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 Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) A provisional network was generated from integration of two microarray data sets. Node color represents increases (red), no significant changes (yellow), and decreases (green) in gene expression in murine prostate tissue after cholesterol alteration as ascertained by cDNA microarray. Changes in RNA expression levels of the corresponding nodes in LNCaP cells are shown as colored node boundaries (donut shape) and the color represents increases (red), no significant change (yellow), and decreases (green) in gene expression under CDM conditions compared to control. Arrows indicate direct activation, T-shaped lines direct repression, dashed arrows indirect activation, and lines physical interaction. ( B ) Gene expression under Normo and Hyper conditions ( in vivo ). To verify in vivo microarray data obtained from SCID experiments, mRNA levels of the indicated genes were determined. GAPDH expression was used to normalize gene expression. Error bars represent SD (n = 3). ( C ) Gene expression under Control and Cholesterol-depleted conditions ( in vitro ). LNCaP cells were incubated in CDM for 0, 3 or 16 h, and mRNA levels of the indicated genes were measured by RT-PCR analysis to validate cDNA microarray data. Error bars represent SD (n = 3). * p <0.05 (Student’s t-test).

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Generated, Microarray, Gene Expression, RNA Expression, Control, Activation Assay, In Vivo, Expressing, In Vitro, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) RT-PCR analysis in vivo . ATF3 levels are reduced in all prostatic lobes from Hyper mice, compared to those from the Normo group (AP = anterior prostate; VP = ventral prostate; DLP = dorsal prostate). ( B ) Immunoblot analysis. Immunoblot of PrEC lysates showed induction of ATF3 protein by CDM (left panel) and by β-cyclodextrin (right panel). MG132, a proteasome inhibitor, also increased ATF3 expression. ( C ) Immunofluorescence analysis. Induction of ATF3 protein by CDM in LNCaP cells as shown by IF. LNCaP cells were treated with CDM for 18 h, stained with anti-ATF3 antibody and nuclei were counterstained with DAPI (left panel: ATF3; middle panel: DAPI; right panel: overlay). ( D ) RT-PCR analysis. ATF3 mRNA levels in LNCaP cells treated with CDM were normalized to levels of GAPDH. RT-PCR analysis shows induction of ATF3 mRNA levels by CDM. ( E–F ) Promoter reporter analysis. A full-length ATF3 promoter was cloned into a luciferase reporter vector and transfected into LNCaP (D) or PrEC (E). Cells were then incubated in Control and CDM medium. ATF3 promoter activity was plotted as arbitrary units (± SD) after normalization with total protein concentration.

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Control, Activity Assay, Protein Concentration

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: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) The Cancer Genome Atlas (TCGA) database analyses demonstrate RBM3 mRNA levels are significantly higher in colon cancer (T) (n=286) than in normal colon (N) (n=41) (p<0.001). (B) RBM3 mRNA expression from a cDNA array of colon tumor (T) samples (n=24) and matched adjacent normal colon tissue (N) (n=24) normalized to β-actin, shows a significant increase of RBM3 expression in tumor tissue (p=0.023). (C) Immunohistochemistry (IHC) of a colon cancer tumor microarray shows that RBM3 is upregulated in colon adenocarcinoma along with lymph node metastasis and liver metastasis as compared to the normal colon, lymph node, and liver. (D) Composite score of colon cancer TMA shows significantly higher expression of RBM3 in tumor (n=28) (p=0.002) and metastasis (n=30) (p=0.036) as compared to normal tissue. RBM3 expression is also increased in the different stages of colon cancer (Stage I (n = 3), Stage II (n = 12) (p=0.015), Stage III (n= 11) (p=0.07), Stage IV (n = 2) and metastasis (n=30) (p=0.036)) as compared to normal colon, liver and lymph node (n=3 each). (E) Western blot analysis of RBM3 protein expression in established colon cancer cell lines HCT116, DLD1, SW480, SW620, HT29, RKO and LST17T as compared to normal colon epithelial cells (FHC cell line). Data in are represented as ± SEM. Also, see Supplementary figure 1

