Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: HL60 granulocytoid-C. albicans interaction. GFP-expressing C. albicans were cultured either alone (right column) or with HL60 granulocytoids at the indicated MOIs, as described in Materials and Methods. Photographs were taken 1.5 h later at 400× magnification. Each of the panels in the first column is a superimposition of three images: phase contrast, blue fluorescence (to visualize DAPI staining), and green fluorescence (to visualize GFP-C. albicans). Panels in the second column show the images corresponding to the green fluorescence in the first column. Panels in the last column show green fluorescence images to visualize C. albicans cultured alone at densities corresponding to the indicated MOIs.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Expressing, Cell Culture, Fluorescence, Staining

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: C. albicans-induced mortality in the HL60 granulocytoid population. HL60 granulocytoids were cultured either alone (left column) or with GFP-expressing C. albicans at an MOI of 0.3 (right column) as described in Materials and Methods. Photographs were taken 1.5 h (A) and 6 h (B and C) later. In the first and second rows, images are superimpositions of three photographs: phase contrast, green fluorescence (to visualize GFP-C. albicans), and blue fluorescence (to visualize DAPI staining) at a magnification of 400×. GFP-Candida can be seen engulfed by HL60 granulocytoids at both 1.5 h and 6 h postinfection. The third row shows blue fluorescence images at a magnification of 200×. DAPI-stained blue cells are indicative of cell death. (C) Nuclear morphology visualized by DAPI staining. The arrow points to a typical example of nuclear condensation and the arrowhead points to an example of nuclear fragmentation. A portion of the 400× image was further magnified 4× digitally.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Cell Culture, Expressing, Fluorescence, Staining

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: C. albicans hyphal growth in the presence and absence of dimethyl formamide-induced neutrophils. GFP-Candida were cultured with (dotted line) or without (solid line) HL60 granulocytoids in a Bioptechs ΔTC3 petri dish as described in Materials and Methods. Photographs were taken every 60 min with a 20× objective. Hyphal length was measured for all Candida cells in three microscopic fields for each time point. The number of Candida cells counted for each point on the graph is indicated. The figure shows the change in average hyphal length of C. albicans. The standard error is indicated at each time point.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Cell Culture

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: Viability of HL60 granulocytoids exposed to C. albicans

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Positive Control

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: Candida colony formation after 5 h of coculture with HL60 or HL60 granulocytoids. C. albicans was cultured alone or with HL60 or HL60-derived granulocytoids for 5 h at the indicated MOIs as described in Materials and Methods. The figure shows percent killing by HL60 granulocytoids (solid bars) or undifferentiated HL60 (hatched bars). The results presented are the means of three independent experiments. Bars represent the standard error.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Cell Culture, Derivative Assay

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: Quantitative RT-PCR analysis. Relative RNA levels of HNP1, N.E., HBEGF, and PAC1 measured by quantitative RT-PCR in RNA from HL60 granulocytoids or HL60 granulocytoids exposed to different MOIs (0.1, 0.5, and 5) of C. albicans for 1 h. RNA levels were measured relative to the amount of ACTB mRNA as described in Materials and Methods. Results are presented as the increase over expression in uninfected HL60 granulocytoids. Bars represent the standard error.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Quantitative RT-PCR, Over Expression

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: Expression of cell surface markers on 4-day dimethyl formamide-treated HL60 cells. Expression of cell surface markers was detected by binding of fluorescently tagged antibodies recognizing the markers in question. The figure shows the number of cells binding antibody (y axis) and the intensity of fluorescence (x axis) determined by flow cytometry. Specific binding was determined by comparing the binding profile of anti-CD11b, anti-CD116, anti-CD14, anti-mannose receptor, and anti-CD16b (filled profile) to the binding profile of the corresponding isotypic negative controls (described in Materials and Methods).

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Expressing, Binding Assay, Fluorescence, Flow Cytometry

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: Scatter plot analysis of microarray data. (A) Comparison of the levels of expression of 7,000 genes in HL60 granulocytoids (HL60 granulocytoid single) with levels in pooled RNA from three extractions (HL60 granulocytoid pooled). (B) HL60 granulocytoids exposed to Candida at an MOI of 0.5 for 1 h (HL60 granulocytoid+candida single) with pooled RNA from independent extractions of neutrophils exposed to Candida at an MOI of 0.5:1 for 1 h (HL60 granulocytoid+candida pooled). (C) HL60 granulocytoid single compared to HL60 granulocytoid+candida single. (D) HL60 granulocytoid pooled compared to HL60 granulocytoid+candida pooled.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Microarray, Comparison, Expressing

Journal:

Article Title: Gene Expression in HL60 Granulocytoids and Human Polymorphonuclear Leukocytes Exposed to Candida albicans †

doi: 10.1128/IAI.72.1.414-429.2004

Figure Lengend Snippet: Graphic representation of RNA levels. Panel A shows genes whose expression levels were higher in HL60 granulocytoids exposed to C. albicans at an MOI of 0.1 than in HL60 granulocytoids exposed to C. albicans at an MOI of 5. Panel B shows genes whose expression levels remained unaffected in HL60 granulocytoids on exposure to C. albicans (internal controls). Panel C shows genes whose expression levels were lower in HL60 granulocytoids exposed to C. albicans at an MOI of 0.1 than in HL60 granulocytoids exposed to C. albicans at an MOI of 5.

