JunD Antibody - #AF6200

Figure 5 YTHDC1 enhances the mRNA stability of NECTIN4 transcription factor JUND in a m6A-dependent manner (A) The Ominer network tool predicts potential transcription factors for NECTIN4. (B) ChIP-seq from JUND on the NECTIN4 promoter region of the ChIP-Atlas. (C and D) HT1376 and RT112 cells were transfected with the specified shRNA or plasmid for 72 h, and, after puromycin screening, the cells were collected for ChIP-qPCR analysis using immunoglobulin (Ig)G or JUND antibodies. (E) The sequence and location of the JUND binding peak in the YTHDC1 promoter. TSS, transcription start site; WT, wild type, MUT, mutant. (F) HT1376 cells were transfected with a designated plasmid for 24 h, and, after puromycin screening, the cells were harvested and the activity of the YTHDC1 promoters was measured. (G) The KnockTF 2.0 web tool predicts the change in YTHDC1 levels after JUND knockout. (H) Relationship between JUND and NECTIN4 expression in patients with BC. (I and J) HT1376 and RT112 cells were transfected with the specified shRNA or plasmid for 72 h, and, after puromycin screening, cells were collected for western blot analysis and RT-qPCR assay. (K and L) IHC staining of BC tissue microarrays using JUND and NECTIN4 antibodies. Typical images are shown in (K). The correlation between JUND and NECTIN4 is shown in (L). (M) The RNA-seq analysis of EV treatment and YTHDC1 knockdown. (N and O) HT1376 and RT112 cells were transfected with the specified shRNA or plasmids for 72 h, and, after puromycin screening, cells were collected for western blot analysis and RT-qPCR assay. (P) RIP-qPCR was performed in HT1376 and RT112 cells by using IgG or an anti-YTHDC1 antibody. (Q) Methylated RNA immunoprecipitation (MeRIP)-qPCR was performed in HT1376 and RT112 cells. (R and S) HT1376 and RT112 cells were transfected with the indicated shRNA or plasmids for 72 h. Cells were collected, and RNA was extracted from the cytoplasm and from the nucleus. RT-qPCR was performed. (T and U) HT1376 and RT112 cells were transfected with the indicated shRNA or plasmids for 72 h. Then, the cells were treated with actinomycin D (5 μg/mL). Then, the cells were collected at different time points. Total RNA was extracted and analyzed by RT-qPCR. mRNA expression in each group was normalized to β-actin. (V and W) IHC staining of BC tissue microarrays using JUND and YTHDC1 antibodies. Typical images are shown in (L). The correlation between JUND and YTHDC1 is shown in (M). Data are representative of three independent experiments (C, D, F, I, J, and N–U). All data were expressed as the mean ± SD and repeated three times. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5 YTHDC1 enhances the mRNA stability of NECTIN4 transcription factor JUND in a m6A-dependent manner (A) The Ominer network tool predicts potential transcription factors for NECTIN4. (B) ChIP-seq from JUND on the NECTIN4 promoter region of the ChIP-Atlas. (C and D) HT1376 and RT112 cells were transfected with the specified shRNA or plasmid for 72 h, and, after puromycin screening, the cells were collected for ChIP-qPCR analysis using immunoglobulin (Ig)G or JUND antibodies. (E) The sequence and location of the JUND binding peak in the YTHDC1 promoter. TSS, transcription start site; WT, wild type, MUT, mutant. (F) HT1376 cells were transfected with a designated plasmid for 24 h, and, after puromycin screening, the cells were harvested and the activity of the YTHDC1 promoters was measured. (G) The KnockTF 2.0 web tool predicts the change in YTHDC1 levels after JUND knockout. (H) Relationship between JUND and NECTIN4 expression in patients with BC. (I and J) HT1376 and RT112 cells were transfected with the specified shRNA or plasmid for 72 h, and, after puromycin screening, cells were collected for western blot analysis and RT-qPCR assay. (K and L) IHC staining of BC tissue microarrays using JUND and NECTIN4 antibodies. Typical images are shown in (K). The correlation between JUND and NECTIN4 is shown in (L). (M) The RNA-seq analysis of EV treatment and YTHDC1 knockdown. (N and O) HT1376 and RT112 cells were transfected with the specified shRNA or plasmids for 72 h, and, after puromycin screening, cells were collected for western blot analysis and RT-qPCR assay. (P) RIP-qPCR was performed in HT1376 and RT112 cells by using IgG or an anti-YTHDC1 antibody. (Q) Methylated RNA immunoprecipitation (MeRIP)-qPCR was performed in HT1376 and RT112 cells. (R and S) HT1376 and RT112 cells were transfected with the indicated shRNA or plasmids for 72 h. Cells were collected, and RNA was extracted from the cytoplasm and from the nucleus. RT-qPCR was performed. (T and U) HT1376 and RT112 cells were transfected with the indicated shRNA or plasmids for 72 h. Then, the cells were treated with actinomycin D (5 μg/mL). Then, the cells were collected at different time points. Total RNA was extracted and analyzed by RT-qPCR. mRNA expression in each group was normalized to β-actin. (V and W) IHC staining of BC tissue microarrays using JUND and YTHDC1 antibodies. Typical images are shown in (L). The correlation between JUND and YTHDC1 is shown in (M). Data are representative of three independent experiments (C, D, F, I, J, and N–U). All data were expressed as the mean ± SD and repeated three times. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 7 Transcription Factor Network in RASFs after extracellular acid stimulation. A The Venn diagram illustrated the intersection of DEGs between the proteomics and RNA-seq data. B The association between transcriptional and translational processes was analyzed across nine quadrants. C Heatmap presented the subunits of transcription factor AP1 in proteomic and RNA-seq. D Ridge plots displayed the transcription factor target enrichment analysis of RNA-seq by GSEA. E Enrichment of transcription factor targets for upregulated genes in RNA-seq. F Central transcriptional status of the transcription factor AP was identified by network analysis. G The expression of AP1 subunits in RASF was confirmed by Western blotting in the presence of 100 nM PcTx- 1, 1 mM EGTA and 10 μM BAPTA-AM. H The expression and localization of the AP1 subunit in RASF treated with 100 nM PcTx- 1, 1 mM EGTA, and 10 μM BAPTA-AM were confirmed by multiple immunofluorescence (n = 3). Scale bar = 50 μm, magnification = 200x. Data are presented as the mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test: *P