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: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Heatmap generated with qPCR data of DTFs and various genes related to three biological alterations. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black, as generated by Cluster 3.0. b. IHC of proteins related to normal renal function (KNG1, AQP2, SCNN1B). c. Immune function (TLR2, CXCR4). d. Metabolic function (ENO2, CYP2J2,ALDOB). This pattern of expression is in accord with the microarray findings. Sum scores are shown with n as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Generated, Expressing, Microarray

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. A microarray heatmap showing significant downregulation of four developmental transcriptional factors in ccRCC and a table showing fold changes of DTFs and their known renal developmental function. b. IHC validation of decreased expression of TFAP2B, DMRT2, and TFCP2L1. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match. c. Microarray heatmap showing downregulation of TFCP2L1 and its regulated genes in ccRCC. S–Stage, N–normal, T–tumor. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in yellow, as generated by Genetree.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Microarray, Biomarker Discovery, Expressing, Generated

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Heatmap showing adipogenic gene expression signature in ccRCC. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black. b. IHC showing lipid-laden clear cell morphology of ccRCC, increased expression of ADFP, and decreased expression of GATA2 in ccRCC. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Cellular differentiation experiments showing that KIJ-308 and KIJ-265 ccRCC cells are capable of adipogenic differentiation and become lipid-laden in adipogenic media, as indicated by Oil Red ‘O’ staining. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of adipogenic differentiation, as indicated by Oil Red ‘O’ staining, unlike normal renal canine MDCK cells. c. Under adipogenic media conditions, ccRCC also produces glycogen, as shown by a PASH stain.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Cell Differentiation, Staining

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. Cellular differentiation experiments showing that KIJ-308 and KIJ-265 ccRCC cells are capable of osteogenic differentiation in osteogenic media by developing calcium deposits, as shown by Alizarin Red stain. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of osteogenic differentiation, as shown by Alizarin Red stain, unlike normal renal canine MDCK cells.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Cell Differentiation, Staining

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: a. A heatmap showing the increased expression of some markers associated with EMT. Upregulation of genes is indicated in red, downregulation is indicated in green, and similar expression is indicated in black. b. IHC validation of two known markers of EMT: vimentin and N-cadherin. Sum scores are shown with n , as indicated. *p<0.01 when comparing ccRCC to normal match.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques: Expressing, Biomarker Discovery

Journal: PLoS ONE

Article Title: Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

doi: 10.1371/journal.pone.0010696

Figure Lengend Snippet: During normal renal development, mesenchymal stem cells undergo mesenchymal epithelial transition (MET) to develop into normal renal epithelial cells (NREs). In renal carcinogenesis, NREs undergo de-differentiation and epithelial mesenchymal transition (EMT), followed by adipogenic differentiation to develop into ccRCC.

Article Snippet: The human A498 ccRCC cell line and MDCK canine normal renal cells were purchased from ATCC (Manassas, VA) while KIJ-265 (Stage 4) and KIJ-308 (Stage 2) cell lines and primary cells were established in the Copland laboratory and derived from human renal clear cell carcinoma and normal-matched tissues.

Techniques:

Journal: PLoS ONE

Article Title: The WSB1 Gene Is Involved in Pancreatic Cancer Progression

doi: 10.1371/journal.pone.0002475

Figure Lengend Snippet: Microarray signal intensities recorded for WSB1 (arbitrary units)

Article Snippet: The human pancreatic cancer-derived Mia-PaCa2, BxPC3, Panc-1, Capan-1 and Capan-2, and the Phoenix Amphotropic viral packaging cell lines were cultivated as recommended by American Type Culture Collection.

Techniques: Microarray

Journal: PLoS ONE

Article Title: The WSB1 Gene Is Involved in Pancreatic Cancer Progression

doi: 10.1371/journal.pone.0002475

Figure Lengend Snippet: Expression of WSB1 isoforms 1+2 and 3 mRNAs was assessed by qRT-PCR in pancreatic cancer cell lines and in mice xenografted tumors (A), in human pancreatic cancer samples (B), and in Mia-PaCa2 cells after treatment with stress-inducing agents (C). Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate. (*, p<0.05).

