Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: Differential gene expression and functional enrichment analysis of PAAD. (A) Volcano plot highlighting DEGs between PAAD and normal tissues. The most significant upregulated gene in the tumor tissues was TM4SF1 . Red dots signify genes that are highly expressed in the tumor group. Blue dots indicate genes with low expression in the tumor group. Gray dots, labeled as ‘not’, represent genes deemed non-differentially expressed due to not meeting the screening threshold. (B) Bubble plot depicting the GO and KEGG functional enrichment of the up- and downregulated DEGs. (C) GSEA analysis results illustrating the signaling pathways significantly enriched with DEGs. BP, biological process; CC, cellular component; MF, molecular function; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; PAAD, pancreatic adenocarcinoma; DEGs, differentially expressed genes.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 ℃.

Techniques: Expressing, Functional Assay, Labeling

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: Immune cell infiltration analysis between high- and low-TM4SF1 expression groups. (A) Scatter plot correlating TM4SF1 expression with levels of immune cell infiltration. (B) Correlation analysis reinforcing the findings in (A). (C) Differential immune cell infiltration based on ssGSEA analysis between the high- and low-TM4SF1 expression groups. (D) High- and low-risk immune features. *, P<0.05; **, P<0.01; ***, P<0.001. MDSC, myeloid-derived suppressor cell; ssGSEA, single-sample gene set enrichment analysis; L, low; H, high.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 ℃.

Techniques: Expressing, Derivative Assay

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: Gene mutation analysis of the five key prognostic genes. (A) Chromosomal positions of the five key genes. (B) Forest plot illustrating the hazard ratios of the five genes concerning PAAD prognosis. (C) Correlation heatmap of the five key genes. (D) Distribution of the mutation frequencies across patients. (E) Waterfall plot of the genes ranked by mutation count. (F) Tissue samples from 10 patients were obtained to examine the mRNA expression differences of TM4SF1, BPIFB4, CPTP, DVL1, PLEKHN1 , and DDR1 between cancer and normal tissues. **, P<0.01. SNP, single nucleotide polymorphism; INS, insertion; DEL, deletion; SNV, single-nucleotide variant; PAAD, pancreatic adenocarcinoma; mRNA, messenger RNA; HR, hazard ratio.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 ℃.

Techniques: Mutagenesis, Expressing, Variant Assay

Journal: Journal of Gastrointestinal Oncology

Article Title: Analyzing the role of TM4SF1 expression in pancreatic adenocarcinoma: understanding prognostic implications and therapeutic opportunities

doi: 10.21037/jgo-24-564

Figure Lengend Snippet: TM4SF1 expression and its functional effects on pancreatic cancer cell behavior. (A) Box plot depicting the relative expression of TM4SF1 mRNA in pancreatic tumor tissues compared to normal tissues, demonstrating a significant upregulation in tumors (*, P<0.05). (B) Kaplan-Meier survival curves stratifying patients into quartiles based on TM4SF1 expression levels, showing an inverse relationship between high TM4SF1 expression and overall survival. (C) Western blot analysis of TM4SF1 protein levels in normal (N1–N5) and tumor (T1–T5) pancreatic tissue samples, with tumors exhibiting higher expression levels. (D) Western blot comparison of the TM4SF1 protein across a panel of pancreatic cancer cell lines, identifying varying expression levels. (E) Bar graph quantifying TM4SF1 mRNA expression in various pancreatic cancer cell lines, with PANC-1 cells showing the highest expression. (F,G) Bar graph showing TM4SF1 mRNA levels in PANC-1 cells after treatment with NC or TM4SF1 -targeting siRNAs (si TM4SF1 #1, #2 and #3), indicating effective knockdown. Proliferation assay (H) and colony formation assay (1×) (stained with 0.1% crystal violet) (I) of PANC-1 cells following si TM4SF1 treatment, revealing reduced cell growth and colony number compared to NC. (J) Cell viability assay assessing the sensitivity of PANC-1 cells to Gemcitabine post-si TM4SF1 treatment, showing increased drug sensitivity. Transwell migration, invasion (10×) (stained with 0.1% crystal violet) (K) and cellular wound healing (5×) (L) assays in si TM4SF1 -treated PANC-1 cells demonstrated decreased migratory and invasive capacities. (M) Western blot confirming overexpression of TM4SF1 in CFPAC-1 cells transfected with oe TM4SF1 versus NC. Cell proliferation assay (N) and colony formation assay (1×) (stained with 0.1% crystal violet) (O) in CFPAC-1 cells with oe TM4SF1 showed enhanced growth and colony-forming ability. Transwell migration, invasion (10×) (stained with 0.1% crystal violet) (P) and cellular wound healing (5×) (Q) assays in oe TM4SF1 -expressing CFPAC-1 cells indicated increased migration and invasion potential. *, P<0.05; **, P<0.01; ***, P<0.001. TPM, transcripts per million; PAAD, pancreatic adenocarcinoma; HR, hazard ratio; NC, control; IC 50 , half-maximal inhibitory concentration; oeTM4SF1, overexpression of TM4SF1; mRNA, messenger RNA.

