soluble pd l1 recombinant proteins  (R&D Systems)

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 is correlated with B7-H3 expression in human ovarian cancer. (a) The mRNA expression of GOLM1 and B7-H3 was analyzed with the Pearson Correlation analysis GEPIA database |Log2FC| cutoff = 1; q -value cutoff = 0.01; ANOVA differential analysis in 426 ovarian cancer (OV) samples and 88 normal ovarian tissue. (b) The GOLM1 and B7-H3 mRNA levels were retrieved from OV dataset ( n = 379) with ENCORI database, R = 0.421. (c) IHC plot of the typical protein expression of GOLM1 and B7-H3 in two OV patients (ID: 1884 and 2347) from The Human Protein Atlas. (d) Statistical analysis of GOLM1 and B7-H3 protein expression in 12 patients from patients with OV from The Human Protein Atlas. (e) Kaplan–Meier plot of the overall survival of 1402 ovarian cancer patients in TCGA database. The logrank p value was 0.0016.

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: B7-H3 expression is partially regulated by GOLM1. (a) qPCR analysis of GOLM1 knockdown efficiency in SKOV3 cell line after stable introduction of three different shRNAs by lentivirus system. (b) qPCR analysis of B7-H3 mRNA expression in three GOLM1 shRNA stable induced SKOV3 cell lines. (c) Western blot analysis of GOLM1 and B7-H3 protein expression in three GOLM1 knockdown SKOV3 cell lines. (d) Statistics of GOLM1 and B7-H3 protein expression in three GOLM1 knockdown SKOV3 cell lines. (e) qPCR analysis of GOLM1 and B7-H3 expression in SKOV3 cell line with transient exogenous expressed GOLM1 using PEI proreagent. (f) Western blot analysis of GOLM1 and B7-H3 expression in GOLM1 overexpressed SKOV3 cell line. (g) Statistics of GOLM1 and B7-H3 expression in GOLM1 overexpressed SKOV3 cell line. Each experiment was repeated three times and values are presented as means ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed Student's t -test).

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Expressing, shRNA, Western Blot, Two Tailed Test

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 expression was correlated with B7-H3 membrane protein expression and B7-H3 secretion. (a) FACS analysis of comparable B7-H3 membrane expression in GOLM1 knockdown and GOLM1 overexpressed SKOV3 cell lines. Original SKOV3 cell line as the control. (b) Statistics of B7-H3 membrane expression in three SKOV3 cell lines indicated in (a). (c) ELISA assay of soluble B7-H3 expression in three SKOV3 cell lines after cultured in incubator for three indicated times (24 hours, 48 hours, and 72 hours). Three independent experiments are performed, and values are presented as means ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed Student's t -test). (d) The interaction of exogenous expressed GOLM1 and B7-H3 was detected in 293T cells cotransfected with His-GOLM1 and Flag-B7-H3. (e) The interaction endogenously expressed GOLM1, and B7-H3 was detected in SKOV3 cells.

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 regulates ovarian cancer metastasis and invasion ability in soluble B7-H3-dependent manner. (a) Western blot of B7-H3, GOLM1 and metastasis-related protein E-cadherin, MMP-9, and VEGF in GOLM1 knockdown and GOLM1 overexpressed SKOV3 cell lines. Both scramble shRNA knockdown SKOV3 cell line and original SKOV3 cell line are treated as the control. (b) Western blot of lentivirus induced stable knockdown B7-H3 in SKOV3 cell lines compared with original and GOLM1 knockdown SKOV3 cell lines. (c) The Transwell assay to determine the invasiveness of GOLM1 knockdown and B7-H3 knockdown SKOV3 cell lines treated with or without additional soluble B7-H3 (10 μ g/ml) for 48 hours. (d) Statistics of the invaded cell number between each group ( ∗ p < 0.05, ∗∗ p < 0.01). (e) The wound-healing ability of GOLM1 knockdown and B7-H3 knockdown SKOV3 cell lines treated with or without soluble B7-H3 (10 μ g/ml) for 48 hours. (f) Statistics of the percentage of wound area remaining between each group ( ∗ p < 0.05).

