Journal: Cell death & disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression.

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: Fig. 3 B7-H3 promoted the expression of VEGFA in CRC. a The expression of angiogenesis-related genes was detected by RT-qPCR in shB7-H3 HCT116 and RKO cells. b Western blot analysis of B7-H3 and VEGFA in the sh-NC and shB7-H3 CRC cell lines. β-actin served as a loading control. c Representative images of IHC for VEGFA in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. d VEGFA protein expression based on the staining index of CRC specimens and matched normal tissues. e VEGFA protein expression is shown for patients stratified into B7-H3 low (median value) groups. f Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens (n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. NS no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After antigen retrieval with 10mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: Cell death & disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression.

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: Fig. 6 B7-H3 promoted angiogenesis via NF-κB/VEGFA pathway in Matrigel plugs in vivo. a, b CD31 (a) and VEGFA (b) protein expression based on their IHC staining index results in subcutaneous tumors formed by sh-NC-HCT116 and shB7-H3-HCT116 cells. N = 5. c, d CD31 (c) and VEGFA (d) protein expression based on their IHC staining index results in subcutaneous tumors formed by EV-HCT116 and B7-H3-HCT116 cells. N = 5. e, f CD31 (e) and VEGFA (f) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with BAY11–7082 (6 mg/kg). g, h CD31 (g) and VEGFA (h) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with bevacizumab (1 mg/kg). i, j CD31 (i) and VEGFA (j) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors co-treated with 3E8 (5 mg/kg) and BAY11–7082 (6 mg/kg) or bevacizumab (1 mg/kg). N = 5. The data represent the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After antigen retrieval with 10mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500).

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: iRPE cells have similar phenotype and functions as iPSC-RPE cells (A) Schematic for the transforming process. A cocktail of TF-expressing retroviruses was used to transfect De-iPSC-RPE cells. After seven days, iRPE clone was observed in the culture and picked out for subculturing. (B and C) RPE-specific and EMT-associated markers detected by (B) immunostaining and (C) western blotting after cells were cultured for 8 days. The expression pattern of these markers in iRPE cells is more similar to that in iPSC-RPE cells. Scale bar = 50 μm. (D) Electron micrographs of iPSC-RPE cells, De-iPSC-RPE cells, and iRPE cells demonstrated that quite a few microvilli were on the surface of iRPE and iPSC-RPE cells. Scale bar = 0.5 μm. (E and F) The bound and phagocyted POSs (pointed by arrows) in iPSC-RPE, De-iPSC-RPE, and iRPE cells (E) and quantification of phagocytosis (F) as determined by the number of bound and phagocyted POS per field. Scale bar = 50 μm. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n ≥ 5. (G and H) TER analysis (G) and HRP permeability assay (H) showed that iRPE cells maintained the same epithelial integrity as iPSC-RPE cells. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 6. (I) Expression levels of PEDF and VEGF from upper and lower chambers were determined by ELISA. iRPE cells and iPSC-RPE cells demonstrated similar secretion patterns. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Expressing, Subculturing Assay, Immunostaining, Western Blot, Cell Culture, Permeability, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: BMP7 and FOXF2 are critical regulators of EMT in iRPE cells (A) The higher level of BMP7 secreted by iRPE cells compared with De-iPSC-RPE cells was determined by ELISA. Data are mean ± SD, unpaired two-sided t-tests, n = 4. (B and C) The reduced expression level of FOXF2 in iRPE compared with that in De-iPSC-RPE cells was determined by (B) immunostaining and (C) western blotting. Scale bar = 50 μm. (D) The efficiency of bmp7 knockdown was determined by qRT-PCR; shBmp7-1 was slightly more efficient at reducing the mRNA level of bmp7 than shBmp7-2; therefore, it was selected for subsequent experiments. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (E) The shBmp7 construct contained the ZsGreen expression element to indicate successful transfection. Scale bar = 50 μm. (F and G) FLAG-FOXF2 overexpression (ov-FOXF2) in (F) iRPE cells (G) shBmp7-iRPE cells. Scale bar = 50 μm. (H and I) The expression levels of RPE-specific markers and EMT markers were determined by (H) western blotting and (I) quantitative analysis. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3. (J) BMP7 expression levels in shCont-iRPE, shBmp7-iRPE, ov-FOXF2- iRPE, and shBmp7 + ov-FOXF2-iRPE. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (K) The immunostaining of RPE-specific markers and EMT markers. Overexpression of FOXF2 exhibited similar effects to knockdown of bmp7 in iRPE cells by downregulating RPE markers and upregulating EMT marker. Scale bar = 50 μm.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunostaining, Western Blot, Knockdown, Quantitative RT-PCR, Construct, Transfection, Over Expression, Marker