Article Snippet: The human pancreatic cancer-derived Mia-PaCa2, BxPC3, Panc-1, Capan-1 and Capan-2, and the Phoenix Amphotropic viral packaging cell lines were cultivated as recommended by American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: The WSB1 Gene Is Involved in Pancreatic Cancer Progression

doi: 10.1371/journal.pone.0002475

Figure Lengend Snippet: A. Mia-PaCa2 cells were transfected with a siRNA directed against WSB1 mRNA and expression of WSB1 isoforms 1+2+3, 1+2 and 3 mRNAs was analyzed by qRT-PCR. B. Mia-PaCa2 cells were transfected with the WSB1 siRNA and growth was analyzed by direct cell counting (B), MTT analysis (C) or by BrdU incorporation (D) as described in Material and Methods section. E. Mia-PaCa2 cells were transfected with the WSB1 siRNA and caspase 3 activity was measured in untreated and after treatment with staurosporine or doxorubicin. Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate. (*, p<0.05).

Article Snippet: The human pancreatic cancer-derived Mia-PaCa2, BxPC3, Panc-1, Capan-1 and Capan-2, and the Phoenix Amphotropic viral packaging cell lines were cultivated as recommended by American Type Culture Collection.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Cell Counting, BrdU Incorporation Assay, Activity Assay

Journal: PLoS ONE

Article Title: The WSB1 Gene Is Involved in Pancreatic Cancer Progression

doi: 10.1371/journal.pone.0002475

Figure Lengend Snippet: A. Mia-PaCa2 cells were transduced with retrovirus expressing WSB1 isoforms 1, 2 or 3 as EGFP fusion protein or EGFP alone as a control and selected with puromycin. Cell growth was measured by direct counting after plating 10 5 cells in 48-well dishes every day during 4 days. B. After synchronization with 24 h serum-starvation, cells were incubated in serum containing medium for 24 h, stained with propidium iodide and cell cycle was analysed with a flow cytometer. C. S/G1 ratio. Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate (A) or trice in triplicate (B and C) (*, p<0.05).

Article Snippet: The human pancreatic cancer-derived Mia-PaCa2, BxPC3, Panc-1, Capan-1 and Capan-2, and the Phoenix Amphotropic viral packaging cell lines were cultivated as recommended by American Type Culture Collection.

Techniques: Transduction, Expressing, Incubation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: The WSB1 Gene Is Involved in Pancreatic Cancer Progression

doi: 10.1371/journal.pone.0002475

Figure Lengend Snippet: Mia-PaCa2 cells were transduced with retrovirus expressing WSB1 isoforms 1, 2 or 3 as EGFP fusion protein or EGFP as control and selected with puromycin. Cells were treated with gemcitabine (100 and 150 µM) for 48 hours or with doxorubicin (150 and 350 µM) for 6 hours. Cell viability was measured by MTT. Values are expressed as the mean±S.D. of combined results from two independent experiments performed in triplicate. (*, p<0.05).

Article Snippet: The human pancreatic cancer-derived Mia-PaCa2, BxPC3, Panc-1, Capan-1 and Capan-2, and the Phoenix Amphotropic viral packaging cell lines were cultivated as recommended by American Type Culture Collection.

Techniques: Transduction, Expressing

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: (A) HepG2 cells were treated with various doses of Vel as indicated in the presence (2.5, 5.0 mM) or the absence of LC for 24 h, cell viability was detected by MTS assay. Mean+SD (n = 3). Combination index (CI) was shown. Vel: bortezomib; DM: DMSO; LC: L-carnitine. (B) Human hepatic SMMC-7721 cancer cells were treated with LC and/or Vel for 48 h, cell viability was detected by MTS assay and PARP cleavage was detected by Western blot. CI was shown. (C) HepG2 cells were treated with the combination of Vel (50 nM) and LC (5 mM) for 48 h and cell apoptosis was labeled with Annexin V and propidium iodide (PI), and detected by flow cytometry. Representative flow images were shown and cell death data was summarized. Mean+SD (n = 3). * P <0.05, compared with Vel or LC treatment alone; # P <0.05, compared with Veh control. (D) as treated in C, living cells were directly stained with PI and imaged under an inverted fluorescent miscoscope. The phase contrast and fluorescent images were taken and merged. Red indicates PI-positive. Typical images were shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: MTS Assay, Western Blot, Labeling, Flow Cytometry, Control, Staining

