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: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: NOX2 enhances FADDosome-induced gastric epithelial mitochondrial fission and dysfunction in PHG. (A) Two-dimensional hierarchical clustering results for the enzyme NADPH oxidase (NOX) family were presented for the comparison between PHG patients and healthy volunteers (n = 3 per group). The fold changes and P values of the indicated mRNAs in PHG tissues relative to those in normal (Uninvolved) tissues from the microarray experiment were also presented. (B) NOX1, NOX2 and NOX4 IHC staining (brown) showed that NOX2, but not NOX1 or NOX4, was upregulated in the gastric mucosal samples of PHG patients, and the NOX1, NOX2 and NOX4 areas were also analyzed. n = 6 per group. * P < 0.05. (C) Representative images of IHC staining (brown) for NOX1, NOX2 and NOX4 in mouse models revealed that Nec-1 (an inhibitor of RIPK1) repressed NOX2 expression in mice with PVL. n = 6 per group. (D) The NOX1, NOX2 and NOX4 areas detected by IHC staining in (C) were presented. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (E) The colocalization of TOMM20 (red) and NOX2 (green) determined by confocal imaging in GES-1 cells confirmed that TNF-α increased the mitochondrial localization of NOX2 (white arrows indicate colocalization). Nuclei (blue) were counterstained with DAPI. (F) The levels of and interaction between NOX2 and TOMM20 were determined by western blotting, and IP showed that Nec-1 decreased the epithelial expression of total and mitochondrial NOX2 in the primary epithelial cells of mice with PVL. TEM also revealed that Nec-1 partly normalized the abnormal or damaged mitochondria in the mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (G) The ATP concentration in the mouse models verified that Nec-1 and GSK2795039 (GSK, a NOX2 inhibitor) improved the production of ATP in mice with PVL. n = 6 per group. * P < 0.05. (H) Representative 4-HNE IHC staining (brown) and quantification analysis in SO mice and mice with PVL suggested that GSK2795039 (GSK) reduced the level of 4-HNE in mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (I) Representative MitoSox staining (red) and the MitoSox intensity in mouse model revealed that GSK2795039 alleviated mitochondrial ROS (mtROS) accumulation in the primary epithelial cells of mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (J) Related protein levels in primary epithelial cells from mouse models treated with the indicated agents ( shFADD , Nec-1 or BAY) showed interactions among TAK1, NF-κBp65, FADD and NOX2 signaling. (K) GES-1 cells were transfected with either the control vector or shFADD and then treated with TNF-α or vehicle, and the NOX2 luciferase reporter activities were presented (left panel). * P < 0.05 versus the vehicle group and TNF-α-treated cells transfected with shFADD . The NOX2 luciferase reporter activities in GES-1 cells transfected with pcDNA3.1-p65 -vector or pcDNA3.1 -vector were also shown (right panel), * P < 0.05.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Comparison, Microarray, Immunohistochemistry, Expressing, Imaging, Western Blot, Concentration Assay, Staining, Transfection, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: The alteration of glycolysis associated with dysfunction of the mitochondrial electron transport chain contributes to oxidative stress in the PHG. (A) Western blotting showed that the protein levels of LDHA and HK2 were increased in mice with PVL but were decreased by GSK2795039 (GSK, a NOX2 inhibitor) in mice with PVL. The ratios of the normalized LDHA/β-actin and HK2/β-actin densitometric units and the relative level of lactic acid were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL not treated with GSK2795039. (B) Oxygen consumption rate (OCR) analysis of primary epithelial cells isolated from mouse models suggested that increased nonmitochondrial oxygen consumption and reduced maximum respiration were present in mice with PVL but were reversed by GSK2795039. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (C) Examination of the glycolytic rate (for the extracellular acidification rate [ECAR]) showed that GSK2795039 treatment reduced basal and compensatory glycolysis in mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (D) Two-dimensional hierarchical clustering results of the genes of electron transport chain (ETC)-related elements between PHG patients (as PHG) and healthy volunteers (as Uninvolved) (n = 3 per group). (E) Western blotting analysis (left panel) of protein levels of representative ETC complex subunits in the primary epithelial cells of PHG patients and healthy volunteers (as Uninvolved) revealed that the levels of the mitochondrial complex I, II, III, IV and V subunits were decreased in PHG patients. The activity of the antioxidant enzyme SOD and the ratio of reduced (GSH) to oxidized (GSSG) states (GSH/GSSG) detected by assay kits (right panel) were found to be decreased in PHG. n = 6 per group. * P < 0.05. (F) Western blotting analysis of representative ETC complex subunits from primary epithelial cells isolated from mouse models suggested that Nec-1 (an inhibitor of RIPK1) mitigated the defects in ETC subunit expression and decreased ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (G) SOD activity and the GSH/GSSG ratio, as detected by assay kits, were inhibited in mice with PVL but were restored by Nec-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (H) Western blotting analysis and quantification of protein levels for representative ETC complex subunits from primary epithelial cells isolated from mouse models showed that Mdivi-1 reversed the decrease in mitochondrial complex I, II, III, IV and V subunit levels and reduced ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also presented. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment. (I) Decreases in SOD activity and the GSH/GSSG ratio detected by assay kits were found in mice with PVL but were reversed by Mdivi-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Western Blot, Isolation, Activity Assay, Expressing