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Volcano plots for RNA-sequencing (RNA-seq) showing differentially expressed lncRNA in both HCT116 and DLD1 RBM3 overexpressing (RBM3 O/E) cell lines compared to empty vector (EV) cell lines. (B) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-seq. (C) The volcano plots for RNA-immunoprecipitation coupled sequencing (RNA-IP seq) showing differentially expressed lncRNA in both HCT116 and DLD1, RBM3 overexpressing cell lines compared to empty vector cell lines. (D) Venn diagram depicting the number of co-expressed and uniquely expressed lncRNA found in RNA-IP seq. (E-F) Plot from REVIGO software showing the Gene Ontology (GO) terms enriched in lncRNA (RNA-seq) for HCT116 RBM3 (E). The graph has been modified to highlight the GO terms and color key. The box plot drawn highlighting enriched GO terms(F). (G) RT-PCR validation of lncRNA identified through RNA-seq in colon cancer cell lines compared to normal FHC cells. (H) RT-PCR validation of lncRNA identified through RNA-seq, in the RBM3 overexpressing (RKO, HCT116, and DLD1) cells, show increased expression of lnc-HOTAIR, lnc-TUG1, lnc-Flii- 1, lnc-LSAMP-3. (I) Western blot analysis for the expression demonstrating levels of RBM3 in the distal colon in representative wild type littermates and RBM3 transgenic mice (RBM3 O/E). (J) RT-PCR analysis demonstrating increased expression of lncRNA lnc-Flii-1 and lnc-LSAMP- 3 compared to GAPDH in representative wild type (WT) littermates and RBM3 overexpressing transgenic mice (RBM3 O/E). Data in are represented as ± SEM. Also, see Supplementary figure 2.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: RNA Sequencing, Plasmid Preparation, RNA Immunoprecipitation, Sequencing, Software, Modification, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Transgenic Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) Quantitative PCR shows increased mRNA levels of VEGFA, ZEB1, TWIST, and SNAI2 in RBM3 overexpressing cells compared to empty vector cell lines. (E) Plot for tube length of the endothelial tubular network formed by HUVEC cell in (G). Increased tube formation in HUVEC cells treated by condition media from HCT116 (p=0.038) and DLD1 (p=0.004) RBM3 overexpressing compared to empty vector cells (F) Plot showing increase in migration of HCT116 (p=0.001), DLD1 (p=0.02) and RKO (p=0.04) RBM3 overexpressing cells compared to empty vector performed by wound healing assay. (G) Plot showing increase in migration of HCT116 (p=0.006), DLD1 (p=0.002) and RKO (p=0.006) RBM3 overexpressing cells compared to empty vector performed by transwell migration assay. (H) Plot showing increase in invasion of HCT116 (p<0.001), DLD1 (p<0.01) and RKO (p<0.01) RBM3 overexpressing cells compared to empty vector performed by transwell invasion assay. Data in are represented as ± SEM. Also, see Supplementary figure 3.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Migration, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A-D) The plot of tumor weights (A, C) and tumor volumes (B, D) from HCT116 and DLD1 empty vector and RBM3 overexpressing xenografts. The tumor volume and weight were significantly increased in RBM3 overexpression compared to control (p<0.05) (E) RT-PCR validation of lncRNA in the tumor xenograft tissues, in both HCT116 and DLD1 xenografts. RBM3 overexpressing xenografts show an increase in lnc-Flii-1 and lnc-LSAMP-3 expression compared to empty vector. (F) RT-PCR validation for increased expression of TWIST1, SNA2 and VEGFA in HCT116 and DLD1 empty vector and RBM3 overexpressing xenograft tumors tissues. (G) Western blot analysis of tumors from HCT116 and DLD1 xenograft tissues for VEGF expression. (H) Increassed RBM3 and PCNA levels in HCT116 and DLD1 RBM3 overexpressing xenograft tissues as compared to empty vector as seen by immunohistochemistry analysis. (I) Increase in percentage of PCNA positive nuclei in RBM3 overexpressing HCT116 (p=0.022) and DLD1 (p=0.025) xenograft tissues immunohistochemistry. The data for are presented as the means ± SEM, (n=10). Also, see Supplementary figure S5.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Plasmid Preparation, Over Expression, Control, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Western Blot, Immunohistochemistry

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A) Protein expression of RBM3 decreases in shRNA knockdown clones (2,3,5) compared to scramble (scr1,scr2) in the HCT116 cell line by Western blot analysis. (B) RT-PCR validation for the decreased expression of lnc-LSAMP-3 and lnc-Flii-1 in HCT116 RBM3 shRNA knocked down clone compared to scramble. (C) Plot for decrease in percentage proliferation of the RBM3 shRNA knockdown clones over empty vector for sh2 (proliferation= 55.06%, p=0.04), sh3 (proliferation= 53.8%, p=0.0023), sh5 (proliferation= 47.45%, p=0.0001). (D) The plot showing decrease in percent migration in HCT116 shRNA knocked down clones (sh2 p=0.044 and sh5 p=0.048) compared to scramble performed by Transwell migration. (E) The plot showing decrease in percent invasion in HCT116 shRNA knocked down clones (sh2 p=<0.001 and sh5 p=0.001) compared to scramble performed by Transwell invasion assay. (F and G) The plot of tumor weights (F) and tumor volumes (G) from HCT116 scramble (scr2) and RBM3 shRNA knockdown (shRNA-2) xenografts. Both tumor volume and weight were significantly reduced in RBM3 knockdown compared to scramble control (p<0.05). (H) Kaplan-Meier analysis showing increased percent survival after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout (KO) compared to wild type (WT) mice. (I) Plot showing decreased intestinal permeability in RBM3 knockout mice as compared to the wild type littermates (p=0.04). (J) The plot showing decreased number of tumors in the distal colon observed after the AOM/DSS induced carcinogenesis in cre inducible RBM3 knockout compared to wild type mice (p=0.007). (K) Representative image of H and E staining of the distal colon with tumors after the AOM/DSS induced carcinogenesis in RBM3 knockout and wild type mice. (L) Decrease in expression of lnc-LSAMP-3 and lnc-Flii-1 seen by PCR analysis in RBM3 knockout mice as compared to the wild type mice. The data for are presented as the means ± SEM, (mice n=10). Also, see Supplementary figure 6.