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: Four TFs transcriptionally regulate bmp7 , foxf2 , lin7a , pard6b , and ppm1a in a direct or indirect manner (A) ELISA analysis demonstrated that BMP7 levels was reduced in 4TFs−nr2e1-RPE, 4TFs−mitf-a-RPE, 4TFs−c-myc-RPE, and 4TFs−crx-RPE cells compared with that in iRPE cells. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (B and C) The levels of FOXF2, LIN7A, PARD6B, and PPM1A were determined by (B) western blotting and (C) quantitative analysis. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3. (D) Generation of FLAG-MITF-A-iRPE, FLAG-CRX-iRPE, FLAG-NR2E1-iRPE, and FLAG-C-MYC-iRPE cells. Scale bar = 50 μm. (E–I) Enriched peaks of CRX binding to the target genes and fold enrichment of CRX immunoprecipitation compared with IgG control, as determined by qRT-PCR. Data are mean ± SD, unpaired two-sided t-tests, n = 3. (J–M) Enriched peaks of MITF-A and NR2E1 binding to the target gene lin7a and fold enrichment of MITF-A and NR2E1 immunoprecipitation compared with IgG control, as determined by qRT-PCR. Data are mean ± SD, unpaired two-sided t-tests, n = 3. (N) Schematic model for the regulation of EMT and MET processes by the four TFs. CRX, MITF-A, NR2E1, and C-MYC directly or indirectly regulated the expression of bmp7 , lin7a , pard6b, ppm1a , and foxf2 to inhibit EMT and promote MET.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Immunoprecipitation, Control, Quantitative RT-PCR, Expressing

Journal: Stem Cells International

Article Title: Heme Oxygenase-1-Modified Bone Marrow Mesenchymal Stem Cells Combined with Normothermic Machine Perfusion Repairs Bile Duct Injury in a Rat Model of DCD Liver Transplantation via Activation of Peribiliary Glands through the Wnt Pathway

doi: 10.1155/2021/9935370

Figure Lengend Snippet: Transformation of PBGs from a static phenotype to an active phenotype. (a) The morphology of PBGs in the sham group was intact, and cells were round with large and round nuclei located in the center of the cells (white ellipse). The morphology of PBGs in the NMP group was destroyed (red ellipse), and continuity was broken, with necrotic cells. PBGs were relatively well preserved in the BP and HBP groups, which were expanded, and a large number of proliferating cells appeared at the connecting tubes between the PBGs and the lumen of the bile ducts (blue ellipse). (b) Relative protein levels of VEGF, PCNA, caspase-3, and cleaved caspase-3 in each group were detected using western blotting ( n = 3). (c) Immunofluorescence staining showed that PBGs expressed VEGFR1 and VEGFR2. (d) Expression of PCNA and caspase-3 in each group was marked by immunohistochemistry staining. Compared with the sham group (5.50 ± 2.29%), the number of PCNA-positive cells was higher in the NMP group (18.90 ± 3.00%, P = 0.0002) and even higher in the BP group (37.10 ± 4.64%, P < 0.0001 vs. the NMP group). The increase in the HBP group was the most significant (53.50 ± 4.24%, P < 0.0001 vs. the BP group). Compared with the sham group (2.70 ± 1.48%), numbers of caspase-3-positive cells (56.90 ± 5.34%, P < 0.0001) were significantly increased in the NMP group and decreased in the BP group (42.30 ± 3.96%, P < 0.0001 vs. the NMP group) and further decreased in the HBP group (35.10 ± 2.66%, P = 0.0306 vs. the BP group). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. PBG: peribiliary gland; NMP: normothermic machine perfusion; HBP: HO-1/BMMSCs plus NMP; BP: BMMSCs plus NMP; VEGF: vascular endothelial growth factor; PCNA: proliferating cell nuclear antigen; VEGFR1: vascular endothelial growth factor receptor 1; VEGFR2: vascular endothelial growth factor receptor 2.