Article Snippet: Primary antibodies against β-actin (#AF0003, Beyotime) and TM4SF1 (#NBP1-76549, NOVUS, Colorado, USA) were incubated on the membranes overnight at 4 ℃.

Techniques: Expressing, Functional Assay, Western Blot, Comparison, Knockdown, Proliferation Assay, Colony Assay, Staining, Viability Assay, Migration, Over Expression, Transfection, Control, Concentration Assay

Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 6. Effects of Inhibiting BMP, Canonical Wnt, and Nodal signaling on Germ Lineage Differentiation. A-B) Representative images of Brachyury (green), Otx2 (red), and DAPI (blue) immunostaining of WT and Dvl TKO EBs on day 7 of differentiation after inhibition with BMP (LDN-193189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB-431542) drug treatments. Scale bar ¼ 100 μm. C-D) Quantification of Brachyury and Otx2-positive cells in WT and Dvl EBs differentiated for 5–7 days (n ¼ 20/group). Brachyury-positive cells are not detectable in Dvl TKO EBs, and there are no significant differences in Otx2-positive cells. E-F) Quantitative RT-PCR analysis of Brachyury (mesoderm) and GATA4 (endoderm) mRNA expression to determine differences in differentiation potential in WT and Dvl TKO EBs over a 6-day time course (n ¼ 3). Expression is normalized to the untreated controls in Fig. 5G (Brachyury) and 5H (GATA4). E) Brachyury expression in WT EBs is attenuated with inhibition of BMP, Wnt, and Nodal signaling, similar to Dvl TKO EBs. F) Inhibition of BMP and Wnt signaling significantly enhances endoderm differentiation in WT and Dvl TKO EBs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Immunostaining, Inhibition, Quantitative RT-PCR, Expressing

Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 7. Effects of Developmental Pathway Modulation on Germ Lineage Differentiation. Activation of BMP (BMP4), canonical Wnt (Wnt3a and CHIR-99021) and Nodal (Activin A) pathways in WT EBs enhances mesoderm (Bra) differentiation, while endoderm (GATA4) differentiation remains low, and ectoderm (Otx2) dif- ferentiation diminishes over time. Treatment with these activators in Dvl TKO EBs does not rescue mesoderm induction. The relative level of endoderm marker expression is higher in Dvl TKO EBs compared to WT EBs treated with Wnt activators, Wnt3a and CHIR, but not BMP4 or Activin A. Dvl loss also delays the decrease in ectoderm lineage expression. Inhibition of BMP (LDN-198189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB431542) pathways in WT EBs results in the suppression of mesoderm differentiation, recapitulating the Dvl TKO phenotype. Pathway inhibitor treatment in WT EBs, leads to similar trends of differentiation as Dvl TKO EBs. Endoderm differentiation is greatly enhanced in Dvl TKO EBs with LDN-193189 and IWP L6 treatment.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Activation Assay, Marker, Expressing, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A and B ) Graphs represent the percentage of B, CD4 + , CD8 + , and CD11b + cells in BM ( A ) and spleen ( B ) of WT and Egfl6 mice. ( C ) Gating and quantification of Ly6G and Ly6C subsets of CD11b + BM and splenic cells from healthy C57BL/6J (WT) and Egfl6 mice. ( D ) Volcano plot showing differentially expressed genes (DEGs) between BM CD11b + cells of Egfl6 mice versus C57BL/6J (WT). P values determined via t test are plotted on the y axis. DEGs are colored in red. ( E ) Gating and quantification of BM-derived CD11b + Ly6G + Ly6C – cells stimulated with rGM-CSF ± rEgfl6. ( F ) qPCR analyses of indicated genes in sorted BM CD11b + cells stimulated with rGM-CSF + rEgfl6. Stimulation with rGM-CSF alone was used as control. ( G and H ) ELISA of Granzyme B (GZMB) in IL-2 + CD3/CD28 activated CD8 + T cells and cultured directly with rEgfl6-stimulated BM-derived MDSC cells or MDSC control at different ratio ( G ) or with the conditioned media (CM) of rEgfl6-stimulated BM-derived MDSC cells or MDSC control ( H ). Unstimulated CD8 + T cells were used as negative control. Results were analyzed using unpaired 2-tailed t test or 2-way ANOVA. Experiments were performed in triplicate. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Tumor volume changes (mm 3 ) and images of 2F8c and 2F8c-Egfl6 subcutaneous tumors resected and measured 3 weeks after tumor cell inoculation ( n = 6 mice per group). ( B ) Time-dependent body weight gain in mice i.p. injected with ID8-CV and ID8-Egfl6 tumors ( n = 8 mice per group). ( C ) Evaluation of peritoneal metastases of ID8-CV and ID8-Egfl6 that had a weight increase of over 35% of their original weight on the day of tumor cell injections ( n = 6 mice per group). ( D and E ) Kaplan-Meier overall survival analysis for 2F8c+/–Egfl6 ( D ) and ID8+/–Egfl6 ( E ). Survival statistics were calculated using log-rank analysis from Kaplan-Meier survival plots. ( F and G ) Flow cytometric evaluation and summary of PMN-MDSC (CD11b + Ly6G + Ly6C – ) ( F , top panel), M-MDSC (CD11b + Ly6G – Ly6C + ) ( F , bottom panel), and TAM (CD11b + F4/80 + CD206 + ) ( G ) in ID8+/–Egfl6 tumors. ( H ) Flow cytometric evaluation and quantification of CD8 T (CD45 + Thy1.2 + ) cells and their expression of IFN-γ in ID8+/–Egfl6 tumors. ( I and J ) ELISA of Granzyme B (GZMB) ( I ) and IFN ( J ) in IL-2 + CD3/CD28 activated CD8 T cells (Pos Control) and cultured directly with F4/80 + or Ly6G + cells isolated from ID8 and ID8-Egfl6 ascites at ratio of 1:1. ( K and L ) Time-dependent volume changes (mm 3 ) of 2F8c and 2F8c-Egfl6 tumor cells ( K ) or body-weight gain in mice i.p. injected with ID8 and ID8-Egfl6 tumor cells ( L ) and treated with anti-Ly6G/Ly6C Ab or IgG isotype control ( n = 6 mice per group). P values were calculated using unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s post test for multiple comparisons. Experiments were performed in triplicate. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, **** P < 0.0001.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Volcano plot showing differentially expressed genes (DEGs) between CD11b + cells infiltrating 2F8c-Egfl6 versus 2F8c tumors. Negative Log 10 P values determined via t test are plotted on the y axis. ( B ) IPA protein analysis of Egfl6 treatment associated DEG pathways identified as significantly ( P < 0.05) upregulated (left panel) or downregulated (right panel). ( C and D ) Summary of PD-L1 expression determined by flow cytometry in infiltrating TAMs ( C ) and by qPCR in BM-derived macrophages polarized with different stimuli as indicated D . ( E ) Western blotting analysis of IL-10 and Cxcl2 in TAMs and PMN-MDSCs isolated from ID8+/–Egfl6 ascites. Actin was used as loading control. ( F ) ELISA of IFN-γ in CD8 + T cells cultured with the Ly6G + cells isolated from ID8+/–Egfl6 ascites in the absence/presence of IL-10 or Cxcl2 NAbs. ( G ) Western blotting showing the indicated protein expression in BM-isolated CD11b + cells treated with GM-CSF and rEgfl6 for 0, 7.5, and 15 minutes. β-Actin was used as loading control. Results are representative of at least 3 independent experiments. ( H and I ) ELISA showing IL-10 and Cxcl2 protein secretion in GM-CSF-treated BM CD11b + cells +/– rEgfl6 and/or Syk inhibitor (R406) ( H ), and GM-CSF-treated BM CD11b + cells +/– rEgfl6 and/or the integrin inhibitor Cyclo-RGD (c-RGD) ( I ). ( J ) Graph represents a ChIP assay performed with anti-Jun Ab followed by qPCR to measure IL-10 promoter in ID8+/–Egfl6 ascites. Data are presented as mean ± SEM. P values were calculated using unpaired 2-tailed t test or 1-way ANOVA with Tukey’s post test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. All results are representative of 3 independent experiments.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) 2F8c and 2F8c-Egfl6 tumor growth in mice treated with anti-PD-L1 Ab or IgG isotype control Ab ( n = 8 mice per group). * P < 0.05, 2F8c + IgG versus 2F8c-Egfl6 + IgG; *** P < 0.001, 2F8c + anti-PD-L1 versus 2F8c + IgG. ( B ) Kaplan-Meier survival analysis for the indicated treatment groups. *** P < 0.001, 2F8c + anti-PD-L1 versus 2F8c + IgG. Survival statistics were calculated using log-rank (Mantel-Cox) analysis from Kaplan-Meier survival plots. ( C ) Flow cytometry quantification of intratumoral PMN-MDSCs (CD11b + Ly6G + Ly6C – ), M-MDSCs (CD11b + Ly6G – Ly6C + ), CD206 + TAMs, and CD8 + T cells in the indicated tumors. ( D – F ) qPCR analysis of mRNA expression of S100A9 , IL-10 , and Cxcl2 gene expression in ( D ) 2F8c-Egfl6 versus 2F8c, ( E ) anti-PD-L1–treated 2F8c versus IgG-treated 2F8c, ( F ) anti-PD-L1–treated 2F8c-Egfl6 versus IgG-treated 2F8c-Egfl6 tumor samples. ( G ) Representative images of IHC staining showing Cxcl2-expressing cells in control and a-PD-L1–treated tumor tissue sections. Graph represents the number of Cxcl2 + cells in the indicated tumors. Scale bars: 20 μm. Error bars show SEM. Experiments were performed in triplicate. Statistical significance was determined by unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Control, Flow Cytometry, Expressing, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) Volume changes (mm 3 ) and representative images of 2F8c-Egfl6 subcutaneous tumors treated with IgG isotype Ab (Control), a-PD-L1 Ab, and a-Egfl6 Ab, alone or in combination, were resected and measured 2 days after the last treatment ( n = 8 mice per group). ** P < 0.01, IgG Ab versus a-Egfl6 Ab; *** P < 0.001, anti-PD-L1 Ab versus a-Egfl6 Ab and IgG Ab versus anti-PD-L1+ a-Egfl6 Abs. ( B and C ) Kaplan-Meier overall survival analysis for 2F8c-Egfl6 ( B ) and ID8 p53–/– Brca2–/— -Egfl6 ( C ) mice receiving the indicated treatment. Survival statistics were calculated using the Log-rank (Mantel-Cox) test analysis. ( D – G ) Flow cytometric gating and quantification of CD206 + TAMs ( D ), PMN-MDSC (CD11b + Ly6G + Ly6C – ) ( E ), MHCII + TAMs ( F ), and CD8 + T (CD45 + Thy1.2 + ) ( G ) cells in 2F8c-Egfl6 and ID8 p53–/– Brca2–/— -Egfl6 tumors. Error bars show SEM. Experiments were performed in triplicate. Statistical significance was determined by unpaired 2-tailed t test or 2-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A ) qPCR analyses of IL-10 and Cxcl2 in the indicated treated Egfl6 + 2F8c tumors. ( B ) IF images and quantification of IL-10 expression in the indicated treated Egfl6 + 2F8c tumors. P values were calculated using unpaired 2-tailed t test. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 , **** P < 0.0001. All results are representative of 3 independent experiments. Scale bar: 30 μm.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Egfl6 promotes ovarian cancer progression by enhancing the immunosuppressive functions of tumor-associated myeloid cells