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Expressing, shRNA, Knockdown, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Migration, Transwell Invasion Assay, Control, Knock-Out, Permeability, Staining

Journal: bioRxiv

Article Title: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

doi: 10.1101/2021.12.14.472669

Figure Lengend Snippet: (A and B) RT-PCR validation of lnc-LSAMP-3 and lnc-Flii-1 knocked down in HCT116 (A) and DLD1 (B) RBM3 O/E and empty vector cells. (C and D) RT-PCR validation of VEGFA, ZEB1 and TWIST in HCT116 (C) and DLD1 (D) RBM3 O/E and empty vector cells, after lncRNA (lnc-Flii-1 and lnc-LSAMP-3) knockdown. (E) Plot showing decreased percent migration of HCT116 empty vector and RBM3 overexpressing cells having knockdown of lncRNA lnc-LSAMP-3 (Empty vector p=0.017, RBM3 O/E p=0.031) and lnc-Flii-1(Empty vector p=ns, RBM3 O/E p=0.011) performed by scratch plate assay. (F) Plot showing decreased tube length of the endothelial tubular network formed by HUVEC cell treated by condition media from knockdown of lncRNA lnc-LSAMP-3 and lnc-Flii-1(Empty vector p=0.012, RBM3 O/E p=0.022) in HCT116 cells. (G-J)The plot showing decrease in tumor weight (G, I) and tumor volumes (H, J) from HCT116 and DLD1 xenografts treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control (p< 0.05). (K and L) RT-PCR validation for the decreased expression of lncRNA (lnc-LSAMP-3 and lnc- Flii-1) in the tumor xenograft of HCT116 (K) and DLD1(L) treated with intratumoral injection of si+ LNA gapmer for lnc-Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. (M and N) Western blot showing decreased protein expression of SNAI2 and VEGF in HCT116 (M) and DLD1 (N) tumor xenograft treated with intratumoral injection of si+ LNA gapmer for lnc- Flii-1 knockdown and lnc-LSAMP-3 knockdown compared to control. The data for are presented as the means ± SEM. Also, see figure Supplementary figure

Article Snippet: Human colon cancer cells HCT116, DLD1, RKO and endothelial cell line HUVEC (all cell lines obtained from American Type Culture Collection, at passage 4).

Techniques: Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Plasmid Preparation, Knockdown, Migration, Injection, Control, Expressing, Western Blot

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 inhibits tube formation and influences osteomimicry. (A) Tube formation assay with constitutively transfected A673 (sh.control and sh. CHM 1) and transiently transfected MHH ‐ ES 1 (si.control and si. CHM 1_1) cells demonstrated CHM 1 to clearly inhibit endothelial differentiation potential (scale bar 0.5 mm). (B) Analysis of osteolysis of A673 sh. CHM 1 and negative controls (sh.control) in an orthotopic bone xenotransplantation model (five to eight mice per group). Affected bones were assessed by histology ( TRAP staining, scale bar 0.25 mm or 0.05 mm). Left panel: quantitative summary of the average number of osteoclasts (mm 2 ) in unaffected bone marrow, tumor samples, and attached to the bone in tumor tissues (bone). Data are mean ± SEM of at least two independent samples (at least 40 segments counted); t ‐test. Right panel: Representative pictures are shown. CHM 1 knockdown significantly enhanced the amount of TRAP ‐positive osteoclasts attached to the bone (b) in the area of tumor (arrow) and thus increased the osteolytic phenotype. (C) Different ES cell lines with constitutive CHM 1 knockdown and respective controls were analyzed by qRT ‐ PCR for expression of osteolytic genes such as HIF 1A , IL 6 , JAG 1 , and VEGF . Data are mean ± SEM of two independent experiments; t‐ test. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: Tube Formation Assay, Transfection, Control, Staining, Knockdown, Quantitative RT-PCR, Expressing

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 delayed proliferation in ES in vitro . (A) Analysis of contact‐dependent growth of constitutively sh. CHM 1‐ and sh.control‐infected ES cell lines with xCELL igence. Left panel: Cellular impedance was measured every four hours (relative cell index). Data are mean ± SEM (hexaplicate/group); t ‐test. Right panel: doubling time of constitutive A673, SK ‐N‐ MC , and TC ‐71 CHM 1 sh RNA infectants. Data are mean ± SEM of two independent experiments/cell line (hexaplicate/group); t ‐test. B. Effect of CHM 1 knockdown on anchorage‐independent growth in A673, SK ‐N‐ MC , and TC ‐71 cells using methylcellulose matrices. Left panel: A representative experiment with SK ‐N‐ MC cells was shown as macrograph. Right panel: The average number of colonies of at least two different experiments with three different ES cell lines was shown after stable CHM 1 suppression. (C) Left panel: evaluation of tumorigenicity of constitutive A673 and TC ‐71 CHM 1 sh RNA infectants in immunodeficient Rag2 −/− γc −/− mice (3–5 mice per group). Right panel: post ex vivo CHM 1 expression using qRT ‐ PCR . Data are mean ± SEM , t ‐test. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: In Vitro, Control, Infection, Knockdown, Ex Vivo, Expressing, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: The endochondral bone protein CHM 1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