Article Snippet: The present study used Dulbecco's modified eagle medium (DMEM)/F12 medium (Solarbio, Beijing, China); fetal bovine serum (Biowest, Loire Valley, France); BMMSC surface marker-related antibodies (anti-rat cluster of differentiation 34- (CD34-) fluorescein isothiocyanate (FITC), anti-rat CD29- (integrin subunit beta 1-) phycoerythrin (PE), anti-rat protein tyrosine phosphatase receptor type C- (CD45-) PE, anti-rat CD90- (Thy-1 cell surface antigen-) FITC, anti-rat RT1A- (rat MHC class I antibody-) PE, and anti-rat RT1B- (rat MHC class II antibody-) FITC (BioLegend, San Diego, CA, USA)); adipogenic and osteogenic differentiation medium (Sigma-Aldrich, St. Louis, MO, USA); Oil Red O (Dingguo Changsheng Biotechnology, Beijing, China); von Kossa cell staining kit (Genmed, Shanghai, China); rat green fluorescent protein genomic adenovirus (GFP-Adv, GeneChem, Shanghai, China); mouse antibodies recognizing cystic fibrosis transmembrane conductance regulator (CFTR) (Santa Cruz, Dallas, TX, USA), SRY-box transcription factor 9 (SOX9) (Santa Cruz), Nanog (Santa Cruz), proliferating cell nuclear antigen (PCNA) (Santa Cruz), and Wnt3 (Santa Cruz); rabbit antibodies recognizing β -catenin (Santa Cruz), vascular endothelial growth factor (VEGF) (Proteintech, Wuhan, China), and occludin (Proteintech); tight junction protein 1 (TJP1, also known as ZO-1) (Proteintech, Wuhan, China), caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and active β -catenin (Cell Signaling Technology); β -actin mouse antibodies (Beyotime, Shanghai, China); and XAV-939 (MedChemExpress, Monmouth Junction, NJ USA).

Techniques: Transformation Assay, Western Blot, Immunofluorescence, Staining, Expressing, Immunohistochemistry

Journal: Stem Cells International

Article Title: Heme Oxygenase-1-Modified Bone Marrow Mesenchymal Stem Cells Combined with Normothermic Machine Perfusion Repairs Bile Duct Injury in a Rat Model of DCD Liver Transplantation via Activation of Peribiliary Glands through the Wnt Pathway

doi: 10.1155/2021/9935370

Figure Lengend Snippet: Changes in the proliferative activity and stemness of PBG cells after inhibition of the Wnt signal pathway. (a) Relative levels of VEGF, PCNA, caspase-3, and cleaved caspase-3 in each group were detected using western blotting ( n = 3). Relative levels of Nanog, SOX9, and CFTR in each group were detected by western blotting (b) and PCR (c) ( n = 3). Immunofluorescence staining showed that XAV-939 effectively degraded β -catenin and downregulated expression of Nanog (d) and VEGF (e) at the same time. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. PBG: peribiliary gland; VEGF: vascular endothelial growth factor; PCNA: proliferating cell nuclear antigen; SOX9: SRY-box transcription factor 9; CFTR: cystic fibrosis transmembrane conductance regulator.

Article Snippet: The present study used Dulbecco's modified eagle medium (DMEM)/F12 medium (Solarbio, Beijing, China); fetal bovine serum (Biowest, Loire Valley, France); BMMSC surface marker-related antibodies (anti-rat cluster of differentiation 34- (CD34-) fluorescein isothiocyanate (FITC), anti-rat CD29- (integrin subunit beta 1-) phycoerythrin (PE), anti-rat protein tyrosine phosphatase receptor type C- (CD45-) PE, anti-rat CD90- (Thy-1 cell surface antigen-) FITC, anti-rat RT1A- (rat MHC class I antibody-) PE, and anti-rat RT1B- (rat MHC class II antibody-) FITC (BioLegend, San Diego, CA, USA)); adipogenic and osteogenic differentiation medium (Sigma-Aldrich, St. Louis, MO, USA); Oil Red O (Dingguo Changsheng Biotechnology, Beijing, China); von Kossa cell staining kit (Genmed, Shanghai, China); rat green fluorescent protein genomic adenovirus (GFP-Adv, GeneChem, Shanghai, China); mouse antibodies recognizing cystic fibrosis transmembrane conductance regulator (CFTR) (Santa Cruz, Dallas, TX, USA), SRY-box transcription factor 9 (SOX9) (Santa Cruz), Nanog (Santa Cruz), proliferating cell nuclear antigen (PCNA) (Santa Cruz), and Wnt3 (Santa Cruz); rabbit antibodies recognizing β -catenin (Santa Cruz), vascular endothelial growth factor (VEGF) (Proteintech, Wuhan, China), and occludin (Proteintech); tight junction protein 1 (TJP1, also known as ZO-1) (Proteintech, Wuhan, China), caspase-3 (Cell Signaling Technology, Danvers, MA, USA), and active β -catenin (Cell Signaling Technology); β -actin mouse antibodies (Beyotime, Shanghai, China); and XAV-939 (MedChemExpress, Monmouth Junction, NJ USA).