Article Snippet: HL60, a human promyelocytic leukemia cell line, was obtained from the American Type Culture Collection and was maintained in RPMI 1640 (Gibco-Invitrogen, Burlington, Ontario, Canada) supplemented with 20% heat-inactivated fetal bovine serum (HyClone, Logan, Utah) (growth medium).

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) Protocol for analysis of collagen antibody-induced arthritis (CAIA) model. PBS (Ctrl P ) or LPS (Ctrl L ) was administered as a control. ( B ) PCA using microarray data obtained from ankle tissue. ( C ) Heatmap of differentially expressed gene probes in ankle tissue (log 2 FC > |1|, P < 0.01). Log 10 transformed read counts are scaled from 1.0 to 3.0. ( D ) Expression of genes related to epigenetic regulation classified by GSEA. Log 10 transformed read counts are scaled to minimum to maximum values. ( E ) Relative probe counts detected in CAIA ankle compared with Ctrl P or Ctrl L ankles. ( F ) RT-qPCR of Uhrf1 mRNA expression in CAIA ( n = 4) and STA ( n = 3) ankles. ( G ) UHRF1 mRNA expression in synovium biopsies from healthy individuals, patients with OA, and patients with RA by RNA-Seq. Data are registered in the Gene Expression Omnibus (GEO GSE89408). ( H ) Left, representative images of immunofluorescence staining for Uhrf1 (red); Pdpn, Fap, Thy-1, CD45, F4/80, and CD3 (green); and DAPI (blue) in WT STA ankle tissue. Scale bar: 50 μm. Right, quantification of Uhrf1 + marker cells in hyperplastic synovium. Cell number in 1 field per similar region of independent mice was calculated. ( I ) Uhrf1 mRNA expression in SFs treated with 20 ng/mL Tnf-α for 24 hours. Mean ± SD is shown. * P < 0.05 and ** P < 0.01 by ANOVA followed by Tukey’s test in F (left), G , and H, and unpaired t test in F (right) and I . Data in A – F and H – I were obtained from 3 to 5 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Control, Microarray, Transformation Assay, Expressing, Quantitative RT-PCR, RNA Sequencing, Gene Expression, Immunofluorescence, Staining, Marker

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) Quantification of methylated DNA after enrichment from genome DNA using MBD beads. ( B ) Distribution of Uhrf1-mediated methylated DNA annotated using given intervals and scores with genome features by CEAS. ( C ) Venn diagram to compare Uhrf1-mediated methylated DNA loci between SFs and chondrocytes using MBD-Seq data from this study and GEO GSE99335. ( D ) Venn diagram for 171 genes having upregulated expression in Uhrf1 ΔCol6a1 SFs and 105 genes having Uhrf1-mediated methylated DNA peaks within the transcriptional start site region (± 50 kb). ( E ) Representative Uhrf1-mediated methylated DNA peaks visualized by Integrative Genomics Viewer. ( F ) KEGG pathway analysis of 105 upregulated with peaks assigned using DAVID Bioinformatics Resources. Significantly enriched pathways illustrated by gene counts and P values. Representative mRNA expression of genes included in the ( G ) KEGG pathways “Rheumatoid arthritis” and “Cytokine-cytokine receptor interaction” and ( H ) GO biological process “Negative regulation of apoptotic process” in SFs from Uhrf1 fl/fl and Uhrf1 ΔCol6a1 mice ( n = 3) as measured by RT-qPCR. ( I ) ChIP-qPCR assay of Uhrf1 for Ccl20 locus in Uhrf1 ΔCol6a1 and Uhrf1 fl/fl SFs. ( J ) Quantification of Ccl20 serum levels in Uhrf1 fl/fl and Uhrf1 ΔCol6a1 on day 0 ( n = 10) and day 10 ( n = 16) after STA induction. ( K ) Left, flow cytometry analysis of the population of Th17 cells (CD45 + , CD4 + , Ccr6 + ) in Uhrf1 fl/fl and Uhrf1 ΔCol6a1 derived from STA mice on day 4 ( n = 6–8) and day 10 ( n = 9–10). Right, quantification of CD45 + CD4 + Ccr6 + cells among CD45 + cells. Mean ± SD shown. NS, not significant versus Uhrf1 fl/fl . * P < 0.05 and ** P < 0.01 by unpaired t test in G – J and ANOVA followed by Tukey’s test in K . Data in A , G , and H obtained from 3 independent experiments. Data in B – F obtained from combined read data from 3 independent experiments. Data in I were technically replicated 3 times. Data in J and K obtained from 6–10 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Methylation, Expressing, Quantitative RT-PCR, ChIP-qPCR, Flow Cytometry, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) Expression levels of UHRF1 and DNMTs ( DNMT1 , DNMT3A , and DNMT3B ) mRNA in synovium obtained from patients with OA ( n = 32) and RA ( n = 30). ( B ) Spearman’s correlation between UHRF1 mRNA expression in RA synovium ( n = 30) and disease activity score 28-CRP (DAS28) as well as levels of C-reactive protein (CRP) and age. ( C ) Correlation of UHRF1 expression in RA synovium with 6-month response to DMARD treatment measured by ΔDAS28-CRP ( https://peac.hpc.qmul.ac.uk/ ). ( D ) Top, Western blot analysis of OA synovium ( n = 5) and RA synovium ( n = 5). Bottom, quantification of relative UHRF1 protein levels. ( E ) H&E staining and immunofluorescence staining for UHRF1 (red); PDPN, FAP, CD45 (green); and DAPI (blue) in specimens from multiple patients with RA (P1–P4). Scale bar: 100 μm. Arrow and arrowhead indicate UHRF1 + cells in cells positive for SF markers PDPN and FAP and leukocyte marker CD45, respectively. ( F ) Quantification of UHRF1 + cell number in CD45 + cells and CD45 – cells among total UHRF1 + cells ( n = 19). ( G ) Spearman’s correlation between DAS28 and number of UHRF1 + cells per tissue area ( n = 19). Mean ± SD is shown. * P < 0.05 and ** P < 0.01 by Mann-Whitney U test in A and D , and by Wilcoxon signed-rank test in F . All data were obtained from 5–32 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Expressing, Activity Assay, Western Blot, Staining, Immunofluorescence, Marker, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) mRNA expression levels of UHRF1 and CCL20 in RASFs transfected with UHRF1 siRNA ( n = 4–5). ( B ) Left, flow cytometry to measure proportion of Th17 cells (CD45 + CD4 + CCR6 + ) in OA ( n = 14) and RA ( n = 21) synovium tissue. Right, quantification of total CD45 + cells, CD45 + CD4 – CCR6 – cells, CD45 + CD4 – CCR6 + cells, CD45 + CD4 + CCR6 – cells, and CD45 + CD4 + CCR6 + cells among 7-AAD – cells. ( C ) Spearman’s correlation between proportion of Th17 cells and UHRF1 mRNA expression level in OASFs ( n = 10) and RASFs ( n = 12) obtained from synovium of the same patients. ( D ) Schematic protocol of consecutive UHRF1 knockdown and experimental apoptosis induction in RASFs. ( E ) Left, phalloidin (green) and DAPI (blue) staining of RASFs transfected twice with UHRF1 siRNA ( n = 9) after treatment with 0.5 μg/mL anti-FAS antibody for 16 hours. Scale bar: 200 μm. Right, quantification of cell numbers after apoptosis induction relative to that for vehicle treatment. Mean ± SD is shown. * P < 0.05 and ** P < 0.01 by ANOVA followed by Tukey’s test in A and E , and Mann-Whitney U test in B . All data were obtained from 4 to 21 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Expressing, Transfection, Flow Cytometry, Knockdown, Staining, MANN-WHITNEY