doi: 10.1002/1878-0261.12057

Figure Lengend Snippet: CHM 1 enhances metastasis in ES in vivo . (A) Analysis of invasiveness of ES cell lines through Matrigel after transfection with specific CHM 1 sh RNA constructs. Data are mean ± SEM of two independent experiments; t ‐test. (B) Upper panel: qRT ‐ PCR of MMP 9 expression after stable CHM 1 knockdown. Data are mean ± SEM of two independent experiments; t ‐test. Lower panel: analysis of the invasive potential of A673 and SK ‐N‐ MC cells after transient transfection with two specific MMP 9 si RNA s 48 h before seeding. Data are mean ± SEM ; t ‐test. (C) Analysis of metastasis using A673 and TC ‐71 cells with stable CHM 1 suppression and respective controls (four to five mice per group). Left panel: Representative lungs with corresponding H&E staining of A673‐injected mice are shown (scale bar 5 or 2 mm). Right panel: Average number of apparent metastases per mouse in lung and liver tissues is illustrated; t ‐test. (D) DotBlot of relative CHM 1 expression in ES osseous tumor samples compared to ES lung metastases samples using microarray analysis of 14 patient tumor samples. * P < 0.05; ** P < 0.005; *** P < 0.0005 (see 2.15. Statistical analyses).

Article Snippet: A673 was purchased from ATCC (LGC Standards, Teddington, UK).

Techniques: In Vivo, Transfection, Construct, Quantitative RT-PCR, Expressing, Knockdown, Staining, Injection, Microarray

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: ( a ) Cell proliferation and ( b ) cell viability alterations of HT-29 and SW480 colorectal cancer cell lines following different folic acid (FA) supplies. Sulforhodamine B (SRB) was used for cell proliferation detection (* p ≤ 0.05, *** p ≤ 0.001), while cell viability data were obtained by alamarBlue assay (** p ≤ 0.01). FA-depleted cells were kept in media containing 0 ng/mL FA, whereas treated cells were exposed to 100 and 10,000 ng/mL FA for 72 h. The percentages of cell proliferation and viability were given relative to samples kept in the normal growth media. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Alamar Blue Assay

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genomic stability detection of HT-29 and SW480 cells exposed to different folic acid (FA) concentrations (0, 100, 10,000 ng/mL). ( a ) Micronucleus (MN) scoring was performed on DAPI- and anti-γ-H2AX-stained slides. Left: We obtained the results by proportioning the cells with MN with all cells counted (** p ≤ 0.01, *** p ≤ 0.001). Right: Representative γ-H2AX-positive micronuclei are indicated with arrows. ( b ) DNA integrity was evaluated with comet assay, additionally. Left: Graphs show the changes in genomic stability in consideration of comet tail DNA percentage (* p ≤ 0.05). Right: Characteristic comets of both cell lines were captured following different treatments. FA: folic acid.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Staining, Single Cell Gel Electrophoresis

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: DNA methylation analysis of HT-29 and SW480 cell lines exposed to different folic acid (FA) concentrations. The methylation levels of long interspersed nuclear element 1 (LINE-1) CpG positions (pos 1, pos 2, pos 3) were ( a ) summarized and also ( b ) visualized individually to detect global DNA methylation changes. With the use of Reduced Representation Bisulfite Sequencing (RRBS) method, a genome-wide methylome profile of 10,000 ng/mL FA-treated cells was established in the comparison of cells kept in FA-free (0 ng/mL FA) media. ( c ) Firstly, the number of genes with altered methylation in the investigated CpG sites was assessed. “Hyper” and “hypo” sections indicate the number of genes with methylated and unmethylated CpG sites, respectively. The intersection of these two categories refers to the genes that possess both methylated and unmethylated CpG dinucleotides. ( d ) Heatmap shows the top 10 significantly ( p ≤ 0.05) enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with the number of differentially methylated genes. ( e ) Pie charts represent the localization of differentially methylated sites (DMS) in distinct chromatin states. FA: folic acid; pos: CpG position; hyper: hypermethylation; hypo: hypomethylation; DMSs: differentially methylated sites; heterochrom/lo: heterochromatin or low signal region; txn: transcription; CNV: copy number variation; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: DNA Methylation Assay, Methylation, Methylation Sequencing, Genome Wide, Comparison

