Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: M2 macrophage promote PCa cell proliferation and invasion through direct contact. (A, B) Flow cytometry (FCM) detection of THP-1 and PBMC-induced M2 macrophages makers. (C) The strategy and schematic diagram for direct coculture and separation of TAMs and mCherry-luciferase-labeled DU145 and 22RV1 cells. FACS was used to separate tumor cells and TAMs according to the color channel. (D) FCM identified the separated tumor cells by detecting mCherry and CD206. (E) Bioluminescence assay of DU145-luc and 22RV1-luc. TAMs (THP-1 cells and PBMCs derived) in direct contact increased the luminescence values of DU145-luc and 22RV1-luc cells compared with those of the noncontact and control groups (left). (F) Quantification data of the cell luminescence value (right). (G) Matrigel-based Transwell invasion assay of DU145-luc and 22RV1-luc cells. TAMs (THP-1 and PBMC-derived) in direct contact increased the invasive ability of DU145-luc and 22RV1-luc cells compared with the noncontact and control groups. (H) The quantification data of invasive assay (right) (*p<0.05, **p<0.01).

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Flow Cytometry, Luciferase, Labeling, ATP Bioluminescent Assay, Derivative Assay, Transwell Invasion Assay

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: M2 macrophages contact activates NOTCH signaling in PCa cells. (A-C) RNA-sequence data of M2 macrophage contact (experimental group) and noncontact (control group). A, Gene cluster analysis of DU145-luc and 22RV1-luc. B, KEGG enrichment analysis of DU145-luc and 22RV1-luc showed that M2 macrophage direct contact increased NOTCH signaling pathway enrichment. C, GSEA showed that direct M2 macrophage contact promoted NOTCH-related gene enrichment. (D) Western blot assay of NOTCH signaling-related protein expression. M2 macrophages (THP-1 and PBMC-derived) in direct contact increased N1ICD, HES1, and HEY1 expression in DU145-luc and 22RV1-luc cells compared with that in the noncontact and control groups.

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Sequencing, Western Blot, Expressing, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: M2 macrophage contact activates EMT signaling in PCa cells. (A) GSEA showed that M2 macrophages direct contact promoted EMT-related gene enrichment in DU145-luc and 22RV1-luc cells. (B) Western blot assay of EMT signaling-related protein expression. TAMs (THP-1 and PBMC-derived) directly contacted increased N-cadherin, vimentin, and Snail and decreased E-cadherin expression in DU145-luc and 22RV1-luc cells compared with the noncontact and control groups. (C) Immunofluorescence assays showed that TAMs in direct contact increased N1ICD (far red) and vimentin (green) expression of DU145-luc and 22RV1-luc compared with the noncontact and control groups. (Scale bar=50 µm). (D) Western blot assay detected γ-secretase component protein expression. M2 macrophages (THP-1 and PBMC-derived) directly contact increases Nicastrin, PEN2, Presenilin1 and Presenilin2 expression in DU145-luc and 22RV1-luc cells compared with the noncontact and control groups.

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Derivative Assay, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: Inhibiting NOTCH1 singaling impaired M2 macrophages-mediated cell proliferation and invasion. (A) Bioluminescence assays showed that inhibiting NOTCH signalling impaired M2 macrophage direct contact-mediated DU145-luc and 22RV1-luc cell proliferation. (B) Matrigel-based Transwell invasion assays showed that γ-secretase inhibition in NOTCH1-depleted DU145-luc and 22RV1-luc cells abolished M2 macrophages direct contact-mediated cell invasive ability. (C) Western blot assay showed that inhibiting γ-secretase activity significantly reduced N1ICD, N-cadherin, vimentin and Snail expression in NOTCH1-depleted DU145-luc and 22RV1-luc cells (*p<0.05, **p<0.01).

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Inhibition, Western Blot, Activity Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: MAML2 is essential for TAM-mediated NOTCH1-dependent transcription. (A) Western blot assay showed that M2 macrophages (THP-1 and PBMC-derived) in direct contact increased MAML2 expression in DU145-luc and 22RV1-luc cells compared with the noncontact and control groups. (B) Lentivirus containing the shMAML2 fragment was infected into DU145 and 22RV1 cells, and Western blotting was used to detect the knockdown effect. (C) Bioluminescence assays showed that MAML2 depletion in DU145 and 22RV1 cells abolished M2 macrophages direct contact-mediated cell proliferation. (D) Matrigel-based invasion assays showed that MAML2 depletion in DU145 and 22RV1 cells abolished M2 macrophages direct contact-mediated cell invasion. (E) Immunofluorescence assays showed that MAML2 depletion inhibited M2 macrophages direct contact-induced EMT by decreasing N-cadherin (far-red) expression in DU145 and 22RV1 cells. (F) Western blot assay showed that knocking down MAML2 did not alter M2 macrophages-mediated N1ICD release but reduced HES1 and HEY1 expression (scale bar=50 µm, *p<0.05, **p<0.01).