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: ( A ) p21 cip1 gene expression. HepG2 cancer cells were either treated with various doses of LC (2.5, 5, 10 mM) for 24 h, or with 50 nM of Vel for 9 h and 24 h. A mixture of three cell samples were collected and DNA microarray assay was performed. Fold increase of the LC-treated versus control was shown. ( B ) p21 cip1 mRNA expression. HepG2 cells were exposed to either LC (5 mM) and Vel (50 nM) or the combination for 18 h; a mixture of three cell samples were extracted for mRNA assay by real-time-PCR. Fold increases of p21 cip1 and p27 kip1 were shown. ( C ) p21 cip1 protein expression and histone acetylation. HepG2 cells were treated with the combination of LC (5 mM) and various doses of Vel as indicated for 24 h, protein levels were detected by Western blot. Antibodies against p21 cip1 , p27 kip1 , histone and acetylated histones were used. GAPDH was used as a loading control. At least three repeats were performed and representative images were shown. ( D ) ChIP assay. HepG2 cells were treated with either vehicle or Vel (50 nM) for 24 h; cells were collected for ChIP assay. Fold enrichment of p21 cip1 and p27 kip1 promoter gene was summarized.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Gene Expression, Microarray, Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: ( A ) HepG2 cells were treated with LC (5 mM) and/or Vel (25, 50, 75 nM) for 24 h, and ubiquitinated proteins were detected by Western blot. ( B ) HepG2 cells were treated with either vehicle or LC (5 mM) for 24 h, then various doses of Vel were added to the culture medium for 8 h and chymotrypsin-like (CT-like) activity in situ was detected. Mean+SD (n = 3). * P <0.05, compared with the control at each point. ( C ) The natural bond Orbital (NBO) charge and geometric optimization were calculated by the DFT method and the NBO charges in the hydroxyl O atom of threonine or acetylthreonine was shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Western Blot, Activity Assay, In Situ, Control

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: (A) HepG2 cells were treated with LC (5 mM), Vel (25, 50, 75 nM) and the combination of them for 24 h. Western blot was performed for the detection of the related proteins including caspases, PARP and ER-related proteins. GAPDH was used as a loading control. (B) As treated and analyzed in , ER-stress-related gene expression as indicated was shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Western Blot, Control, Gene Expression

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: ( A ) As treated and analyzed in , Bax and Bcl-2 gene expression was shown. ( B ) As treated and analyzed in , Bax mRNA was detected by real-time PCR and fold increase of Bax mRNA was shown. ( C ) As treated in , Bax and Bcl-2 protein levels were detected. Representative images were shown. ( D ) HepG2 cells were transfected with Bax-siRNA (#1) for 48 h and then treated with the combination of LC (5 mM) and Vel (50 nM) for 24 h, Western blot was performed to detect Bax and PARP cleavage. GAPDH was used as a loading control.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Control

Journal: PLoS ONE

Article Title: HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

doi: 10.1371/journal.pone.0052576

Figure Lengend Snippet: Nude mice bearing HepG2 tumor were i.p injected with vehicle or LC (400 mg/kg, i.p. once/day except day 8), Vel (0.75 mg/kg, i.v. once/3 days) alone and the combination respectively for 15 days. A, Tumor images, tumor weight, body weight, tumor growth curve including Box-plot image and the relative tumor volume in all the four groups (Vehicle-, LC-, Vel- and LC+Vel-) were shown. * P <0.05, compared with the Veh control; # P <0.05, compared with the treatment alone, ## P >0.05, compared with the treatment alone. B, p21 cip1 and acetylated H3 proteins in tumor tissues of various groups were detected by immunochemistry and the results were shown respectively. All the immunostaining were repeated in three mouse tumor tissues and the typical images were shown.

