Fig 1: LINC00673 silencing inhibits prostate cancer cell proliferation by impairing the methylation of the KLF4 gene promoter. A, The transfection efficiency of overexpressed LINC00673 and KLF4 in cell lines detected by RT-qPCR. B, C, Cell proliferation of DU145 cells treated with pcDNA-KLF4, pcDNA-LINC00673 or both detected by EdU assay (×400). D, Cell cycle of DU145 cells treated with pcDNA-KLF4, pcDNA-LINC00673 or both detected by flow cytometry. E, Cyclin D2 expression in DU145 cells treated with pcDNA-KLF4, pcDNA-LINC00673 or both detected by immunofluorescence staining (×400). F, mRNA expression of Cyclin D2 and Cyclin B1 in DU145 cells treated with pcDNA-KLF4, pcDNA-LINC00673 or both determined using RT-qPCR. G and H, Western blot analysis of Cyclin D2 and Cyclin B1 proteins in DU145 cells treated with pcDNA-KLF4, pcDNA-LINC00673 or both. * P < .05 vs DU145 cells without treatment. Data (mean ± SD) among multiple groups were compared using one-way ANOVA. The experiment was repeated in triplicate to obtain the mean value
Fig 2: circKLF4 promotes the odontoblastic differentiation of mDPCs. (A) The existence of circKLF4 was validated by gel electrophoresis using the products of quantitative real-time PCR. (B) mRNA levels of Alp, Dmp1, Dspp, circKLF4, and Klf4 by real-time reverse transcription polymerase chain reaction in the mDPCs cultured in differentiation medium. (C) Western blot and quantification of the relative protein levels of DMP1, DSP, KLF4 cultured in differentiation medium. (D) In situ hybridization of circKLF4 in PN1 mouse molar. (E) mRNA levels of Alp, Dmp1, Dspp, circKLF4, and Klf4 after transfection with circKLF4 siRNA (circKLF4-si) compared with the control group (NC). (F) DMP1, DSP, KLF4 levels and quantified density after circKLF4 was knocked down. (G) mRNA levels of Alp, Dmp1, Dspp, Klf4, and circKLF4 after transfected with circKLF4 overexpression plasmid (P-circKLF4) compared with the empty vector (P-vector). (H) The protein levels of DMP1, DSP, and KLF4 after overexpression of circKLF4. Right panel shows the quantified data. Significant difference vs. day 0, *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars = 100 µm for (D).
Fig 3: DPYSL2A, but not DPYSL2B induces monocytic differentiation of AML cells. (A) Schematic illustration of the DPYSL2A and DPYSL2B genes. DPYSL2A has unique 118 amino acids at its N terminus (shown in red) with common 559 amino acids (shown in white). (B) Immunoblot analysis of DPYSL2A, DPYSL2B, and GAPDH in THP-1 cells transduced with lentiviruses encoding DPYSL2A, DPYSL2B, or control cassette. Cells were treated with 3 µM doxycycline for 48 h and then lysed for protein extraction. (C) Cell proliferation curves of THP-1 cells used in (B). Cells were cultured in the presence of 3 µM doxycycline (n = 3). (D) Cell surface expression of CD11b and CD14 was determined by flow cytometry in THP-1 cells used in (B). Cells were treated with 3 µM doxycycline for 48 h and then harvested for flow cytometric analysis. (E) Heatmap showing the gene expression profiles of THP-1 cells with exogenous expression of KLF4 or DPYSL2A. The expression levels of the top 500 up- and down-regulated transcripts in KLF4-overexpressing THP-1 cells were examined in DPYSL2A-overexpressing THP-1 cells. Cells were incubated with 3 µM doxycycline for 24 h before total RNA extraction (GSE101751). (F) GSEA was performed in THP-1 cells with exogenous DPYSL2A expression to compare the differences in expression of the top 500 transcripts following exogenous expression of KLF4. Each sample was treated with 3 µM doxycycline for 24 h before being lysed for RNA extraction. Data are presented as mean ± SEM. *P < 0.05, two-tailed Student’s t test.
Fig 4: KLF4 binds to the regulatory regions of GRHL genes.(A) Quantitative ChIP-PCR analysis of KLF4 occupancy of GRHL1, GRHL2 and GRHL3 regulatory regions was performed in HEK293 cells transfected with pcDNA3.1-HA-KLF4 FL. ZNF333 was used to identify non-specific interactions. Chromatin was immunoprecipitated with anti-KLF4 antibody or nonspecific antibody. The amount of DNA amplified from immunoprecipitated DNA was normalized to that amplified from input DNA. Data are shown as means ± SEM from experiments independently performed in triplicate, *significantly different at p= 0.05. (B) EMSA analysis performed with probes including KLF4 binding sequences: left panel–in the region -409/-400 (lane 2) or -409/-400 with the minor frequency allele of SNP rs115898376 (C/T) (lane 5) of the GRHL1 promoter; central panel–in the region -650/-641 (lane 2) of the GRHL2 promoter; right panel–in the region -1302/-1293 (lane 2) of the GRHL3 promoter. Lane 1: probe only. Cold probe: unlabeled double-stranded oligonucleotides including -409/-400 (left panel, lane 3) or -409/-400 with SNP rs115898376 (C/T) (left panel, lane 6) or -650/-641 (central panel, lane 3) or -1302/-1293 (right panel, lane 3) regions of GRHL genes (100-fold molar excess of competitors). Where indicated, 3 µg anti-KLF4 antibody was added per lane (ab106629, Abcam).
Fig 5: Identification of Etv6 direct transcriptional targets in the somites. a Experimental design. RNA-seq was performed on wild type (WT) and Etv6-deficient (etv6 MO-injected embryos) somite explants dissected from stage 22 embryos. Differentially expressed genes (DEGs) were identified by comparing the transcriptome of these tissues. b Spearman correlation analysis on triple biological RNA-seq replicates. c MA plot (magnitude of the difference versus amplitude of the signal) showing the fold-change in gene expression of all genes in WT versus Etv6-deficient somites (log2FC) compared to their expression levels (log2CPM). Black dots, non-significant change; red dots, differentially expressed genes (DEGs). The number of positively and negatively regulated DEGs is indicated. d WISH showing the expression of DEGs in stage 22 WT and Etv6-deficient embryos. Meox2 expression is upregulated in the somites (arrows) in Etv6-deficient embryos whereas expression of crim1, sox18 and vegfa is downregulated. Note that vegfa expression in the hypochord is unaffected (arrowheads). Embryos are shown in lateral view with anterior to the left and dorsal to the top. Numbers in top right corner indicate the number of embryos exhibiting the phenotype pictured (scale bars: 0.5 mm). e The intersection between DEGs and genes harbouring Etv6 ChIP-seq peaks in their TSS region reveals 540 putative direct target genes. f Foxo3 and klf4 (arrows), known transcriptional regulators of vegfa, are amongst the 18 TFs and chromatin modifiers identified as potential direct transcriptional targets of Etv6
Supplier Page from Abcam for Anti-KLF4 antibody