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: Genome-wide transcriptome alterations of HT-29 and SW480 cells following 10,000 ng/mL folic acid (FA) supplementation detected by Human Transcriptome Array 2.0 (HTA 2.0). ( a ) Pie charts represent the proportion of up- and downregulated genes. ( b ) Visual networks of protein–protein interactions were generated by the StringApp of Cytoscape software based on the list of genes with significant ( p ≤ 0.05) expression alterations and ≥|1.5| fold change (FC). Colors refer to the expression level of protein-coding genes (dark blue: FC ≤ −2, light blue: FC ≥ −2 and ≤−1.5, light red: FC ≥ 1.5 and ≤2, dark red: FC ≥ 2). ( c ) Top 10 genes showing significant ( p ≤ 0.05) up- and downregulation visualized with volcano plots. Gray points represent all the transcripts detected by HTA 2.0 microarray, while significantly ( p ≤ 0.05) altering genes with FC ≥ |1.5| value were marked with red and blue. P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, Protein-Protein interactions, Generated, Software, Expressing, Microarray

Journal: Cancers

Article Title: Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines

doi: 10.3390/cancers14071820

Figure Lengend Snippet: The intersection of genome-wide DNA methylation and gene expression data obtained by Reduced Representation Bisulfite Sequencing (RRBS) and Human Transcriptome Array (HTA) 2.0 analyses. Values represent the methylome and transcriptome pattern changes of 10,000 ng/mL folic acid (FA)-treated HT-29 and SW480 cells compared to non-treated samples (0 ng/mL FA). Only genes with promoter methylation status alteration in accordance with their expression level ( p ≤ 0.05 and fold change ≥|1.5|) were listed (left) and also visualized in volcano plots (right). Gray points represent all the transcripts detected by the microarray, while blue ones highlight down- and red ones show upregulating genes from the list. met. status: DNA methylation status; met. diff.: DNA methylation difference; expr. status: gene expression status; P-val: p -value.

Article Snippet: HT-29 (ATCC HTB-39) and SW480 (ATCC CCL-228) human colon adenocarcinoma cell lines were cultured in RPMI 1640 medium (LM-R1641, Biosera, Ringmer, UK) containing 10% fetal bovine serum (Biosera), 80 mg/2 mL gentamycin (Sandoz GmbH, Kundl, Austria), and 2 mM L-glutamine (Biosera).

Techniques: Genome Wide, DNA Methylation Assay, Gene Expression, Methylation Sequencing, Methylation, Expressing, Microarray

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Structures and cytotoxic activities of magnolol, M2M, and MM1. ( a ) Chemical structures of magnolol, M2M, and MM1. ( b and c ) A375 and A431 skin cancer cell lines were treated with various concentrations of magnolol, M2M, or MM1 for 24 h. Their cytotoxic effects and the viabilities of the treated cells were detected with ( b ) SRB assays and ( c ) MTT assays. ( d ) Flow cytometric analysis of cell cycle. A375 and A431 cells were treated with 75 μ M of magnolol (Mag) or MM1 for 24 h. The treated cells were stained with propidium iodide and analyzed by flow cytometry. The percentages of cells in the sub-G1 region (M1) are indicated. Data shown in b and c are expressed as the mean±S.D. of two independent experiments. Symbols: * P <0.05; ** P <0.01; and *** P <0.001, as analyzed by unpaired t -tests. Data shown in d are from one of two similar results

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effects of magnolol and MM1 on the clonogenic ability and anchorage-independent growth of skin cancer cells. ( a ) A375 (left panel) and A431 (right panel) cells were treated with various concentrations of magnolol or MM1 for 6 days and then cultured for an additional 8 days in the absence of drugs. The numbers of foci were scored, and the data are presented as the relative focus-forming ability (FFA). ( b ) A375 cells were treated with different concentrations of magnolol and MM1. The anchorage-independent growth was assessed as described in the Materials and Methods section. Data are expressed as the mean±S.D. of three independent experiments. Symbols: * P <0.05, as analyzed by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effect of magnolol and MM1 on the induction of apoptosis in A375 cells. ( a ) A375 cells were treated with magnolol or MM1 for 24 h, and western blotting was used to assess cell lysates for the cleavages of PARP and procaspase-7, -8, and -9. ( b and c ) Effect of caspase inhibition on the apoptosis of MM1-treated A375 cells. A375 cells were treated with 75 μ M MM1 in the presence or absence of the broad-spectrum caspase inhibitor, z-VAD-fmk (40 μ M), for 24 h. Phase-contrast images of MM1-treated cells treated with or without z-VAD-fmk are shown in b . Western blot analysis was used to assess cell lysates for the cleavages of procaspase-7, -8, and 9, with β -actin used as a loading control ( c )