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Western Blot, Derivative Assay, Expressing, Infection, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: M2 macrophages depletion and NOTCH1 signaling inhibition impaired PCa cell-derived xenograft growth. (A) Xenografts (n=5) were obtained from nude mice that were injected with 1×10 6 DU145-luc and 22RV1-luc cells (mixed with 2×10 5 THP-1-derived M2 macrophages), and pharm treatment was used (including PBS, DAPT, liposomal clodronate or combination) until tumor xenografts were formed. An IVIS-200 bioluminescence system was used to monitor tumor growth once a week, and node mice were sacrificed after 4 weeks. (B) Tumor xenografts were displayed as indicated. (C) The curve of tumor volume and weight of each group (n=5) was analyzed; group differences were analyzed via ANOVA. (D) Histological staining of CD31, Ki67 and N1ICD is displayed as indicated (*p<0.05, **p<0.01).

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Inhibition, Derivative Assay, Injection, Staining

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: M2 macrophages depletion and NOTCH1 signaling inhibition impaired PCa cell lung metastasis. (A) A lung metastasis model (n=3) was established by tail injection of 1×10 6 DU145-luc and 22RV1-luc cells (mixed with 2×10 5 THP-1-derived TAMs) into nude mice. Pharm treatment was used (including PBS, DAPT, liposomal clodronate or combination) once tail injection was finished. An IVIS-200 bioluminescence system was used to monitor lung metastasis once a week, and node mice were sacrificed after 6 weeks. (B) Quantification of lung metastasis nodes is displayed as indicated. (C) Metastatic lungs are displayed as indicated (left, fluorescence; right, brightness, n=3). (D) Alizarin red staining was used to observe the metastatic node of each group. (E) DU145-luc and 22RV1-luc cells (mixed with 2×10 5 THP-1-derived M2 macrophages) (1×10 6 ) were used to establish a lung metastasis model (n=3). Adenovirus carrying NOTCH1 shRNA was injected into mouse lung tissue until tumor metastasis formation. An IVIS-200 bioluminescence system was used to monitor lung metastasis diminishing. (F) Kaplan-Meier survival curves are displayed as indicated. (G) Intralung injection of shNOTCH1 adenovirus suppressed metastatic MET-associated tumor cell plantation (N-cadherin, far-red; E-cadherin, green). (H) Immunofluorescence assays showed that intralung injection of shNOTCH1 adenovirus significantly reduced F4/80 + CD206 + TAM recruitment (F4/80, far-red; CD206, green) (*p<0.05, **p<0.01).

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Inhibition, Injection, Derivative Assay, Fluorescence, Staining, shRNA, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway

doi: 10.7150/ijbs.73141

Figure Lengend Snippet: Recruitment of M2 macrophages in PCa predicts poor prognosis. Tissue immunofluorescence triple staining of CD68, CD206 and N1ICD revealed that CD68/CD206/N1ICD expression was positively associated with high TNM stage. (B) Cox regression analysis showed a significant difference in the CD68/CD206/N1ICD expression status of PCa (p<0.0001). (C) Multivariable Cox analysis including prostate cancer-relevant factors (pTNM stage) suggested that CD68/CD206/N1ICD expression was an independent marker for poor prognosis and that upregulation of N1ICD was associated with poor prognosis. (D) Kaplan-Meier survival analysis revealed that PCa patients with high CD68/CD206/N1ICD expression had the lowest survival rate. (E) Schematic illustration of TAMs directly contacting PCa cells.

Article Snippet: For PBMC-derived M2 macrophages, an appropriate amount of complete M2 macrophage Generation Medium DXF (Promo Cell, Heidelberg, Germany) was incubated with the cells according to the manufacturer's instructions.

Techniques: Immunofluorescence, Staining, Expressing, Marker

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: Relationship between pS118-ER and DNA binding. (A) Western blot analysis of ER and pS118-ER in MCF-7 cells treated with 10 nM E2 for various amounts of time. β-Actin is shown as a loading control. (B) Western blot analysis of MDA-MB-231 cells containing doxycycline-inducible wt ER or S118A ER. MDA-MB-231 cells were treated with 5.0 µg/ml (wt ER) or 0.5 µg/ml (S118A ER) dox for 24 h, followed by 30 min of treatment with vehicle (Veh; 0.1% EtOH) or 10 nM E2. β-Actin serves as a loading control. (C) ER ChIP-qPCR of MDA-MB-231 wt ER or S118A ER-expressing cells. Data are displayed as percentages of input. n = 3. Means ± standard deviations (SD) are shown. *, P < 0.05. (D) HEK293 cells transfected with either vector, wt ER, or the DNA binding ER mutant C202/205H were treated for 30 min with either vehicle or 10 nM E2 and analyzed via immunoblotting.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: pS118-ER ChIP-seq with multiple antibodies. (A) Venn diagrams displaying the overlap in ER or pS118-ER ChIP-seq sites between vehicle (0.1% EtOH)- and E2-treated MCF-7 cells. Three separate pS118-ER antibodies were used for ChIP-seq analysis and are denoted as pS118-ER #1, pS118-ER #2, and pS118-ER #3. (B) ChIP-qPCR validation of pS118-ER occupancy sites. The number after a gene name denotes the approximate distance, in base pairs if not otherwise specified, between the region tested and the transcription start site of the gene, with a negative number being upstream and a positive number being downstream. Data are displayed as a percentage of input. n = 3. Means ± SD are shown. *, P < 0.05 versus vehicle. (C) ChIP-seq track display from the UCSC Genome Browser showing ER and pS118-ER occupancy sites at the GREB1 promoter. For each track, vehicle-treated (yellow) and E2-treated samples (blue) are overlaid. Overlapping tracks are displayed in green. The y axis displays tag density normalized to 107 tags.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: ChIP-sequencing