Journal: Communications Biology

Article Title: Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration

doi: 10.1038/s42003-022-04305-9

Figure Lengend Snippet: A Relative binding of VEGF to 52 different synthetic glycosaminoglycans measured on a microarray. B An illustration of the microarray. C Relative binding of HGF in the microarray to show the difference in binding pattern depending on the glycosaminoglycan. For A and C data is shown in a box and whisker (as Tukey) of 36 binding spots for each synthetic glycosaminoglycan. D Six synthetic glycosaminoglycans with different binding patterns were chosen for further analyses using surface plasmon resonance (SPR) to determine their binding in a quantitative way compared to the semi quantitative analysis that the microarray allows. E Heatmap showing the binding of six selected synthetic glycosaminoglycans on the x -axis and the tested growth factors on the y -axis. The glycosaminoglycans are ordered in increasing degree of sulfation, GAG nr 3, 43, 34, 10, 26, and 19. The color shows the k off value, where red illustrates a slow release while a blue color a fast release. The size of the circle illustrates the equilibrium dissociation constant, K d , where a larger circle is for higher binding while a smaller circle is for lower binding. There are also two points with no data indicated in gray and two points that did not show any release at all, indicated in green. For convenience, we have highlighted the growth factor FGF2 (gray line) and GAG nr 19 used in later experiments (orange highlight). Factors tested that did not show any interactions in the SPR analysis were: IL- 6, IL-11, TGF beta 2, TGF beta 3, CCL2, CCL3, CCL4, CCL23, Wnt2, G-CSF, NOV, EGF, IGF1, GLP, SCF, CXCL2, CXCL7.

Article Snippet: The slides were rinsed in PBST with 1% BSA for 3 min and then in deionized water for 3 min. Antibodies towards the protein were dissolved in PBST 10% BSA according to the manufacturer’s recommended concentration, added to the slide and incubated in a humified chamber for 60 min (HGF Ab, MAB694, 0.4 μg/ml, Mouse anti Human, RnD systems) (VEGF Ab, MAB293, 0.4 μg/ml, Mouse anti Human, RnD systems) (BMP4 Ab, MAB7571, 0.4 μg/ml, Mouse anti Human, RnD systems) (CXCL12 Ab, MAB350, 0.4 μg/ml, Mouse ant Human, RnD Systems) (IL-6 Ab, MAB2063, 0.4 μg/ml, Mouse ant Human, RnD systems) (FGF2 Ab, ab92337, 0.5 μg/ml, Rabbit anti Human, Abcam) (PDGF-AA Ab, MAB221, 0.4 μg/ml, Mouse anti Human, RnD systems) (KGF Ab, MAB251, 0.4 μg/ml, Mouse anti Human, RnD systems) (TGF beta 1 Ab, MAB240, 0.4 μg/ml, Mouse anti Human, RnD systems).