doi: 10.1172/JCI175147

Figure Lengend Snippet: ( A and B ) Gating and quantification of human CD11b + CD66b + ( A ) and CD11b + CD163 + CD64 + ( B ) cells in CD33 + cells isolated from ascites of patients with HGSOC and stimulated with rEGFL6 +/– c-RGD. ( C ) Cytokine array and densitometry of the CM of CD33 + ascites from patients with HGSOC stimulated with GM-CSF +/– rEGFL6. Spot intensities were calculated using ImageJ software. ( D ) Representative immunofluorescence images showing EGFL6 expression (red) and CD68 cell (green) localization in HGSOC tumor tissue sections ( n = 6 per group). DAPI stained nuclei. Graph represents the number of CD68-positive cells in tissues expressing high or low levels of EGFL6. Scale bar: 100 μm. ( E ) Spatial feature plots of EGFL6 and CD163 markers and spatial autocorrelation of selected genes. Moran’s I test, implemented in the Seurat FindSpatiallyVariableFeatures function, was applied to compute spatial autocorrelation of the expression of each gene. Data are from a previously published dataset . ( F – H ) Sorted correlation plots between mRNA expression of EGFL6 in CD45 – cells and mRNA expression of cytokines and surface proteins in the indicated immune cells. Correlation was computed using the Spearman’s correlation with the sample-wise averaged gene expression. Each dot represents the Spearman’s correlation coefficients of a gene, and the dots were sorted in ascending order. P values were calculated using unpaired 2-tailed t test, 1-way, or 2-way ANOVA with Tukey’s post test for multiple comparisons. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.

Article Snippet: Human MDSCs were isolated from ascites of high-grade serous cancer patients using CD33 + magnetic microbeads (Miltenyi) and stimulated with 50 ng/mL human GM-CSF (R&D Systems) and 200 ng/mL recombinant human EGFL6 (rEGFL6) (Sino Biological).

Techniques: Isolation, Software, Immunofluorescence, Expressing, Staining