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Inhibition, Control

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: Effects of magnolol and MM1 on the growth of xenograft tumors in nude mice. A375 cells were injected subcutaneously into the flank of each mouse. When the tumor volumes reached about 50 mm 3 , the mice were i.p. injected with magnolol (1 mM in 100 μ l acetone), MM1 (1 mM in 100 μ l acetone), or DMSO (control) three times per week ( n =6 per group). The tumor volume ( a ) was determined twice weekly, while body weight ( b ) was measured daily. The xenograft tumors were excised from the mice at the end of experiment in 15 days. The sizes of representative tumors excised from the different groups are shown at the top of a , while hematoxylin–eosin staining (magnification, × 200, upper panel) and IHC staining for activated caspase-3 (magnification, × 200, lower panel) are shown in c . The results shown in a and b are presented as the means±S.D. of six mice. Symbols: * P <0.05 by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Injection, Control, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: MM1 upregulates lncRNA GAS5. A375 cells were treated with or without 50 μ M MM1 for 24 h, and total RNA was subjected to microarray analysis as described in the Materials and Methods section. The heatmap shown in a presents a hierarchical clustering of the transcriptome profiles from duplicate cultures of untreated and MM1-treated A375 cells. The relative expression of each RNA transcript is indicated by color, and ranges from red (higher expression) to green (lower expression). ( b ) Functional classification of the differentially expressed genes in MM1-treated A375 cells, as assessed using the DAVID Database web server. The differentially expressed proteins are linked to a number of biological processes. ( c ) Upregulation of lncRNA GAS5 in magnolol- or MM1-treated A375 cells. A375 cells were treated with the indicated concentrations of magnolol or MM1 for 24 h, and the expression levels of lncRNA GAS5 were determined by real time RT-PCR, as described in the Materials and Methods section. The expressions of lncRNA GAS5 in the magnolol- and MM1-treated cells were normalized to that of the untreated cells, and are presented as relative expression levels. The data shown represent the mean±S.D. of three independent experiments. Symbol: ** P <0.01, as analyzed with the unpaired t -test

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Microarray, Expressing, Functional Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: List of the 20 highest-scored lncRNA differentially expressed in MM1-treated A375 cells

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: 2-O-Methylmagnolol upregulates the long non-coding RNA, GAS5, and enhances apoptosis in skin cancer cells

doi: 10.1038/cddis.2017.66

Figure Lengend Snippet: lncRNA GAS5 plays a critical role in the MM1-mediated induction of apoptosis. ( a ) Effect of lncRNA GAS5 depletion on cell apoptosis. Left panel: A375 cells were transfected with siRNA against lncRNA GAS5 (si-lncRNA GAS5) at 50 nM or with nontargeting siRNA(−). After 48 h, the transfected cells were treated with 75 μ M of MM1 for 24 h. The levels of lncRNA GAS5 were assessed by real-time RT-PCR and expressed as relative to that of non-MM1-treated control (Left panel). The cleavage of PARP and caspase-9 was analyzed by western blotting (Right panel). β -actin served as an internal control. ( b and c ) Effect of lncRNA GAS5 overexpression on cell proliferation and apoptosis. A375 cells were transfected with pCDNA3.1-lncRNA GAS5 or the empty vector (control). After 48 h, the transfected cells were assayed for expression of lncRNA GAS5 ( b , left panel), for viability by MTT method ( b , right panel), and for the induction of apoptosis by phosphatidylserine exposure with Annexin V-FITC using flow cytometry ( c ). Data are expressed as the mean±S.D. of three independent experiments. Symbols: ** P <0.01; and *** P <0.001, as analyzed by unpaired t -tests

Article Snippet: A375 (melanoma) and A431 (squamous cell carcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Quantitative RT-PCR, Control, Western Blot, Over Expression, Plasmid Preparation, Expressing, Flow Cytometry

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Hypoxia‐induced alterations in mRNA and protein expression of G2 checkpoint regulators. A. Gene expression of positive G2 checkpoint regulators in U2OS cells. The ratio of mRNA expression in cells treated with hypoxia (0.2% O2, 24 h) relative to mRNA expression in cells cultured at normoxia (21% O2) is shown. Data were obtained from genome wide microarray analysis. The positive G2 checkpoint regulators were found from published studies as described in Table 1. B. Gene expression of negative G2 checkpoint regulators similar as in A. C. Immunoblot analysis of protein extracts from U2OS cells exposed to hypoxia or normoxia for 24 h. The samples are from the same experiment as the microarray results shown in A and B. HIF1α was shown to confirm hypoxia. H4 was used as loading control.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Gene Expression, Cell Culture, Genome Wide, Microarray, Western Blot, Control

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Protein levels of G2 checkpoint regulators in individual G2 cells following hypoxia. A. Cell cycle profiles of U2OS cells after hypoxia treatment as in Figure 1 (24 h 0.2% O2). Flow cytometric analysis was performed after staining with anti phospho‐H3Ser10 (H3P) to mark mitotic cells, and the DNA stain Hoechst. Numbers indicate fraction of mitotic cells. B. Flow cytometric barcoding analysis for accurate measurement of protein levels in G2 phase cells. U2OS cells treated with four different conditions, as indicated in the right column, were labeled with different concentrations of Pacific Blue and combined into a single sample. The single sample of cells was then stained with antibodies to Cyclin B and phospho‐H3 and with the DNA‐stain FxCycle Far Red, and analyzed by flow cytometry. Gating of the Pacific Blue‐SSC plot (left) was used to separate the four original samples. The G1, S, G2 and M cell cycle phase populations were gated from the scatter plot of phospho‐Histone H3(Ser10) (H3P) versus DNA content, and the median signal for Cyclin B levels in each cell cycle phase could thus be obtained. C.Median values of G2 phase levels of the indicated proteins obtained as in B, after subtraction of background values obtained as in Figure S1. U2OS cells were grown at 21% O2, or exposed to 24 h hypoxia at 0.2% O2, or first exposed to 0.2% O2 for 24 h followed by subsequent incubation at 21% O2 for 90 min (90 min reox) or 6 h (6 h reox). Average results from at least 3 independent experiments are shown. Error bars indicate SEM.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Staining, Labeling, Flow Cytometry, Incubation

