Journal: International Journal of Biological Sciences

Article Title: VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk

doi: 10.7150/ijbs.86085

Figure Lengend Snippet: VEGFA is highly expressed in M2 type TAMs and TNBC cells and generates TNBC cells with CSC phenotype. (A) Volcano plot representing the differentially expressed genes between THP-1 and M2-type macrophages. (B) Venn diagram representing the differentially expressed genes overlapping between the GEO database and GeneCard database. Blue: secretory factor gene sets in the GeneCard database. Red: differentially expressed genes between THP-1 monocytes and M2-like macrophages in the GEO database. (C) VEGFA expression in M0-, M1- and M2-type macrophages of breast cancer using the GEPIA2021 database. (D) Western blotting analysis of VEGFA expression in THP-1 cells and M0- and M2-type macrophages. (E) ELISA detection of the secretion of VEGFA in THP-1 cells and M0- and M2-type macrophages. (F-I) Migration and invasion of TNBC cells after coculture with the indicated conditional medium were determined by Transwell assay (200 ×). The error bar indicates the mean ± SD. (J) Western blotting of breast cancer stem cell markers (CD24, CD44, OCT-4, Nanog and SOX-2) in TNBC cells after coculture with the indicated conditioned medium in the presence or absence of 10 ng/ml hVEGF 165 . (K) VEGFA mRNA expression in nonpaired (left panel, adjacent noncancerous tissue n=113, cancer tissue n=1113) and paired (right panel, n = 113) breast cancer samples from the TCGA database. The error bar indicates the mean ± SD. (L) The secretion of VEGFA from different breast cancer cell lines was assessed by ELISA. (M) VEGFA expression in different breast cancer cell lines from the CCLE database. (N) Kaplan-Meier analysis to compare the OS (high n=298, low n=106) and RFS (high n=298, low n=548) of TNBC patients with high and low VEGFA mRNA expression using the TCGA database. (O-R) Representative images of the microspheres formed after treatment of TNBC cells with 10 ng/ml hVEGF 165 (Treatment). The number of microspheres was counted and plotted, and the percentage of microspheres with diameters of < 50 μm, 50-100 μm and > 100 μm was calculated and plotted (200 ×, scale bars = 100 μm). (S) Western blotting of breast cancer stem cell markers (CD24, CD44, OCT-4, Nanog and SOX-2) in TNBC cells after treatment with 10 ng/ml hVEGF 165 . (T-W) Representative images of the microspheres after VEGFA knockdown (shVEGFA) in TNBC cells. The number of microspheres was counted and plotted, and the percentage of microspheres with diameters of < 50 μm, 50-100 μm and > 100 μm was calculated and plotted (200 ×, scale bars = 100 μm). (X) Western blotting of breast cancer stem cell markers (CD24, CD44, OCT-4, Nanog and SOX-2) in TNBC cells after VEGFA knockdown. The images show representative data, and data are expressed as the mean ± SD of each group of cells from three separate experiments. n.s., no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the controls.

Article Snippet: The lentiviral vectors NRP-1 shRNA, VEGFA shRNA, GAPVD1 shRNA and nontargeting control were designed and synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, Knockdown

Journal: International Journal of Biological Sciences

Article Title: VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk

doi: 10.7150/ijbs.86085

Figure Lengend Snippet: VEGFA promotes the CSC phenotype via NRP-1. (A-B) The mRNA and protein levels of NRP-1 in different breast cancer cell lines were detected by RT‒qPCR and Western blotting. (C) NRP-1 mRNA expression in different breast cancer cell lines from the CCLE database. (D-G) Representative images of microspheres after NRP-1 knockdown (shNRP-1) in the presence or absence of 10 ng/ml hVEGF 165 . The number of microspheres was counted and plotted, and the percentage of microspheres with diameters of < 50 μm, 50-100 μm and > 100 μm was calculated and plotted (200 ×, scale bars = 100 μm). (H) Western blotting of breast cancer stem cell markers (CD24, CD44, OCT-4, Nanog and SOX-2) in TNBC cells after NRP-1 knockdown in the presence or absence of 10 ng/ml hVEGF 165 . The images show representative data, and data are expressed as the mean ± SD of each group of cells from three separate experiments. n.s., no significance, ** P < 0.01, **** P < 0.0001 vs. the controls.

Article Snippet: The lentiviral vectors NRP-1 shRNA, VEGFA shRNA, GAPVD1 shRNA and nontargeting control were designed and synthesized by GeneChem (Shanghai, China).

