Journal: Journal of Biological Chemistry

Article Title: The Glycan Domain of Thrombopoietin (TPO) Acts intrans to Enhance Secretion of the Hormone and Other Cytokines

doi: 10.1074/jbc.m201297200

Figure Lengend Snippet: FIG. 1. Diagram of TPO muteins. The TPO and TGD constructs used in our studies are illustrated. In the center of the figure is TPO 1–335, the native murine (m) TPO, with glycan domain derivatives (lacking the entire receptor binding domain) immediately below, and two truncated yet biologically active TPO forms above. Locations of O- and N-linked residues as identified and proposed by Hoffman et al. (20) are denoted by the letters O and N. The filled diamond region denotes the secretory leader, the unfilled region denotes the receptor binding or erythropoietin (EPO)-like domain, and the gray shaded region denotes the glycan domain. The TGD mutein includes the secretory leader of TPO, the first three coding residues of the RBD, followed by residues 173–335 of mTPO, the majority (all but the first 20 residues) of the glycan domain. TGD-SL encodes the same sequence as TGD, but the secretory leader is deleted and replaced by a single Met initiation codon, and TGD-KDEL encodes the same sequence as TGD but adds an endoplasmic retention signal (Lys-Asp-Glu-Leu) to the carboxyl terminus of the construct. TPO 1–238 encompasses the RBD plus the first 86 residues of the TGD. TPO 1–152 encompasses the entire RBD, a spacer (show in diagonal markings, containing Ser, Ala, and Gly residues without identity or homology to TPO) and a poly(Tyr) and poly(His) (pYpH) tail. Note that TPO 1–152 shares no glycan domain residues with TGD. Restriction mapping and DNA sequencing verified the cDNA sequence of each TPO glycomutein and truncation, and the cDNA was subcloned into the mammalian expression vector pDX (39).

Article Snippet: Immune Assay—Supernatants were tested in paired samples using a commercial ELISA for mTPO (MTP00, QuantikineTM M from R & D Systems, Minneapolis, MN); in this immunoassay the lower limit is 62.5 pg/ml.

Techniques: Construct, Glycoproteomics, Binding Assay, Sequencing, DNA Sequencing, Expressing, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: A novel form of docetaxel polymeric micelles demonstrates anti-tumor and ascites-inhibitory activities in animal models as monotherapy or in combination with anti-angiogenic agents

doi: 10.3389/fphar.2022.964076

Figure Lengend Snippet: Ex vivo analysis of plasma and ascites samples from the H22 and ES-2 ascites-bearing mice. The ascites and plasma from H22 or ES-2 ascites bearing mice sacrificed 7 or 8 days after treatment as in were analyzed. (A,E) Ascites weight of H22 and ES-2 ascites-bearing mice. The numbers on the column represents number of mice with ascites formed/all analyzed mice. (B,F) Cell counts in ascites from H22 and ES-2 ascites bearing mice. (C,G) VEGF levels in ascites from H22 and ES-2 ascites-bearing mice. (D,H) VEGF levels in plasma from H22 and ES-2 ascites-bearing mice. Error bars represent SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: In addition, VEGF levels in plasma and ascites were measured by QuantikineTM mouse VEGF immunoassay kit (R&D Systems, United States).

Techniques: Ex Vivo, Clinical Proteomics

Journal: Frontiers in Pharmacology

Article Title: A novel form of docetaxel polymeric micelles demonstrates anti-tumor and ascites-inhibitory activities in animal models as monotherapy or in combination with anti-angiogenic agents

doi: 10.3389/fphar.2022.964076

Figure Lengend Snippet: Effects of HT001 and Endostar combination on H22 ascites-bearing mice. The H22 bearing ascites mice were intraperitoneally administrated with normal saline, 5 mg/kg CDDP, 1.5 mg/kg HT001, 5 mg/kg HT001, 8 mg/kg Endostar, 1.5 mg/kg HT001+8 mg/kg Endostar, or 5 mg/kg HT001+8 mg/kg Endostar ( n = 16/group). HT001 and CDDP were administrated once a week and Endostar was administrated daily for 3 weeks. After 8 days of administration, 6 mice from each group were sacrificed for analysis and the remaining mice were used for monitoring survival. (A) Kaplan-Meier survival analysis of H22 ascites-bearing mice. (B) Ascites weight of H22 ascites mice. The numbers on the column represent number of mice with ascites formed/all analyzed mice. (C) Cell counts in ascites of H22 ascites-bearing mice. (D) VEGF levels in ascites from H22 ascites-bearing mice. (E) VEGF levels in plasma from H22 bearing ascites mice. Error bars represent SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: In addition, VEGF levels in plasma and ascites were measured by QuantikineTM mouse VEGF immunoassay kit (R&D Systems, United States).