Article Snippet: Human hepatoma HepG2, SMMC-7721 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% FBS in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Injection, Control, Immunostaining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, Human samples of normal urothelium (N=10) and urothelial carcinoma of the bladder (N=8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma compared to the normal urothelium. B, Upregulation of these genes was validated by two-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N=3) and normal urothelium (N=3). 10 of 13 genes (asterisked) demonstrated statistical significance (T-test P-value < 0.05) for differential expression in urothelial carcinoma compared to normal urothelium.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: MLN8237 induces cell cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 demonstrated specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, Propidium iodide staining with flow cytometry analysis was performed to assess cell cycle changes. T24, UM-UC-3, and RT4 cells were treated with 10 nM to 1 μM MLN8237 for 48 h. All three cell lines demonstrated dramatic cell cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also demonstrated a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Cellular phenotypes of T24 and RT4 cells differ after MLN8237 treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, Immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells demonstrate a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic aurora A expression, whereas RT4 cells lacked cytoplasmic aurora A expression. E, Real-time imaging of T24 and RT4 cells treated with MLN8237 was performed over 48 h. T24 cells exhibited dramatic increases in cell size as a result of repeated cell cycle progressions without separation of daughter cells. RT4 cells appeared to become arrested after one failed mitotic attempt, preventing repeated cell cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Fluorescence, Microscopy, Expressing, Imaging

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: MLN8237 induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 h. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 nM and 45 nM, respectively) and lowest potency in RT4 cells (IC50 of 120 nM). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 h after initiation of treatment. C, Annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 h after initiation of MLN8237 treatment. D, Clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nM MLN8237.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Flow Cytometry, Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 h (left column), or sequentially, with one drug for 48 h, followed by washout and the other drug for 48 h (middle and right columns). MTS assay was utilized to quantify the effect on cell viability of these combination treatments. MLN8237 demonstrated synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right columns), and antagonistic effects when dosed simultaneously (left column).