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Hypoxia‐induced changes in CDK activity and G2 checkpoint activation. A. CDK activity in G2 phase cells as measured by phosphorylation of BRCA2‐Ser3291. U2OS cells were grown at 21% O2, or incubated at 0.2% O2 for 24 h and harvested inside the hypoxia chamber and at 1 and 4 h after reoxygenation, or treated with Roscovitine for 2 h at 21% O2. Flow cytometry barcoding analysis of phospho‐BRCA2‐Ser3291 was performed as in Figure 2 and S2. B. Immunoblot analysis of U2OS cells treated as in A with antibodies to total BRCA2 and γ‐tubulin (loading control). C. G2 checkpoint activation after IR (0.2%O2 24 h). Flow cytometric analysis of G2 checkpoint arrest after X‐ray irradiation (0, 0.5, 1 Gy) of normoxic U2OS cells (21%O2) or U2OS cells exposed to 24 h of hypoxia at 0.2%O2 and irradiated 15 min after reoxygenation. Nocodazole was added to all samples 1 h after IR, and the samples were harvested 5 h later. The relative mitotic fraction was determined as the fraction of phospho‐H3 positive cells in irradiated samples divided by the fraction of phospho‐H3 positive cells in non‐irradiated samples. Average values from 3 independent experiments are shown. Error bars indicate SEM. D. Phosphorylation of BRCA2‐Ser3291 in H460 cells treated with hypoxia and analyzed as in A. E. G2 checkpoint activation in H460 cells treated with hypoxia and IR and analyzed as in C.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Incubation, Flow Cytometry, Western Blot, Control, Irradiation

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: IR‐induced G2 checkpoint and expression of G2 checkpoint regulators in U2OS cells after severe hypoxia (∼0.03% O2 20 h) and prolonged mild hypoxia (0.2%O2, 72 h). A. Similar G2 checkpoint measurement after IR as in Figure 3C following incubation at severe hypoxia (∼0.03% O2 20 h). B. Similar as in A following incubation at prolonged mild hypoxia (0.2% O2 72 h). C. Similar flow cytometric barcoding analysis of protein levels in G2 phase cells as in Figure 2C following incubation at severe hypoxia (∼0.03% O2 20 h). D. Similar as in C following incubation at 0.2% O2, 72 h.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Incubation

Journal: Scientific Reports

Article Title: Identification of KIAA1199 as a Biomarker for Pancreatic Intraepithelial Neoplasia

doi: 10.1038/srep38273

Figure Lengend Snippet: ( a ) Expression of KIAA1199 in human cancers via Oncomine analysis. ( b , c ) Expression of KIAA1199 in human pancreatic adenocarcinoma via Oncomine analysis. ( d – f ) Expression of KIAA1199 in human pancreatic adenocarcinoma. Fluorescence immunohistochemical analysis of KIAA1199 of human pancreatic cancer tissue microarray (TMA; US Biomax PA207), using anti-KIAA1199 antibody ( d ). Quantitative analysis of KIAA1199 expression in TMA ( e ). Chromogenic immunohistochemical analysis of human pancreatic cancer tissue microarray (US Biomax BIC14011a, PA242b) using-KIAA1199 antibody and DAB substrate ( f ). ( g , h ) mRNA expression of KIAA1199 in PDAC cell lines (AsPC-1, BxPC-3, and Panc-1). Quantitative RT-PCR ( g ) and semi-quantitative RT-PCR ( h ). KIAA1199 expression in PDAC cell lines compared to HPNE (normal pancreas epithelial cell line) and normalized to 18 S rRNA . Gel images shown have been cropped to show the relevant band. Full-length gels are presented in . ( i , j ) Endogenous and secreted protein expression of KIAA1199 in PDAC cell lines (AsPC-1, BxPC-3, and Panc-1). Molecular weight of KIAA1199 was approximately 150 kDa. Blot images shown have been cropped to show the relevant band. Full-length blots are presented in .