Techniques: Saline, Clinical Proteomics

Journal: Development (Cambridge, England)

Article Title: A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels.

doi: 10.1242/dev.01393

Figure Lengend Snippet: Fig. 8. The spatial expression pattern of Vegfa expression is not altered in Hhex–/– embryos. (A) and (B) Low-power views of sagittal sections of E9.5 embryos stained with Hoecsht (blue staining of cell nuclei). Higher-power views of the areas boxed in (A) and (B) are shown as indicated. Green staining represents presence of Vegfa. In the heart of both Hhex+/+ (C) and Hhex–/– (D) embryos, Vegfa is expressed in both the myocardium and endocardium. Vegfa is also highly expressed in the developing gut endoderm of both Hhex+/+ (E) and Hhex–/– (F) embryos.

Article Snippet: Whole hearts were isolated from E9.5-E13.5 Hhex+/+ and Hhex–/– embryos, sonicated in PBS and Vegfa levels were assayed using the mouse Vegfa ELISA detection kit QuantikineM (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion

doi: 10.1101/2023.11.29.569032

Figure Lengend Snippet: Representative Opal Multiplex immunohistochemical stains for F4/80 (red), CD204 (yellow), and CD206 (green) in tumors derived from SCC 31, SCC 40, SCC 35, SCC 37, and SCC 32 on day 10 after inoculation. 4 areas from each tumor and 3 tumors from each single cell clone were examined. Scale bars represent 20 μM. DAPI staining is not shown. B) Representative Opal Multiplex IHC stains for CD3 (green) and CD8 (red) in tumors derived from SCC 31, SCC 40, SCC 35, SCC 37, and SCC 32 on day 10 after inoculation. 4 areas from each tumor and 3 tumors from each single cell clone were examined. Scale bars represent 20 μM. DAPI staining is not shown. C) Larger magnification of representative Opal Multiplex immunohistochemical stain for F4/80 + CD204 + CD206 + cells in tumor derived from SCC35 (left) and CD3 + CD8 + cells in tumors derived from SCC 31 (right) on day 10 after inoculation, to demonstrate markers co-localization. Scale bars represent 10 μM. DAPI staining is not shown. D) Quantification of the percentage of F4/80+CD204+CD206+ cells described in (A). Data are mean ± SEM. The rejected and non- rejected groups were compared using Mann-Whitney U test with a p-value of 2.55*10 -8 . E) Quantification of the percentage of CD3+CD8+ cells described in (B). Data are mean ± SEM. The rejected and non- rejected groups were compared using Mann-Whitney U test with a p-value of 7.094*10 -7 . F) CFSE-based T cell proliferation assay following 48 hours co-culture with non-rejected SCC35 tumor-derived macrophages isolated 10 days post-inoculation. Representative histogram plot and quantification of CFSE intensity (n = 5). CD8 + /CD4 + T cells isolated from healthy mouse spleens cultured w/o macrophages served as control. CD8: The Kruskal-Wallis (KS) chi-squared test shows Chi-square 12.5, P = 0.001930454. Pairwise comparison by Wilcoxon test with Bonferroni correction are shown in the figure. * Pvalue < 0.05. CD4: The Kruskal-Wallis (KS) chi-squared test shows Chi-square(2) 12.5, P = 0.00193. Pairwise comparison by Wilcoxon test with Bonferroni correction are shown in the figure. * Pvalue < 0.05. T cell mediated IFNγ secretion to the culture media, 48 hours post co-culture of T cells with or without tumor- derived macrophages, measured using an ELISA. n=6. Data are mean ± SEM. The Kruskal-Wallis (KS) chi-squared test shows Chi-square(2) 15.726, P = 0.0003847. Pairwise comparison by Wilcoxon test with Bonferroni correction are shown in the figure. *** Pvalue < 0.01.

Article Snippet: To measure IFNγ secretion by the T cells co-cultured with the tumor-derived macrophages in the media of the T cell suppression assay we used QuantikineTM ELISA Mouse IFN-γ Immunoassay (R&D Systems, catalog number #MIF00-1) according to manufacturer instructions.

Techniques: Multiplex Assay, Immunohistochemical staining, Derivative Assay, Staining, MANN-WHITNEY, Proliferation Assay, Co-Culture Assay, Isolation, Cell Culture, Control, Comparison, Enzyme-linked Immunosorbent Assay