Techniques: Binding Assay, Microarray, Whisker Assay, SPR Assay

Journal: Communications Biology

Article Title: Controlled release of growth factors using synthetic glycosaminoglycans in a modular macroporous scaffold for tissue regeneration

doi: 10.1038/s42003-022-04305-9

Figure Lengend Snippet: Growth factors tested and the molecular weight according to the manufacturer, the theoretical pI, the highest concentration tested for the SPR experiment as well as the lowest concentration tested.

Article Snippet: The slides were rinsed in PBST with 1% BSA for 3 min and then in deionized water for 3 min. Antibodies towards the protein were dissolved in PBST 10% BSA according to the manufacturer’s recommended concentration, added to the slide and incubated in a humified chamber for 60 min (HGF Ab, MAB694, 0.4 μg/ml, Mouse anti Human, RnD systems) (VEGF Ab, MAB293, 0.4 μg/ml, Mouse anti Human, RnD systems) (BMP4 Ab, MAB7571, 0.4 μg/ml, Mouse anti Human, RnD systems) (CXCL12 Ab, MAB350, 0.4 μg/ml, Mouse ant Human, RnD Systems) (IL-6 Ab, MAB2063, 0.4 μg/ml, Mouse ant Human, RnD systems) (FGF2 Ab, ab92337, 0.5 μg/ml, Rabbit anti Human, Abcam) (PDGF-AA Ab, MAB221, 0.4 μg/ml, Mouse anti Human, RnD systems) (KGF Ab, MAB251, 0.4 μg/ml, Mouse anti Human, RnD systems) (TGF beta 1 Ab, MAB240, 0.4 μg/ml, Mouse anti Human, RnD systems).

Techniques: Molecular Weight, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: The MBD3/NuRD complex promotes H3K27 deacetylation on STAT1 promoter to inhibit STAT1 expression in GSCs. (A) The UCSC Genome Browser shows the acetylation of H3K27 on the promoter of STAT1 in GM12878 (B-lymphocyte), hESC (human embryonic stem cells), HSMM (skeletal muscle myoblasts), HUVEC (human umbilical vein endothelial cell), K562 (leukemia), NHEK (epidermal keratinocytes), and NHLF (lung fibroblasts) cells. (B and C) ChIP analyses on STAT1 promoter in GSCs/NSTCs or GSCs expressing shNT/shMBD3s. Assays were performed with the H3 antibody, and immunoprecipitates were subjected to qPCR analyses ( n = 3). (D) IB analysis of the indicated genes in 4 GSCs and matched NSTCs derived from 4 human GBM tumors. (E) Liquid chromatography-tandem MS (LC MS/MS) analysis of the purified MBD3/NuRD complex in GSC. Flag IP was performed in T387 GSC expressing Flag-MBD3. The components of NuRD complex were identified with MS. (F) IP of MBD3 was performed in T4121GSCs (left) and T387GSCs (right). The IB for CDH4, HDAC1, MBD3, and MBD2 are shown. IgG was used as an antibody control for IPs. Asterisks indicate nonspecific bands. (G) Real-time qPCR analysis of mRNA levels of MBD3, STAT1, and STAT3 in T4121GSCs or T387GSCs expressing shNT or shMBD3s ( n = 3). (H) Knockdown of MBD3 increased the expression of STAT1 in both mRNA and protein in D456 GSCs ( n = 3). (I) IB analysis of STAT1, p21, and H3K27ac in GSCs treated with SAHA for the indicated times. (J and K) T387 GSCs (J, n = 3) and T4121 GSCs (K, n = 3) were treated with the indicated dose of IFN-α/IFN-β in the absence or presence of SAHA (2 mM) for 3 d. Cell viability was assessed and normalized to the untreated control. Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as assayed by unpaired Student’s t test or Welch’s t test. ns, not significant.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing, Derivative Assay, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Purification

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: The MBD3/NuRD complex promotes H3K27 deacetylation on STAT1 promoter to inhibit STAT1 expression in GSCs. (A) ChIP analyses on STAT1 promoter. Assays were performed with the H3K27ac (left, n = 3) and H3K27me3 (right, n = 3) antibodies, and immunoprecipitates were subjected to qPCR analyses. (B) ChIP analysis with MBD3 antibody showing the enrichment of MBD3 at the promoter of STAT1 (around primer 6) in T4121GSCs and T387GSCs. Schematic showing the ChIP primer location with respect to the TSS of the STAT1 promoter (top). (C and D) ChIP analysis on the promoter of STAT1 in T4121GSCs ( n = 3) and T387GSCs ( n = 3) expressing shNT or two independent shMBD3s. Assays were performed with the indicated antibodies, and immunoprecipitates were subjected to qPCR analyses (primer 6). (E) IB analysis of STAT1, STAT3, and MBD3 in T387GSCs and T4121GSCs expressing shNT or two independent shMBD3s. (F) ChIP analyses on STAT1 promoter in GSCs and matched NSTCs. Assays were performed with the HDAC1 (left, n = 3) and CHD4 (right, n = 3) antibodies, and immunoprecipitates were subjected to qPCR analyses (primer 6). (G) Proposed model for MBD3/NuRD-mediated regulation of STAT1 transcription. In GSCs, MBD3 is highly expressed and binds to STAT1 promoter, recruits the NuRD complex (including CHD4 and HDAC1) to suppress STAT1 expression by H3K27 deacetylation. Loss of MBD3 disassembles the NuRD complex, increases H3K27 acetylation, and promotes STAT1 transcription. Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as assayed by unpaired Student’s t test or Welch’s t test.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: MBD3 is preferentially expressed in GSCs. (A) IB analysis of MBD3, MBD2, SOX2, and GFAP in GSCs and matched NSTCs derived from five human GBM tumors. (B) IB analysis of MBD3, MBD2, SOX2, and GFAP during GSC differentiation. (C) IB analysis of MBD3, STAT1, SOX2, and Olig2 in GSCs and NHAs. (D) Co-IF staining of MBD3 (green) and SOX2/Olig2 (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue). (E) IHC staining of MBD3 in brain tumor tissue microarray. Section was counterstained with hematoxylin (left). Box plot of histoscore of MBD3 (right). Normal brain tissue ( n = 5), low-grade gliomas (I–II, n = 15), and high-grade gliomas (III–IV, n = 39). One-way ANOVA; *, P < 0.05. (F) IHC staining of MBD3 (left) and STAT1 (right) in serial sections of human GBM specimens. Sections were counterstained with hematoxylin. (G) Co-IF staining of STAT1 (green) and MBD3 (red) in human GBM specimen and mouse GBM orthotopic xenograft. Nuclei were counterstained with Hoechst (blue).