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV envelope protein (ZIKV-E, green) and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h post-infection (p.i.) with ZIKV. Scale bar, 50 μm. (B) Quantification of infection efficiency in four GSC and NPC lines 48 h p.i. with ZIKV. (C) Quantification of ZIKV+ cells in a panel of human GSCs and NPCs. (D) Kinetics of viral RNA copies p.i. with ZIKV by measuring viral RNA copies by qRT-PCR in NPC C4–7 and GSC3565. (E) ZIKV infection efficiency of GSCs and NPCs was measured by direct measurement of viral RNA copies. (F) Representative bright-field images 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. Scale bars, 50 μm. (G) Cell viability normalized to day 5 mock, as measured 5 days p.i. with ZIKV for GSCs, NPCs, and primary astrocytes. (H) GSCs (GSC3565), differentiated GSCs, NPCs (NPC C4–7), and differentiated NPCs were assayed for cell viability 72 h p.i. with ZIKV. (I) Apoptosis of GSCs (387, 3565) and primary (NPC194, fetal human [fh] NPC) or iPSC-derived NPCs (WT83, C4–7) p.i. with ZIKV was measured by cleaved caspase-3 (CC3) staining. (J) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (K) Representative immunostaining for ZIKV-E (green), CC3 (red), and DAPI (blue) of GSCs and forebrain-specific NPCs 72 h p.i. with ZIKV. Scale bars, 50 μm. (L) Quantification of the percentage of CC3+ cells in DAPI+ cells for GSCs and NPCs 72 h p.i. with ZIKV. (M) Cell viability of patient-derived cultures from GBM (387 and 3565), pontine glioma (3752 and 007), meningioma (CH-157MN, IOMM-LEE), ependymoma (EP1), and medulloblastoma cell lines (DAOY, D283, HDMB03, D341) 72 h after ZIKV infection. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. NS, no significance. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Quantitative RT-PCR, Derivative Assay, Staining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative immunostaining for ZIKV-E (green), SOX2 (red), and DAPI (blue) of GSCs and forebrain-specific hiPSC-derived NPCs 48 h p.i. with ZIKV. Scale bar, 50 μm. (B) Quantification of the percentage of SOX2+ cells in DAPI+ cells for GSCs and NPCs 48 h p.i. with ZIKV. (C) Representative immunostaining for ZIKV-E (green), SOX2 or AXL (red), and DAPI (blue) of GSCs (GSC3565) without transduction (shRNA) or transduced with control shRNA (shCONT), AXL shRNA (shAXL.2), or SOX2 shRNA (shSOX2.53) for 72 h and then 48 h with ZIKV infection. Scale bars, 100 μm. (D) Quantification of the percentage of ZIKV+ cells in DAPI+ cells in GSCs 1517 and 3565 under conditions for (C), with a range of ZIKV infection. (E) Viral copy number by qRT-PCR of GSCs (GSC3565 or GSC1517) or NPC C4–7 transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53) for 72 h and then either exposed to mock conditions or infected with ZIKV for another 72 h. All comparisons are versus shCONT. (F) Gene set enrichment (GSE) bubble plots showing pathways positively (top, r > 0.4) or negatively (bottom, r < −0.4) correlated with SOX2 expression in the TCGA GBM HG-U133A microarray dataset. Each circle represents an enriched pathway, with the border color indicating the false discovery rate (FDR)-corrected p value. (G) GSE graph showing the top pathway enrichments positively or negatively correlated with SOX2 as described in (F). (H) Correlation of mRNA levels of SOX2 with IFNAR1, IRF1, promyelocytic leukemia (PML), and IFITIM1 from the TCGA GBM HG-U133A microarray dataset. (I) Correlation between SOX2 with ISGs from the TCGA GBM HG-U133A microarray dataset. The size and color of the dots indicate the degree of correlation (p < 0.001). Blank cells indicate a non-significant correlation. (J) qPCR of ISGs (IFNAR-1, ISH20, IRF1, IFITM1, TLR3, and OAS2) in GSCs (GSC3565) transduced with either shCONT or SOX2 shRNA (shSOX2.52 or shSOX2.53). Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. **p < 0.001, ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Derivative Assay, Transduction, shRNA, Control, Infection, Quantitative RT-PCR, Expressing, Microarray

