Fig 1: Inhibition of tumour-derived LAMA5 inhibits the growth of colorectal liver metastases and promotes endothelial Notch signalling. (A) Western blotting for LAMA5 in Hct-116 and HT29 colon cancer cell lines transfected with non-targeting control (ctrl) or one of two LAMA5 shRNA constructs (sh1 or sh2). (B) Liver metastasis growth in Hct-116 ctrl and Hct-116-sh1 or Hct-116-sh2 cell lines. (C) Liver metastasis growth in HT29ctrl and HT29-sh1 or HT29-sh2 cell lines. (D) Immunofluorescence staining for the human LAMA5 protein in liver metastases developed using HT29ctrl or HT29-sh2 cell lines. Scale bars represent 40 µm. (E) Immunofluorescence staining for CD31 in liver metastases developed using HT29ctrl or HT29-sh2 cell lines. Scale bar represents 40 µm. (F) Quantification of total CD31+ area, total and average vessel length and vessel junction density in liver metastases developed using HT29ctrl or HT29-sh2 cell lines. (G) Immunofluorescence staining for vessel perfusion (tail vein injection of anti-CD31) in red and endothelial cells (immunohistochemistry for CD31) in green in HT29ctrl and HT29-sh2 liver metastases. Scale bars represent 40 µm. (H) Quantification of the percentage of perfused vessels in liver metastases developed using HT29ctrl and HT29-sh2 cell lines. (I) RNA expression of endothelial and immune cell genes in endothelial (CD31+) and non-endothelial (CD31neg) cells MACS-separated from liver metastases generated in mice using HT29ctrl or HT29-sh2 cell lines. (J) Endothelial cell expression of genes involved in the regulation of angiogenesis presented as fold-change in expression in CD31+ cells MACS-separated from HT29-sh2 metastases relative to those from HT29ctrl metastases. (K) Western blotting for Hey2 protein in lysates derived from hepatic metastases developed using HT29ctrl or HT29-sh2 cell lines. (L) Blotting for LAMA5 in matrices derived from HT29 control and shLAMA5 cells following 7-day serum-starved culture with and without TNFa supplementation at 100 ng/mL. (M) Representative immunocytochemistry images of 2H11 endothelia following culture on matrices from (L) stained for the indicated Notch pathway proteins. (N,O) Quantification of HEY2 and NCID nuclear staining intensity in endothelia from (M). Analysis performed on at least 40 cells across experiments in triplicate. Student’s t-test throughout. p < 0.05 considered significant. * 0.01 < p = 0.05, ** 0.001 < p = 0.01, *** p = 0.001, ns: not significant.
Fig 2: SETD1A activates Notch signaling pathway. A Volcano plot of differently changed genes associated with SETD1A. B KEGG pathway of differently changed genes associated with SETD1A. C The protein levels of Nothc1, Hes1, Hey1, Hey2, and Heyl in SETD1A overexpression RMGI and OVCAR5 cells analyzed by western blotting assays. D,E The mRNA levels of Notch1, Hes1, Hey1, Hey2, and Heyl in SETD1A overexpression RMGI D and OVCAR5 E cells analyzed by RT-PCR assays. Unpaired two-tailed t-test. Data are expressed as mean ± SD. ***p < 0.001, ****p < 0.0001. F,G The protein levels of Nothc1, Hes1, Hey1, Hey2, and Heyl in SETD1A knockdown SKOV3 F and Caov3 G cells analyzed by western blotting assays. H,I The mRNA levels of Notch1, Hes1, Hey1, Hey2, and Heyl in SETD1A knockdown SKOV3 H and Caov3 I cells analyzed by RT-PCR assays. Unpaired two-tailed t-test. Data are expressed as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 3: Knockdown of SETD1A suppresses OV cell tumorigenesis. A,B The tumor size A and weight B in SETD1A knockdown and control SKOV3 xenograft tumor (n = 6 per group). Unpaired two-tailed t-test. Data are expressed as mean ± SD. ****p < 0.0001. C The numbers of Ki67-positive tumor cell in SETD1A knockdown and control SKOV3 xenograft tumor tissue sections analyzed by IHC staining. Unpaired two-tailed t-test. Data are expressed as mean ± SD. ***p < 0.001. D The mRNA levels of Notch target genes, including Hes1, Hey1, Hey2, and Heyl in SETD1A knockdown and control SKOV3 xenograft tumor. Unpaired two-tailed t-test. Data are expressed as mean ± SD. ** p < 0.01, ***p < 0.001. E The proposed model of SETD1A promoting OV development
Fig 4: SETD1A increases H3K4me3 levels at the promoters of Hes1 to enhance Notch transactivation. A-C The recruitments of Notch1 A, SETD1A B, and the H3K4me3 levels C at Hes1 promoter in JAG overexpression SKOV3 cells analyzed by ChIP assays. Unpaired two-tailed t-test. Data are expressed as mean ± SD. **p < 0.01, ***p < 0.001. D-F The recruitments of SETD1A D and Notch F, and the H3K4me3 levels E at Hes1 promoters in SETD1A knockdown SKOV3 cells analyzed by ChIP assays. Unpaired two-tailed t-test. Data are expressed as mean ± SD. **p < 0.01, ***p < 0.001. G The protein levels of Notch1 and its target genes (Hes1, Hey1, Hey2 and Heyl) in JAG1 overexpressed SETD1A knockdown SKOV3 cells analyzed by western blotting assays. H-J The recruitments of Notch1 H, SETD1A I and the H3K4me3 levels J at Hes1 promoters in JAG1 overexpressed SETD1A knockdown SKOV3 cells analyzed by ChIP assays. Unpaired two-tailed t-test. Data are expressed as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001
Supplier Page from Abcam for Anti-HEY2 antibody