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Derivative Assay, Staining, Immunohistochemistry, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: MBD3 is preferentially expressed in GSCs. (A) Co-IF staining of MBD3 (green) and SOX2, NESTIN (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue). (B and C) Co-IF staining of MBD3 (green) and SOX2, Olig2, NESTIN (red) in mouse GBM xenografts. Nuclei were counterstained with Hoechst (blue). (D) Pairwise correlation analysis of the indicated genes in TCGA GBM database. Pearson correlation coefficient (r) value and P value are shown ( n = 538). (E–H) IHC staining of SOX2, MBD3, and STAT1 in the serial sections of human glioma tissue microarrays. Sections were counterstained with hematoxylin (E, F, and H). IHC score of MBD3 in brain tumor tissue microarray (E). Boxplot (G, left) and correlation analysis (G, right; n = 35) of histoscores of the tissue microarray stained for indicated proteins are shown. Low-grade gliomas (I–II, n = 13) and high-grade gliomas (III–IV, n = 42). SOX2 + cells were quantified to imply the fraction of GSCs in tumor (G; low GSCs, n = 21; high GSCs, n = 19). The scale bar represents 50 μm (F). *, P < 0.05; ***, P < 0.001, as assayed by unpaired Student’s t test. (I) Co-IF staining of STAT1 (green) and MBD3 (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue).

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Staining, Immunohistochemistry, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Depletion of MBD3 leads to upregulation of IFN signaling and growth inhibition in GSCs. (A) Overrepresented gene ontology terms among upregulated gene sets (left) and downregulated gene sets (right) in shMBD3-GSCs compared with the shNT-GSCs. (B) Gene set enrichment analysis shows the enrichment of gene sets positive related to immune response (left) and negative related to cell cycle process (right) in shMBD3-GSCs compared with the shNT-GSCs. (C) Heatmap representation of upregulated genes involved in IFN response (left) and downregulated genes involved in cell cycle process (right) in shMBD3-GSCs compared with the shNT-GSCs. (D) Real-time qPCR analysis of mRNA level of IRGs in T387 or T4121 GSCs expressing shNT or shMBD3 ( n = 3). (E) Real-time qPCR (left) and IB (right) analysis of p21 expression in T387 or T4121 GSCs expressing shNT or shMBD3 ( n = 3). (F) p21 promoter (WWP-Luc) luciferase reporter assay showed that knockdown of MBD3 induced the transcription activation of p21 in GSCs ( n = 3). (G) IHC staining of p21 in GBM xenografts derived from T387 GSCs expressing shNT or shMBD3 (left). The percentage of p21 + cells was quantified (right; n = 3). (H) Knockdown of MBD3 impaired GSC proliferation assessed by EdU incorporation assay and Ki67 staining in T387 GSCs. Representative images are shown (left). The percentage of EdU + or Ki67 + cells was quantified (right; n = 3). (I and J) Knockdown of MBD3 with two shRNA sequences inhibited GSC sphere formation (I) and cell viability (J; n = 3). (K) Knockdown of MBD3 had no obvious effect on cell viability of NHA ( n = 3). (L) T4121 GSCs expressing shNT or shMBD3 were treated with indicated dose of IFN-α or IFN-β for 3 d, and cell viability was assessed and normalized to the untreated control ( n = 3). Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as assayed by unpaired Student’s t test or Welch’s t test.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Inhibition, Expressing, Luciferase, Reporter Assay, Activation Assay, Immunohistochemistry, Derivative Assay, Staining, shRNA

