Journal: Scientific Reports

Article Title: The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes

doi: 10.1038/s41598-020-65353-x

Figure Lengend Snippet: HE4 regulates immune-related gene expression in peripheral blood mononuclear cells ( A,B ) Scatter plots of gene expression determined by quantitative PCR array in control or rHE4-treated PBMC. qPCR was performed to validate genes changes, revealing upregulation of CSF3 , IL8 , and HIF1A with rHE4 treatment of PBMC (C–E) . No change in STAT3 or VEGFA levels were observed with rHE4 treatment (F,G) . Error bars represent standard deviation. *p < 0.05. Results are the average of at least three biological replicates.

Article Snippet: Uncropped blots can be seen in Supplemental Fig. . Antibodies and dilutions used are as follows: GAPDH (Cell Signaling, 2118, 1:2000) Phospho-STAT3 (Cell Signaling, 9131, 1:1000) STAT3 (Cell Signaling, 30835, 1:1000) HE4 (Origene, TA307787, 1:2000) HIF1A (Novus, NB-100–134SS, 1:200)

Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Scientific Reports

Article Title: The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes

doi: 10.1038/s41598-020-65353-x

Figure Lengend Snippet: HE4-mediated upregulation of IL8 and HIF1A gene expression is suppressed by STAT3 inhibition in PBMC ( A ) qPCR revealed upregulation of HIF1A in PBMC treated with rHE4 for 6 h, which was blocked by treatment with a STAT3 inhibitor. (B) Upregulation of IL8 in PBMC treated with rHE4 for 6 h, which was blocked by treatment with a STAT3 inhibitor. (C) Upregulation of HIF1A in PBMC treated with rHE4 for 24 h, which was blocked by treatment with a STAT3 inhibitor. (D) Upregulation of IL8 in PBMC treated with rHE4 for 24 h, which was blocked by treatment with a STAT3 inhibitor. Error bars represent standard deviation. *p < 0.05. Results are the average of ≥3 biological replicates.

Article Snippet: Uncropped blots can be seen in Supplemental Fig. . Antibodies and dilutions used are as follows: GAPDH (Cell Signaling, 2118, 1:2000) Phospho-STAT3 (Cell Signaling, 9131, 1:1000) STAT3 (Cell Signaling, 30835, 1:1000) HE4 (Origene, TA307787, 1:2000) HIF1A (Novus, NB-100–134SS, 1:200)

Techniques: Expressing, Inhibition, Standard Deviation

Journal: Scientific Reports

Article Title: The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes

doi: 10.1038/s41598-020-65353-x

Figure Lengend Snippet: HE4-mediated STAT3 activation and upregulation of IL8 and HIF1A is blocked by STAT3 inhibition ( A ) Upregulation of phospho-STAT3 in SKOV3 cells, HUVECs, and PBMCs by rHE4 treatment. Upregulation of HIF1α by rHE4 observed in HUVECs and PBMC. STAT3 activation and HIF1A levels were ablated by treatment with a STAT3 inhibitor. Boxes separate images from the same gel for each cell type. Blots were either subsequently reprobed for various antibodies, or stripped and reprobed in the case of STAT3/p-STAT3. (B) ELISA revealed increased secretion of IL8 by PBMC treated with 20 nM rHE4 from 4–24 h post-treatment, which was suppressed by STAT3 inhibition. (C) ELISA revealed increased secretion of IL8 by PBMC treated with 1 nM rHE4 from 4–24 h post-treatment, which was suppressed by STAT3 inhibition. (D) ELISA revealed IL8 secretion by PBMC was increased as early as 2 h post-treatment with rHE4. Error bars represent standard deviation of ≥3 biological replicates. *p < 0.05, **p < 0.005, ***p < 0.005, ****p < 0.0005.

Article Snippet: Uncropped blots can be seen in Supplemental Fig. . Antibodies and dilutions used are as follows: GAPDH (Cell Signaling, 2118, 1:2000) Phospho-STAT3 (Cell Signaling, 9131, 1:1000) STAT3 (Cell Signaling, 30835, 1:1000) HE4 (Origene, TA307787, 1:2000) HIF1A (Novus, NB-100–134SS, 1:200)

Techniques: Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Scientific Reports

Article Title: The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes

doi: 10.1038/s41598-020-65353-x

Figure Lengend Snippet: HE4-mediated tube formation of endothelial cells is blocked by STAT3 inhibition ( A ) rHE4 promotes tube formation of HUVECs, and STAT3 inhibition blocks tube formation in rHE4-treated cells. Top panel shows representative 4x images with tube features outlined in blue and tube numbers labeled. Bottom panel shows representative 10x images. (B) Quantification of tube formation from ( A ). Error bars represent standard deviation of at least three biological replicates. *p < 0.05, **p < 0.005.

Article Snippet: Uncropped blots can be seen in Supplemental Fig. . Antibodies and dilutions used are as follows: GAPDH (Cell Signaling, 2118, 1:2000) Phospho-STAT3 (Cell Signaling, 9131, 1:1000) STAT3 (Cell Signaling, 30835, 1:1000) HE4 (Origene, TA307787, 1:2000) HIF1A (Novus, NB-100–134SS, 1:200)

Techniques: Inhibition, Labeling, Standard Deviation

Journal: Scientific Reports

Article Title: The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes

doi: 10.1038/s41598-020-65353-x

Figure Lengend Snippet: ( A ) HE4 and IL8 tissue levels are elevated in serous EOC compared to normal adjacent tissue (NAT) ( B ) HE4 and IL8 tissue levels are elevated in serous EOC at all stages compared to NAT. (C) Representative images of HE4 and IL8 staining in EOC tissue and NAT. (D) Tissue HE4 and IL8 levels (mean intensity) are positively correlated in serous EOC. (E) CD8 + T cells in serous EOC tissue. (F) Inverse correlation between HE4 serum levels and CD8 + T cell counts in patient EOC tissue (n = 26). (G) CD34 + microvascular staining in EOC tissue. (H) Correlation between HE4 serum levels and CD34 + area in patient EOC tissue (n = 14). **p < 0.005, ****p < 0.0005.

Article Snippet: Uncropped blots can be seen in Supplemental Fig. . Antibodies and dilutions used are as follows: GAPDH (Cell Signaling, 2118, 1:2000) Phospho-STAT3 (Cell Signaling, 9131, 1:1000) STAT3 (Cell Signaling, 30835, 1:1000) HE4 (Origene, TA307787, 1:2000) HIF1A (Novus, NB-100–134SS, 1:200)

Techniques: Staining