Techniques: Western Blot, Expressing, Knockdown

Journal: International Journal of Biological Sciences

Article Title: VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk

doi: 10.7150/ijbs.86085

Figure Lengend Snippet: GAPVD1 interacts with NRP-1 and is regulated by the VEGFA/NRP-1 axis. (A) GAPVD1 mRNA expression in nonpaired (left panel, adjacent noncancerous tissue n=113, cancer tissue n=1113) and paired (right panel, n=113) breast cancer samples from the TCGA database. The error bar indicates the mean ± SD. (B) GAPVD1 protein expression in breast cancer tissue from the HPA database. (C) Correlation between GAPVD1 and NRP-1, ALDH1, CD44, and CD24 mRNA expression in human breast cancer samples from the TCGA dataset. (D-E) Coimmunoprecipitation of NRP-1 with the GAPVD1 antibody from TNBC whole-cell extracts. Precipitation with normal rabbit IgG was used as a negative control. (F) Western blotting of GAPVD1 after treatment with 10 ng/ml hVEGF 165 in TNBC cells. (G) Western blotting of GAPVD1 after VEGFA knockdown in TNBC cells. (H) Western blotting of GAPVD1 after NRP-1 knockdown in the presence or absence of 10 ng/ml hVEGF 165 . The images show representative data, and data are expressed as the mean ± SD of each group of cells from three separate experiments. n.s., no significance, * P < 0.05, ** P < 0.01 vs. the controls.

Article Snippet: The lentiviral vectors NRP-1 shRNA, VEGFA shRNA, GAPVD1 shRNA and nontargeting control were designed and synthesized by GeneChem (Shanghai, China).

Techniques: Expressing, Negative Control, Western Blot, Knockdown

Journal: International Journal of Biological Sciences

Article Title: VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk

doi: 10.7150/ijbs.86085

Figure Lengend Snippet: The VEGFA/NRP-1 axis promotes TNBC cell progression and stemness via GAPVD1. (A-D) Representative images of microspheres after GAPVD1 knockdown (shGAPVD1#2 and shGAPVD1#3) in TNBC cells. The number of microspheres was counted and plotted, and the percentage of microspheres with diameters of < 50 μm, 50-100 μm and > 100 μm was calculated and plotted (200 ×, scale bars = 100 μm). (E) Western blotting analysis of breast cancer stem cell markers (CD24, CD44, OCT-4, Nanog and SOX-2) in TNBC cells after GAPVD1 knockdown. (F-I) Representative images of microspheres after GAPVD1 overexpression (OE GAPVD1) in control and NRP-1-silencing TNBC cells. The number of microspheres was counted and plotted, and the percentage of microspheres with diameters of < 50 μm, 50-100 μm and > 100 μm was calculated and plotted (200 ×, scale bars = 100 μm). (J) Western blotting analysis of breast cancer stem cell markers (CD24, CD44, OCT-4, Nanog and SOX-2) after GAPVD1 overexpression in control and NRP-1-silencing TNBC cells. The images show representative data, and data are expressed as the mean ± SD of each group of cells from three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the controls.

Article Snippet: The lentiviral vectors NRP-1 shRNA, VEGFA shRNA, GAPVD1 shRNA and nontargeting control were designed and synthesized by GeneChem (Shanghai, China).

Techniques: Knockdown, Western Blot, Over Expression, Control

Journal: International Journal of Biological Sciences

Article Title: VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk

doi: 10.7150/ijbs.86085

Figure Lengend Snippet: The VEGFA/NRP-1/GAPVD1 axis targeted the downstream Wnt/β-catenin signaling pathway. (A) Western blotting analysis of β-catenin and Wnt/β-catenin downstream targets in TNBC cells after treatment with 10 ng/ml hVEGF 165 . (B) Western blotting analysis of β-catenin and Wnt/β-catenin downstream targets in TNBC cells after VEGFA knockdown (shVEGFA). (C) Western blotting analysis of β-catenin and Wnt/β-catenin downstream targets in TNBC cells after NRP-1 knockdown (shNRP-1) in the presence or absence of 10 ng/ml hVEGF 165 . (D) Western blotting analysis of β-catenin and Wnt/β-catenin downstream targets in TNBC cells after GAPVD1 knockdown (shGAPVD1#2 and shGAPVD1#3). (E) Western blotting analysis of β-catenin and Wnt/β-catenin downstream targets in control and NRP-1-silencing TNBC cells after GAPVD1 overexpression (OE GAPVD1). The images show representative data, and data are expressed as the mean ± SD of each group of cells from three separate experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001 vs. the controls.

Article Snippet: The lentiviral vectors NRP-1 shRNA, VEGFA shRNA, GAPVD1 shRNA and nontargeting control were designed and synthesized by GeneChem (Shanghai, China).