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Depletion of MBD3 upregulates IFN signaling and inhibits GSC growth. (A) Real-time qPCR analysis of mRNA levels of MCM10, POLA1, CDK4, and CDC45 in T387GSCs expressing shNT or shMBD3 ( n = 3). (B) CDKN1A promoter (WWP-Luc) luciferase reporter assay showed that MBD3 depletion had no effect on the reporter with STAT1 binding site mutation. Binding sites of STAT1 on CDKN1A promoter was mutated from 5′-TTCCCGGAA-3′ to 5′-AAGCTTGAA-3′ ( n = 3). (C) Knockdown of MBD3 inhibited GSC proliferation assessed by EdU incorporation assay and Ki67 staining in T4121 GSCs expressing shNT or shMBD3. Representative images are shown (left). The percentage of EdU + or Ki67 + cells was quantified (right; n = 3). (D) IB analysis showed the knockdown of MBD3 with two different shRNAs in T4121GSCs and T387GSCs. (E) Knockdown of MBD3 inhibited D456 GSC sphere formation. (F) Cell viability of T387 NSTCs expressing shNT or shMBD3 ( n = 3). (G) Representative images of cross sections (H&E stain) of mouse brains (nu/nu) 38 d after transplantation with T387 GSC expressing shNT, shMBD3#1, or shMBD3#2. (H) T4121 GSCs transduced with Tet-on-shMBD3 were treated with Dox (100 ng/ml) or vehicle control. IB analysis showed the knockdown of MBD3 in T4121 GSCs (left). Inducible knockdown of MBD3 inhibited T4121 GSCs tumorsphere formation (middle) and cell viability (right; n = 3). (I) IF staining of MBD3 (red) in xenograft tissues to assess the efficiency of MBD3 knockdown in vivo in , respectively. (J) IF staining of SOX2 or GFAP (red) in xenografts of T4121 GSCs (Dox-shMBD3) implanting mice (nu/nu) treated with or without Dox. Quantification of SOX2 or GFAP percentage are shown (right, n = 5). (K) Kaplan–Meier survival analysis of patients with high ( n = 76) and low ( n = 79) expression of MBD3 in Gravendeel GBM dataset. Log-rank test. For A–J, data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as assayed by unpaired Student’s t test or Welch’s t test.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing, Luciferase, Reporter Assay, Binding Assay, Mutagenesis, Staining, Transplantation Assay, Transduction, In Vivo

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Highly expressed MBD3 promotes GSC malignant progression . (A) GSCs expressing shNT or shMBD3s were transplanted into brains of nude mice (5 × 10 4 cells/mouse). Kaplan–Meier survival curves of mice implanted with T4121 GSCs (shNT, n = 7; shMBD3#1, n = 6; shMBD3#2, n = 7) or T387 GSCs (shNT, n = 6; shMBD3#1, n = 8; shMBD3#2, n = 7) are shown. Log-rank test. (B) T4121 GSCs expressing shNT or shMBD3 were transplanted into brains of nude mice in a limiting dilution manner (2 × 10 5 or 2 × 10 4 cells/mouse, n = 9 or n = 8, respectively). Kaplan–Meier survival plots are shown. Log-rank test. (C–F) Luciferase-labeled T4121GSCs were transduced with the Tet-on-inducible shMBD3 and then transplanted into the brains of nude mice (2 × 10 4 cells/mouse). Mice were treated with vehicle control or Dox (2 mg/ml in drinking water) to induce expression of shMBD3 from day 0 (C and E) or day 14 (D and F). GBM xenografts were tracked by bioluminescence, and the representative images from animals at the indicated time are shown (C and D, left). Bioluminescent quantification indicated that induced knockdown of MBD3 inhibited GSC tumor initiation and growth (C and D, right). Kaplan–Meier survival plots of mice are shown (E, shNT, n = 8; shMBD3, n = 10; F, n = 7 for each group). Unpaired Student’s t test for C and D. Log-rank test for E and F. (G) Co-IF staining of Ki67 and MBD3 in GBM xenografts derived from T387 GSCs expressing shNT or shMBD3 (left). The percentage of Ki67 + cells was quantified (right, n = 3). Data are represented as mean ± SD (unpaired Student’s t test). (H) Kaplan–Meier survival analysis of patients with high ( n = 93) and low expression ( n = 88) of MBD3 in REMBRANDT GBM dataset. Log-rank test. (I) Knockout of STAT1 rescued the inhibition of MBD3 depletion on GSC viability and tumor initiation. IB of WT and STAT1 KO GSCs transduced with shNT or shMBD3 (left). Cell viability was assessed with GSCs as indicated (middle, n = 3, unpaired Student’s t test). The indicated GSCs were transplanted into brains of nude mice (2 × 10 4 cells/mouse). Kaplan–Meier survival curves of mice are shown ( n = 6 for each group). Log-rank test. Data are represented as mean ± SD (G and I) or mean ± SEM (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001. nu/nu nude mice were used in the animal experiments.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing, Luciferase, Labeling, Transduction, Staining, Derivative Assay, Knock-Out, Inhibition

Journal: Molecular Medicine Reports

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

doi: 10.3892/mmr.2018.9336

Figure Lengend Snippet: lncRNA expressions in HCC tissues. (A) lncRNA expression spectrum clustering map from six paired tumor samples and adjacent normal tissues. Green and red bars indicate downregulation and upregulation, respectively. (B) Relative expression levels of 10 aberrantly expressed lncRNAs were validated by reverse transcription-quantitative polymerase chain reaction. *P<0.05 and **P<0.01. NC, negative control from the corresponding adjacent normal tissue. DGCR5, DiGeorge syndrome critical region gene 5, HCC, hepatocellular carcinoma; lncRNA, long non-coding RNA; NS, not significant; SNHG1, small nucleolar RNA host gene 1.