Journal: Cell Reports Medicine

Article Title: An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases

doi: 10.1016/j.xcrm.2023.101130

Figure Lengend Snippet:

Article Snippet: Incomplete Freund’s Adjuvant , InvivoGen , Catalog #: vac-ifa-60.

Techniques: Blocking Assay, Sequencing, Control, Recombinant, Adjuvant, Cell Isolation, Membrane, Sterility, Microarray, Software

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: Molecular Cancer

Article Title: Up-regulation of lncRNA CASC9 promotes esophageal squamous cell carcinoma growth by negatively regulating PDCD4 expression through EZH2

doi: 10.1186/s12943-017-0715-7

Figure Lengend Snippet: An overview of deregulated lncRNAs in ESCC specimens. a The proportion of deregulated lncRNAs in ESCC specimens. b Differentially expressed lncRNAs in ESCC and adjacent normal tissues. The lncRNA profiles from five paired ESCC tissues were clustered using Cluster 3.0. The red lines represented the up-regulated lncRNAs while the blue lines represented the down-regulated lncRNAs ( P < 0.05 and fold change > 2). c The scatter plots showed the expression of selected lncRNAs in cancer (circle in red) and normal (circle inblue) tissues. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: RNA extraction and microarray hybridization were performed by Kangchen Company (Shanghai, China) using the Human lncRNA microarray v2.0 (8 × 60 K, arraystar, USA).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Up-regulation of lncRNA CASC9 promotes esophageal squamous cell carcinoma growth by negatively regulating PDCD4 expression through EZH2

doi: 10.1186/s12943-017-0715-7

Figure Lengend Snippet: PDCD4 is a target gene of CASC9 and its expression correlates with clinical parameters in ESCC tissues. a Schematic flowchart showing the process of identifying PDCD4 as the target of CASC9. The combination of GO analysis and the gene expression correlation study identified PDCD4 in the cell growth pathways as potential regulatory target of CASC9. b Knockdown of CASC9 caused alterations in multiple pathways associated with cell growth. c Correlation analysis between CASC9 intensities and PDCD4 intensities provided by the microarray data from ESCC tissues. d Western blotting analysis showed that knockdown of CASC9 elevated the expression of PDCD4. e Spearman correlation was used to analyze the relationship between CASC9 and PDCD4 expression in another 37 ESCC tissues. f - h qPCR analysis of PDCD4 expression in ESCC tissues and adjacent normal tissues. PDCD4 expression was significantly lower in tumor tissues and especially lower in advanced pathological tumor samples. PDCD4 expression was higher in smaller tumor samples. Student’s t-test and one-way ANOVA analysis were performed to calculate the statistical difference. i Kaplan-Meier analysis showed that low PDCD4 expression indicated poor prognosis of ESCC patients. * P < 0.05, ** P < 0.01

Article Snippet: RNA extraction and microarray hybridization were performed by Kangchen Company (Shanghai, China) using the Human lncRNA microarray v2.0 (8 × 60 K, arraystar, USA).

Techniques: Expressing, Gene Expression, Knockdown, Microarray, Western Blot

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: 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