Fig 1: PKC-? regulates DUSP4, p300 and H3K27ac in mesenchymal MCF-7/PMASUS cells.Confocal laser scanning microscopy was performed on MCF-7/PMASUS cells and treated with either BIM or C27 PKC-? catalytic inhibitors. Cells were fixed and probed with primary mouse antibodies to DUSP4 and either primary rabbit antibodies to (A) p300 or (B) H3K27ac followed by the corresponding secondary antibody conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Representative images for each antibody dataset pair are shown: green = p300 or H3K27ac; red = DUSP4; yellow = overlap between DUSP4 and p300 fluorescence signals. The PCC was determined as described in methods for both MOCK control and cells treated with either BIM or C27 PKC-? catalytic inhibitors. PCC indicates the strength of relation between the two fluorochrome signals for at least 20 individual cells ± SE. (C). Bar graphs indicate the total NFI of DUSP4, p300, or H3K27ac as measured using ImageJ software to select the nucleus of each cell and measure the total NFI signal minus background for at least 20 individual cells ± SE.
Fig 2: DUSP1 and DUSP4 directly tether to the promoters of mesenchymal genes.MCF-7 cells were incubated with either vehicle alone or with PMA for 60 h. ChIP was performed with DUSP1 and DUSP4 antibodies. ChIP DNA was analysed by SYBR Green real-time PCR. Enrichment across the promoter regions of FN1, PLAUR, and IL6 are shown for: (A) DUSP1 and (B) DUSP4. Data are expressed as percentage enrichment relative to total input control and represent the mean ± SE of three independent experiments. (C) Nuclear extracts were obtained from MCF-7 cells stimulated as previously described and subjected to half-ChIP using DUSP4 pull down or a no antibody control. Immunoblots of the samples were probed with primary rabbit antibodies to RNA-Pol-II-serine2p or RNA-Pol-II-serine5p and detected as described in the methods. Representative images of the immunoblots are depicted along with the Novex loading control (LC).
Fig 3: A novel role for nuclear DUSP4 in the regulation of the key epigenetic protein p300 in the mesenchymal/CSC state in breast cancer.Step 1: Activation of the PKC-? pathway by inflammatory signals leads to DUSP4 phosphorylation and nuclear translocation. Phosphorylation of these proteins, either directly or indirectly by PKC-?, maintains them in the nucleus [52]. PKC-? also directly binds DUSP4 and leads to DUSP4 transcription. Step 2: DUSP4 and p300 co-exist in the epigenomic context. Step 3: DUSP4 dephosphorylates the inhibitory serine-89 phosphorylation mark of p300. Step 4: This allows phosphorylation of p300-1834 by protein kinases, such as PKC-? and Akt [43,44]. Step 5: p300-1834p promotes H3K27 acetylation.
Fig 4: DUSPs are induced during epithelial-to-mesenchymal transition.DUSP1, DUSP4, and DUSP6 transcript levels measured by real-time PCR in (A) MCF-7 and MDA-MB-231 cells. Data are expressed as arbitrary copy numbers normalised to PPIA and are representative of the mean ± SE. (B) MCF-7 cells after incubation with PMA for various periods of time. Spline curves represent mean arbitrary copy numbers normalised to PPIA of two independent experiments. (C) MCF-7 cells after incubation with vehicle alone or in the presence of either PMA or PMA+TGF-ß for 60 h. Data are expressed as relative fold change to MCF-7 normalised to PPIA and are representative of the mean ± SE of four independent experiments. MCF-7 cells were incubated with vehicle alone or in the presence of PMA for 60 h. Transcript levels measured by real-time PCR of: (D) CDH1, VIM, FN1, SNAI1, SNAI2, CD44, and PLAUR and (E) DUSP1, DUSP4, and DUSP6. Data are presented as relative fold change to MCF-7 normalised to PPIA and are representative of the mean ± SE. (F) DUSP1, DUSP4, and DUSP6 transcript levels in PKC-? siRNA transfected cells. Data are expressed as relative fold change to MCF-7 MOCK siRNA normalised to PPIA and are representative of the mean ± SE for four experiments. (G) PKC-? ChIP-seq across DUSP1, DUSP4, and DUSP6 transcripts in MCF-7 and MCF-7/PMA cells. Data are shown in the UCSC Genome Browser. The scale in all UCSC images is indicated on the y-axis as numbers in reads per million mapped reads.
Fig 5: DUSPs show distinct localisation in the mesenchymal state.Confocal laser scanning microscopy was performed on either MDA-MB-231 cells or MCF-7 cells treated with vehicle alone or PMA for 60 h. Cells were fixed and probed with primary rabbit, mouse, or goat antibodies to DUSP1, DUSP4, and DUSP6, respectively, followed by the corresponding secondary antibody conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Fn/c values for: (A) MCF-7 and MCF-7/PMA cells; (B) MDA-MB-231 cells. Data shown represent the mean ± SE. Value 1 = equal cytoplasmic and nuclear fractions; >1 = nuclear bias; <1 = cytoplasmic bias. MCF-7 cells were probed with either: (C) primary anti-rabbit-DUSP1 and anti-mouse-H3K9me1; or (D) primary anti-mouse-DUSP4 and anti-rabbit-H3K27ac or anti-rabbit-H3K4me1. All antibodies were conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. MDA-MB-231 cells probed with either: (E) primary anti-rabbit-DUSP1 and anti-mouse-H3K4me3 or anti-mouse-H3K9me1 or (F) primary anti-mouse-DUSP4 and anti-rabbit-H3K4me1 or anti-rabbit-H3K27ac. All antibodies were conjugated to Alexa-Fluor 568 or Alexa-Fluor 488. Representative images for each dataset are shown: green = DUSPs; red = histone marks; yellow = overlap between DUSPs and histone mark fluorescence signals. The plot-profile feature of ImageJ was used to plot the fluorescence signal intensity along a single line spanning the nucleus (n = 5 lines per nucleus, 5 individual cells). The average fluorescence signal intensity for the indicated pair of antibodies was plotted for each point on the line ±SE. Signal was plotted to compare how the signals for each antibody varied compared to the opposite antibody. The PCC was determined for each plot profile. PCC indicates the strength of relation between the two fluorochrome signals for at least 20 individual cells ± SE. Colours from representative images correspond to plot profiles.
Supplier Page from Santa Cruz Biotechnology, Inc. for MKP-2 siRNA (h)