Article Snippet: The RT reaction was performed at 95°C 15 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 1 min. cDNA was labeled with Cy3 or Cy5 fluorescent probes (Aksomics, Inc., Shanghai, China) and hybridized to the Human LncRNA Expression Microarray v3.0 (format, 8×60 K; Arraystar, Rockville, MD, USA), according to the manufacturer's protocol.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Molecular Medicine Reports

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

doi: 10.3892/mmr.2018.9336

Figure Lengend Snippet: Top 5 upregulated and top 5 downregulated lncRNAs in hepatocellular carcinoma tissues identified by microarray analysis.

Article Snippet: The RT reaction was performed at 95°C 15 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 1 min. cDNA was labeled with Cy3 or Cy5 fluorescent probes (Aksomics, Inc., Shanghai, China) and hybridized to the Human LncRNA Expression Microarray v3.0 (format, 8×60 K; Arraystar, Rockville, MD, USA), according to the manufacturer's protocol.

Techniques: Microarray

Journal: Molecular Medicine Reports

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

doi: 10.3892/mmr.2018.9336

Figure Lengend Snippet: Relative expression levels of lncRNA SNHG1 in plasma samples from patients with HCC, patients with HCH and healthy Control individuals. (A-C) Relative lncRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction for (A) SNHG1, (B) ASLNC12773 and (C) BF896662 in the three groups. (D) A positive correlation was identified between plasma and tissue SNHG1 expression by Pearson correlation analysis. r=0.6210; R 2 =0.3857; P<0.001. (E) Relative plasma expression levels of SNHG1 pre- and post-surgery in 72 patients with HCC. HCC, hepatocellular carcinoma; HCH, hepatitis B virus-positive chronic hepatitis and cirrhosis; lncRNA, long non-coding RNA; NS, not significant; SNHG1, small nucleolar RNA host gene 1.

Article Snippet: The RT reaction was performed at 95°C 15 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 1 min. cDNA was labeled with Cy3 or Cy5 fluorescent probes (Aksomics, Inc., Shanghai, China) and hybridized to the Human LncRNA Expression Microarray v3.0 (format, 8×60 K; Arraystar, Rockville, MD, USA), according to the manufacturer's protocol.

Techniques: Expressing, Clinical Proteomics, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Virus

Journal: Molecular Medicine Reports

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

doi: 10.3892/mmr.2018.9336

Figure Lengend Snippet: Clinicopathological characteristics of the patients, divided into two groups according to the plasma lncRNA SNHG1 levels in HCC.

Article Snippet: The RT reaction was performed at 95°C 15 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 1 min. cDNA was labeled with Cy3 or Cy5 fluorescent probes (Aksomics, Inc., Shanghai, China) and hybridized to the Human LncRNA Expression Microarray v3.0 (format, 8×60 K; Arraystar, Rockville, MD, USA), according to the manufacturer's protocol.

Techniques: Clinical Proteomics, Expressing

Journal: Molecular Medicine Reports

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

doi: 10.3892/mmr.2018.9336

Figure Lengend Snippet: Receiver operating characteristic analysis of lncRNA in HCC patients.

Article Snippet: The RT reaction was performed at 95°C 15 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 1 min. cDNA was labeled with Cy3 or Cy5 fluorescent probes (Aksomics, Inc., Shanghai, China) and hybridized to the Human LncRNA Expression Microarray v3.0 (format, 8×60 K; Arraystar, Rockville, MD, USA), according to the manufacturer's protocol.

Techniques: Control

Journal: Molecular Medicine Reports

Article Title: Diagnostic utility of plasma lncRNA small nucleolar RNA host gene 1 in patients with hepatocellular carcinoma

doi: 10.3892/mmr.2018.9336

Figure Lengend Snippet: Diagnostic performance of plasma SNHG1 and AFP. (A) ROC curve analysis of plasma SNHG1 and AFP expression in differentiating patients with HCC (n=72) from healthy controls (n=50). SNHG1, AUC = 0.92 (95% CI, 0.86–0.96), cut-off value = 2.54; AFP, AUC = 0.85 (95% CI, 0.77–0.90), cut-off value = 187.88 µg/l. (B) ROC analysis of plasma SNHG1 and AFP expression in differentiating patients with HCC (n=72) from patients with HCH (n=50). SNHG1, AUC = 0.74 (95% CI, 0.65–0.83), cut-off value = 3.25; AFP, AUC = 0.79 (95% CI, 0.71–0.86), cut-off value = 268.11 µg/l. (C) ROC analysis of combination of SNHG1 and AFP differentiate patients with HCC from health Controls. (D) ROC analysis of the combination of SNHG1 and AFP differentiate patients with HCC from patients with HCH. AFP, α-fetoprotein; AUC, area under the ROC curve; CI, confidence interval; HCC, hepatocellular carcinoma; HCH, hepatitis B virus-positive chronic hepatitis and cirrhosis; lncRNA, long non-coding RNA; NS, not significant; ROC, receiver operating characteristic; SNHG1, small nucleolar RNA host gene 1.

Article Snippet: The RT reaction was performed at 95°C 15 sec, followed by 30 cycles of 95°C for 5 sec and 60°C for 1 min. cDNA was labeled with Cy3 or Cy5 fluorescent probes (Aksomics, Inc., Shanghai, China) and hybridized to the Human LncRNA Expression Microarray v3.0 (format, 8×60 K; Arraystar, Rockville, MD, USA), according to the manufacturer's protocol.