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: (A) Combination of pS118-ER sites into one data set. E2 peaks from pS118-ER #2 and pS118-ER #3 ChIP-seq were combined into one peak set and overlapped with ER sites. Ab, antibody. (B) Location annotation of pS118-ER and ER occupancy sites. Sites were annotated to either a promoter region, intron, intergenic region, or other. A promoter region was defined as bp −1000 to bp +100 from a transcription start site. The outer ring displays the distribution of ER sites, and the inner ring displays the distribution of pS118-ER sites. (C) ER and pS118-ER sites were annotated to their nearest transcription start site and binned into various distances. (D) Average H3K27ac signal at ER and pS118-ER sites. H3K27ac data for MCF-7 cells treated with E2 were acquired from a previously published data set (58) (GEO accession no. GSE45822). (E) Volcano plot of RNA-seq data from MCF-7 cells treated with 1 nM E2 for 24 h acquired from a previous published data set (59) (GSE89888). Fold change represents E2 treated versus vehicle treated. Each dot represents a gene, and red dots are genes with a pS118-ER occupancy site within 100 kb of its transcription start site. The horizontal dashed line denotes a cutoff of significance at P = 0.001. (F) Proportion of genes upregulated or downregulated by E2 with a pS118-ER site within 100 kb of their respective transcription start sites.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: ChIP-sequencing, RNA Sequencing Assay

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: Motif analysis of ER and pS118-ER sites. (A) De novo motif analysis of ER sites and pS118-ER sites using HOMER. TF, associated transcription factor for each motif. (B) Differential motif analysis of pS118-ER sites. Enriched motifs were searched for using the CentriMo tool in the MEME suite (60). All ER sites (44,050) were used as control (background) sequences, and pS118-ER sites were used as primary input sequences. (C) Proportion of sites containing each specific motif from known motif analysis using HOMER. P values from chi-squared test are displayed when significant. (D) Distribution of specific motifs around ER and pS118-ER sites.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: SNAP array analysis reveals direct and indirect ER binding events. (A) Schematic for identifying direct and indirect binding events. If a site shows binding in both ChIP-seq and the SNAP array, it is categorized as a direct binding event. If a peak identified in ChIP-seq is not detected on the SNAP array, it is categorized as an indirect binding event. (B) Representative examples of a direct and indirect binding event. (Top) ChIP-seq signal from ER and pS118-ER at the TFF1 promoter and a site within the IGF1R gene. The color key and y axis are the same as those in Fig. 2C. (Bottom) Relative ER binding intensity at these regions on the SNAP array. Each region contains multiple DNA probes tiled across the region. (C) Breakdown of regions assayed on the SNAP array. Pie charts display the proportion of regions assayed on the SNAP array and present at either the pS118-ER or ER-only ChIP-seq sites. Analysis revealed that directly bound sites were more likely to be occupied by pS118-ER than ER. (D) Distribution of the number of ER binding events present in the SNAP array genomic regions assayed. The number of ER binding events was calculated for each genomic region and is displayed as a histogram. A relative ER binding intensity of 5 was used as a cutoff for a binding event. (E) Proportion of SNAP array regions present in ER or pS118-ER ChIP-seq sites. SNAP array regions were binned by the number of ER binding events, and these regions were overlapped with ER and pS118-ER sites.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: Binding Assay, ChIP-sequencing

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: Analysis of GRHL2 occupancy at pS118-ER sites. (A) Overlap of GRHL2 sites with pS118-ER sites and ER sites identified by ChIP-seq. (B) Average GRHL2 signal at ER and pS118-ER sites. GRHL2 ChIP-seq data were acquired from a previously published data set (61). (C and D) ChIP-qPCR analysis of ER, pS118-ER, and GRHL2 after 30 min of treatment with E2 at sites cooccupied by pS118-ER and GRHL2 (n = 4) (C) or occupied by GRHL2 only (D). IgG was used as a control. n = 3. Means ± standard errors (SE) are shown. *, P < 0.05.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: ChIP-sequencing