Techniques: Activity Assay, Inhibition, Western Blot, Immunofluorescence, Staining, Expressing

Journal: Journal of Cosmetic Dermatology

Article Title: Protective Effects of Exogenous Donkey Oil on Skin Healing Under Incisional Wound Damage

doi: 10.1111/jocd.70550

Figure Lengend Snippet: Effects of different concentrations of DO on the content of VEGF (A) and MMP‐9 (B) in the skin tissue of mice after incision injury. # p < 0.05, ### p < 0.001 vs. BC group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC group.

Article Snippet: The ELISA kits IL‐1α and VEGF were purchased from ProteinTech (Wuhan, China, Cat No: KE10024 and KE10009); IL‐6 from Multi Sciences (Hangzhou, China, Cat No: EK206/3‐96); PGE2 from Lanpaibio (Shanghai, China, Cat No: LP‐M05161); MMP‐9 from CUSABIO (Wuhan, China, Cat No: CSB‐E08007m).

Techniques:

Journal: Journal of Orthopaedic Translation

Article Title: Tibial cortex transverse transport regulates Orai1/STIM1-mediated NO release and improve the migration and proliferation of vessels via increasing osteopontin expression

doi: 10.1016/j.jot.2024.02.007

Figure Lengend Snippet: Osteopontin (OPN) on the para-osseous tissues of the TTT group was significantly enhanced. (A) Based on the positive dual-staining area of VEGF-A and Orai1 (shown at a magnification of 100 ×), tissues within the planned area were acquired by utilizing laser capture microdissection (LCM). (B) Gene ontology (GO) analysis of the proteins discovered by proteomics. (C) The Venn diagram presents the distinct expression profiles of the TTT and NTT groups. (D) The protein–protein interaction networks (PPI) analysis of the expressed in the TTT group indicated their functional relationship. (E) The expression level of OPN in each group was detected by western blotting. The right chart shows the gray scale statistics. n = 3. (F) Immunohistochemical staining of the para-osseous tissues of the NTT and the TTT groups detected the expression of OPN, shown at a magnification of 100 ×. (G) Double immunostaining of VEGF-A (shown in red) and OPN (shown in green) or STIM1 (shown in green) on the para-osseous tissues of each group. The magnification is 400 ×. DAPI (blue) presents the cellular nuclei. n = 5. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The mixed dual-labeled antibodies (VEGF-A + Oria1/2/3 (calcium release-activated calcium modulator 1/2/3), VEGF-A + STIM1/2, CD31+Orai1, CD31+STIM1, VEGF-A + Osteopontin) were applied, which included Orai1 (28411-1-AP, rabbit, 1:200, Proteintech, Wuhan, China), Orai2 (20592-1-AP, rabbit, 1:200, Proteintech, Wuhan, China), Orai3 (25766-1-AP, rabbit, 1:200, Proteintech, Wuhan, China), STIM1 (11565-1-AP, rabbit, 1:200, Proteintech, Wuhan, China), STIM2 (21192-1-AP, rabbit, 1:200, Proteintech, Wuhan, China), VEGF-A (sc-7269, mouse, 1:100, Santa Cruz, USA), CD31 (sc-376764, mouse, 1:100, Santa Cruz, USA), and Osteopontin (ab63856, rabbit, 1:200, Abcam, Cambridge, UK).

Techniques: Staining, Laser Capture Microdissection, Expressing, Functional Assay, Western Blot, Immunohistochemical staining, Double Immunostaining