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative images of mock- or ZIKV-infected BCOs stained with neuronal markers (CTIP2 and NeuN), a neural progenitor cell marker (SOX2), and DAPI. Scale bars, 100 μm. (B) Quantification of BCO size p.i. with ZIKV. Significance was assessed by two-tailed Student’s t test, and experiments were performed in two batches with 12 organoids per group per batch. (C) BCO size fold change of ZIKV- and mock-treated groups over a period of 1 month. (D) Quantification of SOX2+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (E) Quantification of CC3+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (F) Quantification of SATB2+ cells within MAP2+ cells in ZIKV- versus mock-infected groups. **p < 0.01 by two-tailed Student’s t test. (G) Quantification of GFAP+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (H) Quantification of NeuN+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (I) Quantification of CTIP2+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (J) Bright-field images of engraftment of two patient-derived GSCs (387 and 3565) transduced with GFP into human BCOs over a time course. Scale bars, 1 mm. (K) Engrafted GSCs (GFP+) with normal BCO immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 200 μm. (L) Quantification of integrin αvβ5+ cells in normal BCOs or GSC-BCOs. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (M) Representative images of GFP-labeled GSC-BCOs immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (N) Representative images of GFP-labeled GSC-BCOs immunostained for SOX2 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (O) Images of GFP-labeled GSC-GFP BCOs 13 days p.i. with ZIKV. Scale bars, 1 mm. (P) Representative images of residual GSCs (green) and DAPI staining (blue) of GFP-labeled GSC-GFP BCOs cultured under mock conditions or with ZIKV for 2–4 weeks. Scale bars, 200 μm. The percentage of GFP+ cells among DAPI+ cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-way ANOVA. (Q) Representative immunostaining for integrin αvβ5 (red), GFP (green), ZIKV-E (white), and DAPI (blue) of GFP-labeled GSC-GFP BCOs mock- or ZIKV-infected for 2–4 weeks. Scale bars, 200 μm (left) and 100 μm (center). The percentage of ZIKV-E+ cells among integrin αvβ5 cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (R) Representative images of 387 and 3565 GSC-BCOs with or without ZIKV, respectively, stained with SOX2, ZIKV-E, and DAPI. GFP shows the presence of GSCs (scale bars, 50 μm). ZIKV-E+, GFP+, and ZIKV-E+ cells among GFP+ cells were quantified by counting (two GSCs cell lines, two repeats, n = 12 organoids/group); *p < 0.05 by two-tailed Student’s t test. (S) Schematic of the experiment design. (T) Volcano plot showing differences between GSC-BCO ZIKV versus GSC-BCO mock. 113 genes were differentially expressed (greater than 1.5-fold) between these two groups (*p < 0.05). (U) Network analysis of genes differentially expressed upon ZIKV infection, represented as a bubble plot.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Infection, Staining, Marker, Two Tailed Test, Derivative Assay, Transduction, Labeling, Cell Culture, Immunostaining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Immunostaining of the subventricular zone (SVZ) of mice 72 h following ZIKV infection ZIKV-E (green), SOX2 (red, top panels), and integrin αvβ5 (red, bottom panels). Scale bars, 50 μm. (B) Higher magnification of images from (A), demonstrating ZIKV infection of SOX2+ (top panels) and integrin αvβ5+ cells. Scale bars, 10 μm. (C) Survival of ZIKV-infected NSG mice from (A) was plotted by the Kaplan-Meier method. (D) ZIKV-infected brains from the mice in (A) were collected upon death, and histology was assessed by H&E staining. Scale bars, 20 μm. (E) Survival of NSG mice following implantation of GSCs treated with isotype control, P1F6 antibody, ZIKV, combined P1F6 and ZIKV, combined CRISPR knockout (KO) of integrin β5 (sgRNA1 sgRNA2) with ZIKV inoculation, analyzed by log rank test; p < 0.01. (F) H&E staining of tumor-bearing brains from (E). Scale bars, 50 μm. (G) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection (10e3 FFU). Slices then underwent immunofluorescence staining for ZIKV-E (green), integrin αvβ5 (red), and DAPI (blue). Scale bars, 10 μm. (H) Intraoperative brain slices from GBM patients were pre-incubated with an IgG control antibody or an integrin-blocking antibody under mock conditions or upon ZIKV infection. Slices then underwent a viral RNA copy assay by qRT-PCR. Experiments were performed in two biological replicates with three technical repeats. Values represent mean ± SEM. ****p < 0.0001 by one-way ANOVA.

Article Snippet: After 30 minutes, the reaction cocktail was removed, cells were washed once with 1 mL of 3% BSA in PBS before proceeding to DNA staining (DAPI, Vector Laboratories H-1200) and imaging (Zeiss Apotome). . ZIKV preparation ZIKV human isolates H/PAN/2016/BEI-259634 and PRVABC59 (BEI Resources, NR-50210 and NR-50240) from Panama and Puerto Rico, respectively, were acquired from the ATCC and distributed by BEI.

Techniques: Immunostaining, Infection, Staining, Control, CRISPR, Knock-Out, Incubation, Blocking Assay, Immunofluorescence, Quantitative RT-PCR

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited the proliferation and induced the apoptosis of PCa cells. The IC50 of mannose in ( a ) DU145 and ( b ) PC3 cells was determined using a CCK-8 assay. ( c ) Intracellular mannose concentration in PCa cells. Cell proliferation of ( d ) DU145 and ( e ) PC3 was assessed using growth curves, respectively. ( f ) Colony formation assays were performed and ( g ) colony numbers were counted in PCa cells. ( h ) Flow cytometric analysis was used to assess ( i ) the apoptosis rate of PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; IC50: the half-maximal inhibitory concentration; CCK-8: Cell Counting Kit-8; AAD: Aminoactinomycin D; APC: Allophycocyanin.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: CCK-8 Assay, Concentration Assay, Cell Culture, Cell Counting