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: Western Blot, shRNA, Transwell Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: GOLM1 as a Potential Therapeutic Target Modulates B7-H3 Secretion to Drive Ovarian Cancer Metastasis

doi: 10.1155/2022/5151065

Figure Lengend Snippet: GOLM1 regulates ovarian cancer progression in vivo. (a) In vivo bioluminescence imaging of 107 GOLM1 and B7-H3 knockdown SKOV3 cell lines subcutaneous injected behind the right upper limb in immunodeficient NSG mice, using original SKOV3 as the control. Each group contains three mice. The imaging was performed at day 0 and day 42. (b) Statistics of BLI signal between three groups ( ∗ p < 0.05, ∗∗ p < 0.01). (c) Vernier caliper measurement of tumor volume every 7 days after tumor inoculation. Statistical difference was calculated at day 42 ( ∗ p < 0.05, ∗∗ p < 0.011). (d) ELISA assay of soluble B7-H3 in peripheral blood of each mouse at day 42 ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (e) In vivo bioluminescence imaging of 107 GOLM1 and B7-H3 knockdown SKOV3 cell lines' tail vein injected in immunodeficient NSG mice, using original SKOV3 as the control. Each group contains three mice. The imaging was performed at day 1 and day 42. (f) Statistics of BLI signal between the three groups ( ∗ p < 0.05). (g) ELISA assay of soluble B7-H3 in peripheral blood of each mice at day 42 ( ∗∗ p < 0.01). (h) Survival curve of the mice in the three groups.

Article Snippet: For in vitro metastasis assay, the cells were treated with 10 μ g/ml soluble human B7-H3 (R&D) for indicated times.

Techniques: In Vivo, Imaging, Injection, Enzyme-linked Immunosorbent Assay

Journal: Cancer immunology research

Article Title: Dysregulated NFκB-dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Melanoma Patients

doi: 10.1158/2326-6066.CIR-20-0274

Figure Lengend Snippet: (A) Log2 mRNA expression of ADAM10 and ADAM17 for iDCs, mDCs, and AdV/DC in melanoma patients (n=33). Each end of the box and whisker plots represent the first (25%) and third (75%) quartile, while the middle line represents the mean. Patients were segregated by clinical outcomes, with intergroup significance determined using one-way ANOVA. (B) ICOSL surface expression analyzed from HD DCs (n=3) at baseline (iDCs), stimulated with IFNγ+LPS (24hrs.), or stimulated with IFNγ+LPS with the ADAM10/17 inhibitor, Tapi-2 (20 μM, 24hrs.). Gating is shown from a representative HD, and ICOSL frequencies and histograms are shown for mDC- and Tapi-2-treated cells. (C) ICOSL MFI or sICOSL for mDC controls and Tapi-2-treated cells. Data is displayed as normalized percentages against the mDC control, and an unpaired student t-test (two-tailed) was used to determine significance. *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Soluble ICOSL ELISA Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008).

Techniques: Expressing, Whisker Assay, Control, Two Tailed Test

Journal: Cancer immunology research

Article Title: Dysregulated NFκB-dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Melanoma Patients

doi: 10.1158/2326-6066.CIR-20-0274

Figure Lengend Snippet: (A) ICOSL protein surface expression was analyzed on melanoma patient (n=30) DC subtypes and HDs (n=4) by flow cytometry. Representative histograms from a patient and HD are shown for each DC subtype. Background staining was corrected with isotype (red) controls (left). Quantified ICOSL MFI values are shown (right). Patients were segregated by clinical outcomes, with intergroup significance determined using unpaired Wilcoxon rank sum test. Error bars represent standard deviation. (B) Correlation of mDC and AdV/DC ICOSL and IL10 (n=22). The grey shaded area represents the 95% Confidence Interval bands. (C) Microarray and qPCR analysis of ICOSLG transcripts in patient DCs (n=33, iDC, mDC, AdV/DC). Patients were segregated by clinical outcomes, with intergroup significance determined using unpaired Kruskal-Wallis one-way ANOVA (left and right panels) or paired student t-test (one-tailed)(middle panel). Each end of the box and whisker plots represent the first (25%) and third (75%) quartile, while the middle line represents the mean. (D) Kaplan-Meier (KM) curves showing OS and PFS in patients (n=33) based on iDC mRNA expression of ICOSLG. (E) Correlation of AdV/DC ICOSLG mRNA and IL12B mRNA expression in favorable clinical outcome patients (n=33). The grey shaded area represents the 95% Confidence Interval bands. Correlations were calculated using Pearson’s correlation coefficients, and the data was log transformed to meet normality assumptions (E). *p≤0.05, **p≤0.01.

Article Snippet: Soluble ICOSL ELISA Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008).

Techniques: Expressing, Flow Cytometry, Staining, Standard Deviation, Microarray, One-tailed Test, Whisker Assay, Transformation Assay

Journal: Cancer immunology research

Article Title: Dysregulated NFκB-dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Melanoma Patients

doi: 10.1158/2326-6066.CIR-20-0274

Figure Lengend Snippet: (A-B) DC culture supernatants were tested using a specific ELISA. Error bars represent standard deviation. (A) Soluble ICOSL (sICOSL) was determined in patient (n=16) matched (n=8; iDC and mDC, left) and nonmatched (n=16, right) pairs. Patients were segregated by clinical outcomes, with intergroup significance determined using unpaired (left) or paired (right) Wilcoxon rank sum tests. (B) sICOSL of HD was determined in matched pairs (n=4). (C) KM curves showing OS and PFS in patients (n=15) based on mDC sICOSL. The log-rank p value is indicated. (D) Correlation of mDC sICOSL and Th1 chemokines, MIP-1α and CXCL9 (n=11). The grey shaded area represents the 95% Confidence Interval bands. Correlations were calculated using Pearson’s correlation coefficients. The data was log transformed to meet normality assumptions (right panel). *p≤0.05, **p≤0.01, ns= not significant.

