Fig 1: ECRG4 upregulates OGN via an NFIC-mediated mechanism in BCa cells. (A) The NFIC transcription factor was predicted at -989 to +91 bp of the OGN promoter region using the JASPAR online tool (http://jaspardev.genereg.net/). (B) A schematic representation of the truncated luciferase reporter plasmids with OGN promoter activity. (C) The ChIP assay was performed using the NFIC antibody. (D) ChIP-qPCR detected the promoter occupancy of OGN. (E) Western blotting detected the protein levels of NFIC in BCa cells after transfection with the indicated plasmids. (F) Quantification of the protein levels of NFIC in (E). (G) RT-qPCR detected the mRNA levels of OGN. (H) NFIC mRNA levels in the GEPIA database. (I) Correlation analysis of ECRG4 and NFIC expression was performed using GEPIA in patients with BCa and healthy people. (J) Correlation analysis of OGN and NFIC expression was performed using GEPIA in patients with BCa and healthy people. (B,D,F,G) ANOVA followed by Bonferroni’s post hoc test; (H) Student’s t-test. All data were replicated three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.
Fig 2: ECRG4 inhibits the NF-?B pathway via the upregulation of OGN in BCa cells. (A) NF-?B transcriptional activity was detected using an NF-?B luciferase reporter gene system. (B) Western blotting detected the protein levels of ECRG4, OGN, p65, and p-p65 (S536) in BCa cells after transfection with ECRG4-overexpressing or silencing plasmids. (C,D) Quantification of the protein levels of ECRG4, OGN, p65, and p-p65 (S536) in panel (B). (E) The NF-?B luciferase reporter gene system was used to detect the NF-?B transcriptional activity of BCa cells after transfection with indicated plasmids. (F) Western blotting detected the protein levels of p65 and p-p65 (S536) in BCa cells after transfection with the indicated plasmids. (G) Quantification of the protein levels of p65 and p-p65 (S536) in panel (F). (H) The proposed mechanism of ECRG4 inhibition of the carcinogenesis of BCa cells via NFIC/OGN/NF-?B signaling. (A–G) ANOVA followed by Bonferroni’s post hoc test. All data were replicated three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3: TBX3 and NFIC are amplified in prostate cancer cells.(A) The genomic amplification rates of TBX3 and NFIC are examined in various prostate cancer studies. (B) In one metastatic castration-resistant prostate cancer (mCRPC) study, the rates of TBX3, NFIC, and FOXA1 gains are depicted. (C and D). The expression of TBX and NFIC are compared in one mCRPC study in which the regression line, Spearman’s correlation coefficients, and q-values are depicted.
Fig 4: Left.V5-tagged CREB5 was targeted for immunoprecipitation in cell lysates in vehicle control (DMSO) or enzalutamide treated (ENZ) LNCaP cells. TBX3 and NFIC were detected in the total lysate (input) and precipitates (IP) using immunoblots. IgG was used as a negative precipitation control. Right. FOXA1 was targeted in enzalutamide treated cells in a Co-IP experiment. CREB5 and FOXA1 were detected in the total lysate (input) and precipitates (IP) using immunoblots. IgG was used as a negative precipitation control.
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