Techniques: Western Blot, Knockdown, Control, Over Expression

Journal: International Journal of Biological Sciences

Article Title: VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk

doi: 10.7150/ijbs.86085

Figure Lengend Snippet: TNBC cell-derived VEGFA promotes TAM polarization into M2 type and schematic of the crosstalk between TAMs and TNBC cells. (A-B) Flow cytometry analysis of the expressions of M2 macrophage markers (CD163 and CD206) after co-cultured with conditional medium from control TNBC cells or VEGFA-silencing TNBC cells. (C) RT-qPCR analysis of M1-related markers (CD80 and CD86) and M2-related markers (CD163, CD206 and IL-10) expression after co-cultured with conditional medium from control TNBC cells or VEGFA-silencing TNBC cells. The images show representative data, and data are expressed as the mean ± SD of each group of cells from three separate experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001 vs. the controls. (D) Schematic of the crosstalk between TAMs and TNBC cells mediated by VEGFA in the promotion of breast cancer stemness. VEGFA (secreted by TAMs in a paracrine manner and by TNBC cells in an autocrine manner) binds to NRP-1 and activates the downstream GAPVD1/Wnt/β-catenin signaling pathway to promote the stemness of TNBC. Additionally, VEGFA may provide an immunosuppressive microenvironment for tumor progression by recruiting TAMs and facilitating the M2 polarization of TAMs.

Article Snippet: The lentiviral vectors NRP-1 shRNA, VEGFA shRNA, GAPVD1 shRNA and nontargeting control were designed and synthesized by GeneChem (Shanghai, China).

Techniques: Derivative Assay, Flow Cytometry, Cell Culture, Control, Quantitative RT-PCR, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing

doi: 10.1155/2015/162145

Figure Lengend Snippet: TRITC labeled phalloidin staining showing actin cytoskeleton of VSMCs. Under the excitation wavelength of 488 nm, the cytoskeletons of control and Notch3 siRNA VSMCs were reddish-yellow and the GFP dots were green in the cytoplasm. Notch3 siRNA VSMCs are more rounded and irregular and lack actin cytoskeleton. Upon treatment with PNS, VSMCs became regular.

Article Snippet: To generate adenovirus transfection particles, a set of 4pAd/CMV/V5-DEST vectors (Invitrogen) encoding siRNA targeting the Notch3 gene (Gen Bank accession number NM_000435.2, mRNA, 8089 bp) and a nontarget siRNA control vector (TRC1 library, Sigma-Aldrich, St. Louis, MO, USA) were used to cotransfect HEK293A cells (Invitrogen) using pDONR221 (Invitrogen).

Techniques: Labeling, Staining

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing

doi: 10.1155/2015/162145

Figure Lengend Snippet: Effects of PNS on α -SM-actin in VSMCs transfected with a Notch3 siRNA. α -SM-actin was examined with Western blot. The data represent mean ± S.D. ∗∗∗∗ P < 0.0001 versus Notch3 siRNA VSMCs.

Article Snippet: To generate adenovirus transfection particles, a set of 4pAd/CMV/V5-DEST vectors (Invitrogen) encoding siRNA targeting the Notch3 gene (Gen Bank accession number NM_000435.2, mRNA, 8089 bp) and a nontarget siRNA control vector (TRC1 library, Sigma-Aldrich, St. Louis, MO, USA) were used to cotransfect HEK293A cells (Invitrogen) using pDONR221 (Invitrogen).

Techniques: Transfection, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing

doi: 10.1155/2015/162145

Figure Lengend Snippet: Effects of PNS on SM22 α in VSMCs transfected with a Notch3 siRNA. SM22 α was examined by Western blot. The data represent mean ± S.D. ∗∗∗∗ P < 0.0001 or ∗ P < 0.05 versus Notch3 siRNA VSMCs.

Article Snippet: To generate adenovirus transfection particles, a set of 4pAd/CMV/V5-DEST vectors (Invitrogen) encoding siRNA targeting the Notch3 gene (Gen Bank accession number NM_000435.2, mRNA, 8089 bp) and a nontarget siRNA control vector (TRC1 library, Sigma-Aldrich, St. Louis, MO, USA) were used to cotransfect HEK293A cells (Invitrogen) using pDONR221 (Invitrogen).

Techniques: Transfection, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Panax notoginseng Saponins Attenuate Phenotype Switching of Vascular Smooth Muscle Cells Induced by Notch3 Silencing

doi: 10.1155/2015/162145

Figure Lengend Snippet: Effects of PNS on OPN in VSMCs transfected with a Notch3 siRNA. OPN was examined with Western blot. The data represent mean ± S.D. ∗∗∗∗ P < 0.0001 versus Notch3 siRNA VSMCs.

Article Snippet: To generate adenovirus transfection particles, a set of 4pAd/CMV/V5-DEST vectors (Invitrogen) encoding siRNA targeting the Notch3 gene (Gen Bank accession number NM_000435.2, mRNA, 8089 bp) and a nontarget siRNA control vector (TRC1 library, Sigma-Aldrich, St. Louis, MO, USA) were used to cotransfect HEK293A cells (Invitrogen) using pDONR221 (Invitrogen).

Techniques: Transfection, Western Blot