Article Snippet: Soluble ICOSL ELISA Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Transformation Assay

Journal: Cancer immunology research

Article Title: Dysregulated NFκB-dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Melanoma Patients

doi: 10.1158/2326-6066.CIR-20-0274

Figure Lengend Snippet: (A) Correlation of ICOSL protein surface expression on AdV/DC and in vivo vaccine-specific tyrosinase CD8+ T-cell responses (determined using IFNγ ELISPOT assays)(n=25; 12). The grey shaded area represents the 95% Confidence Interval bands. The data were log-transformed to meet normality assumptions. Pearson’s R correlation was used to determine the correlation significance. (B) Soluble ICOSL detected in supernatants of patient mDCs based on bulk tumor-associated CD8+ T-cell responses in patients (n=12). Error bars represent standard deviation. Patients were segregated by clinical outcomes, with intergroup significance determined using paired student t-tests. (C-D) CD107a MFI from CMV-specific T cells when (C) primed (n=5) or (D) boosted (n=4) with autologous mDCs previously treated with blocking anti-ICOSL (10 μg/mL) or IgG control. Granzyme-B secretion from CMV-specific T cells when primed with autologous mDCs previously treated with blocking anti-ICOSL or IgG control (n=5)(C). Data is displayed as normalized percentages against the IgG control and unpaired Wilcoxon rank sum test was used to determine significance (C, D, E). *p≤0.05, **p≤0.01, ns= not significant.

Article Snippet: Soluble ICOSL ELISA Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008).

Techniques: Expressing, In Vivo, Enzyme-linked Immunospot, Transformation Assay, Standard Deviation, Blocking Assay, Control

Journal: Cancer immunology research

Article Title: Dysregulated NFκB-dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Melanoma Patients

doi: 10.1158/2326-6066.CIR-20-0274

Figure Lengend Snippet: sICOSL is a prognostic index for extended overall survival and progression-free survival of melanoma patients. Univariate Cox regression analysis for sICOSL protein levels (n=15) or surface ICOSL MFI values (n=30) from baseline mDC culture supernatants or mDCs and AdV/DC, respectively. The hazard ratios, significance values, and 95% Confidence Intervals for OS and PFS are listed.

Article Snippet: Soluble ICOSL ELISA Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008).

Techniques:

Journal: Cancer immunology research

Article Title: Dysregulated NFκB-dependent ICOSL Expression in Human Dendritic Cell Vaccines Impairs T-cell Responses in Melanoma Patients

doi: 10.1158/2326-6066.CIR-20-0274

Figure Lengend Snippet: (A) Chromatin immunoprecipitation assays performed on HD (n=4) cells to determine NF-κB p65 binding to the ICOSLG promoter region in IFNγ+LPS-treated (matured) DCs. TNFα was used as a positive control. Error bars represent standard deviation. The grey dotted line seperates the negative control experiments (Exon region) and the experimental group (NF-κB site) for ICOSLG and TNFα relative expression. (B-C) ICOSL surface expression analyzed from HD DCs (n=6) at baseline (day 5 iDCs), stimulated with IFNγ+LPS (15 minutes), IFNγ+LPS with DMSO (15 minutes), or IFNγ+LPS with the NFκB inhibitor, parthenolide (15 μM, 15 minutes). (B) Gating and histograms from two representative donors for total p65, phospho-p65, and ICOSL MFI values. Isotype antibodies were used to control for background staining. (C) The ratio of phospho-p65 to total p65 (left). ICOSL MFI values for each treatment group (right). Each end of the box and whisker plots represent the first (25%) and third (75%) quartile, while the middle line represents the mean. Data is displayed as normalized percentages against the positive control (IFNγ+LPS) and unpaired student t-test (two-tailed) was used to determine significance. *p≤0.05, **p≤0.01.

Article Snippet: Soluble ICOSL ELISA Soluble ICOSL concentrations were determined using the Human B7-H2 Duo Kit ELISA (R&D #DY165–05) and the DuoSet ELISA Ancillary Reagent Kit 2 (R&D #DY008).

Techniques: Chromatin Immunoprecipitation, Binding Assay, Positive Control, Standard Deviation, Negative Control, Expressing, Control, Staining, Whisker Assay, Two Tailed Test