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose inhibited tumor growth in a PCa xenograft model without affecting mice health. ( a ) Subcutaneous tumors from the xenograft model with DU145 cells. ( b ) Tumor growth was monitored for 30 days after mannose treatment. ( c ) The volume of tumor growth. ( d ) The weight of the tumors. ( e ) Intratumoral mannose concentration and ( f ) ATP content in subcutaneous tumors. The weights of ( g ) mice and ( h ) major metabolic organs. ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. PCa: prostate cancer; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Concentration Assay, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted mitochondrial function, led to ROS overproduction, and activated Bax/Bak in PCa cells. ( a ) JC-1 staining and ( b ) rhodamine 123 staining were used to assess the MMP in DU145 and PC3 cells. ( c ) The ATP content in cells. ( d ) Mitochondrial ROS and ( e ) cellular ROS levels in cells. ( f and g ) The protein expression of Bax and Bak in cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine; Bax: BCL2-associated X; Bak: BCL2-antagonist/killer 1; ROS: reactive oxygen species; MMP: mitochondrial membrane potential; ATP: adenosine triphosphate; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Expressing, Cell Culture, Membrane

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Mannose disrupted the balance of mitochondrial dynamics in PCa cells. ( a ) Mitochondria stained with MitoTracker Red were observed by confocal microscopy. ( b ) Mitochondrial structure under a transmission electron microscopy, and the mitochondrial cross-sectional area was quantified. ( c ) The protein expression of FIS1 in cells. Upregulated ( d ) FIS1 and ( e ) increased ATP content in PCa cells. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. ATP: adenosine triphosphate; FIS1: fission, mitochondrial 1; PCa: prostate cancer.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Staining, Confocal Microscopy, Transmission Assay, Electron Microscopy, Expressing, Cell Culture

Journal: Asian Journal of Andrology

Article Title: Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism

doi: 10.4103/aja2021104

Figure Lengend Snippet: Downregulation of MPI expression enhances the anticancer effect of mannose. The expression of MPI in human prostate cancer tissues and its prognostic value. ( a and b ) The expression of MPI was silenced by siRNA-MPI and verified by western blotting. siRNA-MPI with different base sequences including si-1, si-2 and si-3 were used for downregulating the MPI expression in PCa cells. According to the degree of down-regulation of MPI protein, si-3 and si-2 with the best interference effect were applied to DU145 and PC3 cells, respectively. ( c ) Intracellular mannose concentration and ( d ) ATP content in cells. ( e ) Growth curves and ( f ) colony formation assays of cells. ( g ) The IHC scores for MPI expression in PCa tissues. ( h ) Kaplan–Meier curves of BCR-free survival and ( i ) overall survival for the low and high MPI expression groups of patients in the TCGA-PRAD dataset. * P < 0.05, ** P < 0.01. NC: PCa cells cultured in normal medium. MAN: PCa cells cultured in normal medium with 25 mmol l −1 mannose for DU145 or with 50 mmol l −1 mannose for PC3. MPI: mannose phosphate isomerase; si: siRNA, small interfering RNA; IHC: immunohistochemistry; PCa: prostate cancer; BCR: biochemical recurrence; TCGA-PRAD: The Cancer Genome Atlas-Prostate Adenocarcinoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TMA: tissue microarray; ATP: adenosine triphosphate.

Article Snippet: The human PCa cell lines DU145 and PC3 were obtained from the American Type Culture Collection (ATCC® HTB-81 and ATCC® CRL1435, Manassas, VA, USA) and grown in Dulbecco's modified Eagle medium (DMEM; C11965500BT, Gibco, Paisley, UK) containing 10% fetal bovine serum, streptomycin, and penicillin.

Techniques: Expressing, Western Blot, Concentration Assay, Cell Culture, Small Interfering RNA, Immunohistochemistry, Microarray