected cells were used as control (siCtr.). The Rad51 protein level in the respective samples were quantified and normalized to the correspondent actin level (lower graph, n = 3, mean ± SD, Unpaired Student’s t-test, *: p < 0.05) g Elk1 knockdown cells (siElk1A and siElk1B) or Control cells (siCtr.) described in F. were treated for 5 days with different concentrations of Rad51is (B02 or RI-1) (viability analysis in quituplicates, mean ± SD, Multiple t-test according to Holm Sidak method, * for p < 0.05).

Fig 5: The binding site of ELK1 in CEA promoter was confirmed by ChIP and luciferase assays. A, Predicted binding site and sequence of ELK1 within the CEA promoter using UCSC and PROMO databases. The ATG sequence marked in blue indicates the transcription start site (TSS). B, ChIP assay was performed on the LS174T cells treated with DMSO or 1 µmol/L of TPA for 3 h. Compared with DMSO group, the binding of ELK1 to the CEA promoter is evidently increased in the TPA-treated cells, in which ELK1 is hyperactivated. AcH3 is used as a positive control and IgG as a negative control. C, Luciferase activity of the WT CEA promoter-driven luciferase reporter is significantly increased by the co-transfected ELK1 plasmid. ELK1 plasmid transfection is unable to increase the luciferase activity of the MUT CEA promoter-driven luciferase reporter. **P < .01