Techniques: Diagnostic Assay, Clinical Proteomics, Expressing, Virus

Journal: Molecular Cancer

Article Title: METTL3/IGF2BP3 axis inhibits tumor immune surveillance by upregulating N 6 -methyladenosine modification of PD-L1 mRNA in breast cancer

doi: 10.1186/s12943-021-01447-y

Figure Lengend Snippet: PD-L1 expression positively correlates with METTL3 and IGF2BP3 expression in breast cancer. a The expressions of PD-L1, METTL3 and IGF2BP3 were analyzed by IHC in a tissue microarray containing of 140 breast cancer tissues. Four Cases as representative IHC staining with positive- and negative-PD-L1 were shown. Scale bars, 100 μm. b The correlation of PD-L1 with METTL3 and IGF2BP3 in all breast cancer tissues ( n = 140) were analyzed by IHC scores. Proportion scores were recorded as 0, 1, 2, 3, 4 corresponding to < 5%, 5–25%, 25–50%, 50–75%, and ≥ 75%. Intensity scores were recorded as 0, 1, 2, 3 corresponding to negative, weak, moderate, and strong staining. Finally, IHC scores was calculated as “proportion score × intensity score”. c The correlation between PD-L1 and METTL3 or IGF2BP3 were analyzed in HER2+ ( n = 26) and TNBC ( n = 27) subtypes. Spearman’s rank correlation test was used to analyze the P value. d Number of cases of METTL3 and IGF2BP3 were presented in two categories (PD-L1 positive and PD-L1 negative) in 140 tissues. e The differential expression of METTL3 or IGF2BP3 between responders and non-responders in cilnial data sets. The Y-axis represents the log2 Fold change values (responders vs. non-responders). f A schematic model illustrating the mechanism of METTL3/IGF2BP3-mediated N 6 -methyladenosine modification of PD-L1 mRNA in breast cancer

Article Snippet: Hybridization of cRNA was done after merging to Arraystar Human m 6 A Epitranscriptomic Microarray (8 × 60 K, Arraystar, Rockville, MD, USA).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Quantitative Proteomics, Modification

Journal: Cell Reports

Article Title: Notch Signaling Facilitates In Vitro Generation of Cross-Presenting Classical Dendritic Cells

doi: 10.1016/j.celrep.2018.05.068

Figure Lengend Snippet: Notch-Driven Differentiation Yields Functional Human cDC1s DCs generated from CD34 + progenitors in cultures with OP9 or OP9-DL1 were analyzed in parallel to primary DCs from PB. (A and B) Cytokine production by DCs stimulated for 14 hr with a cocktail of TLR agonists (poly-I:C, LPS, CL075, and CpG). (A) Representative flow cytometric analysis of intracellular cytokine production (TNF, IL-12, and IFN-α) in the indicated gated DC subsets from OP9-DL1 cultures. Grey contours represent unstimulated cells; numbers represent the cytokine-positive fraction. (B) Proportion of cytokine-positive cDC1s (purple), cDC2s (red), or pDCs (blue) generated from CD34 + progenitors in culture with OP9 (n = 4) or OP9-DL1 (n = 3) cells compared with PB primary (n = 7) cells following TLR stimulation. Circles, histograms, and bars represent individual experiments, mean, and SEM, respectively; p values are indicated. (C and D) T cell stimulation by DCs cultured with sorted allogeneic blood CD3 + T cells. (C) Representative flow cytometric analysis of T cell proliferation in response to culture with DCs. The data show output of a T cell and cDC1 (generated with OP9-DL1) culture. CD11c + CD141 + cDC1s could be identified (purple gate) and gated out. CD3 + T cells were subdivided by CD8 and CD4 expression. Cell division was indicated by CFSE dilution (turquoise gate). (D) Proportion of CD4 + or CD8 + T cells that underwent division (CFSE dilution) in culture with cDC1 (purple) or cDC2 (red) isolated from PB or generated in culture with OP9 or OP9-DL1 (DL1) cells. T cells cultured alone or with beads coated with anti-CD3 plus anti-CD28 were used as negative (Neg) and positive (Pos) controls, respectively. Responses to blood DCs were generated from 2–3 DC donors and 3 T cell donors (2–6 independent experiments). Responses to cultured DCs were generated from 2 BM donors combined with 3 T cell donors (4–6 independent experiments). Each circle represents an independent experiment (mean of 1–3 technical replicates). Histograms and bars represent mean and SEM, respectively. The p values were derived from unpaired two-tailed Student’s t test.

Article Snippet: Mouse anti-human CD141 (BV510/APC conjugate, clone 1A4/AD5-14H12, 3/5 μl/50 μl sample) , BD/Miltenyi , Cat# 563298, 130-090-907.

Techniques: Functional Assay, Generated, Cell Stimulation, Cell Culture, Expressing, Isolation, Derivative Assay, Two Tailed Test

Journal: Cell Reports

Article Title: Notch Signaling Facilitates In Vitro Generation of Cross-Presenting Classical Dendritic Cells

doi: 10.1016/j.celrep.2018.05.068

Figure Lengend Snippet:

Article Snippet: Mouse anti-human CD141 (BV510/APC conjugate, clone 1A4/AD5-14H12, 3/5 μl/50 μl sample) , BD/Miltenyi , Cat# 563298, 130-090-907.

Techniques: Recombinant, Isolation, Cell Culture, Microarray, Software