Article Snippet: Human PDAC cell lines AsPC-1, BxPC-3, and Panc-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Fluorescence, Immunohistochemical staining, Microarray, Quantitative RT-PCR, Molecular Weight

Journal: Scientific Reports

Article Title: Identification of KIAA1199 as a Biomarker for Pancreatic Intraepithelial Neoplasia

doi: 10.1038/srep38273

Figure Lengend Snippet: ( a ) Detection of KIAA1199 in mouse blood serum. Mouse blood sera from each group (3-, 5-, 7-, and 10-month-old wild-type [WT] or PanIN [ Pdx1-Cre:K-Ras LSLG12D ] mice) were analyzed by immunoblotting for KIAA1199. Immunoglobulin (Ig) served as an internal control. HCT116 colon cancer cells served as a positive control. ( b ) Experimental scheme of KIAA1199 autoantibody detection. Pancreatic ductal adenocarcinoma (PDAC) cells secrete KIAA1199 protein. Host immune system generates autoantibodies against KIAA1199. Blood samples were collected and incubated with KIAA1199 recombinant protein. KIAA1199 autoantibody binds to KIAA1199 recombinant protein. Immune complex is precipitated using magnetic beads. KIAA1199 autoantibody (IgG heavy chain [H.C.] and light chain [L.C.]) is detected by immunoblotting using antibody against the host. ( c ) Purification of KIAA1199 recombinant protein (N- and C-termini). ( d ) Detection of KIAA1199 autoantibody using immunoprecipitation. Mouse blood sera were immunoprecipitated using recombinant KIAA1199 proteins (N- and C-termini). Then, immunoprecipitates were analyzed by immunoblotting using mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP). ( e ) Purification of GST-KIAA1199 protein C-terminus. GST-KIAA1199 (C-terminus) protein was purified using the bacterial protein expression system. Arrow indicates purified KIAA1199 protein (Coomassie staining). ( f ) Detection of KIAA1199 autoantibody using dot blot assays. Different exposure times are indicated. Recombinant KIAA1199 protein (C-terminus) and mouse IgG (a negative control) were transferred onto the membrane for dot blot assays. The sera of mice (WT or PanINs [ Pdx1-Cre:K-Ras LSLG12D ] mice; 5-month-old) were added to probe for the KIAA1199 autoantibody.

Article Snippet: Human PDAC cell lines AsPC-1, BxPC-3, and Panc-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Control, Positive Control, Incubation, Recombinant, Magnetic Beads, Purification, Immunoprecipitation, Expressing, Staining, Dot Blot, Negative Control, Membrane

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Upregulation of the transcription factors POU3F2 and NTF3 during neuronal differentiation of NT2D1. a The protocol for neuronal induction of NT2D1 cells is schematized. b β3-tubulin staining for neuronal cells in NT2D1 cells untreated (non) and treated with neuronal induction medium at the indicated time points. c Quantification of β3-tubulin-positive cells. d Immunoblotting analysis for POU3F2, POU3F3, β3-tubulin, and NTF3 in NT2D1 cells untreated or treated with neuronal induction medium at the indicated time points. The values show the expression relative to that of untreated cells (to which a value of 1 was assigned). e Microarray analysis showed that neuronal induction for 6 h increased the expression of NTF3 and GADD45 in NT2D1 cells. f NTF3 mRNA expression after neuronal induction was analyzed by real-time PCR. The levels of mRNA were calculated as the relative expression compared with that of non-induced NT2D1 cells. GAPDH mRNA was used as a control. * p < 0.05; *** p < 0.001. g Phospho-TrkC (Tyr820) staining in treated and untreated NT2D1 cells. Values are presented as mean ± SEM of three independent experiments for c and f

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

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

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Identification of the POU3F2 binding site on the NTF3 promoter. a Transcription factor response elements predicted by the Transcription Element Search System for the nucleotide sequence of the NTF3 promoter region (− 1823 to + 243). The transcription start site is indicated as + 1. b Comparison of NTF3 promoter sequence conservation between different species. c Biotin-labeled oligonucleotides containing the intact or mutated POU3F2 binding site were hybridized with total lysates prepared from NT2D1 cells. The POU3F2-DNA complexes were precipitated by streptavidin agarose beads. POU3F2 was analyzed by Western blot analyses. The input of nuclear extracts was used as loading control. Three independent experiments were performed. d Chromatin was prepared from NT2D1 cells treated with induction medium for 0, 2, and 6 h. Cell lysates were mixed with antibodies against POU3F2 or IgG and then precipitated. The precipitates were analyzed by PCR for the presence of the NTF3 promoter sequence. The DNA purified from the sonicated chromatin was directly analyzed by PCR using the ChIP primer, which was used as an input control (Input). e The values of the ChIP DNA were normalized to that of the NT2D1 cells at 0 h (as a control). Values of fold-change over the control are presented as mean ± SEM of three independent experiments for d . * p < 0.05 compared with the control

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Binding Assay, Sequencing, Comparison, Labeling, Western Blot, Control, Purification, Sonication

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 on NTF3 promoter activity. a Schematic representation of NTF3-luciferase chimeric constructs. The negative numbers refer to the numbers of bases upstream of the transcription start (+ 1) site of the NTF3 gene. b NT2D1 cells were transiently transfected with the pGL3 basic vector or NTF3 promoter constructs of different lengths. The luciferase activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. c NT2D1 cells were transfected with the pGL3 basic vector, pNTF3-1902, and pNTF3-1902 POU3F2 mut. Approximately 24 h later, cells were treated with neuronal induction medium. The transcriptional activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. Values are presented as mean ± SEM of three independent experiments for b and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 silencing on neuronal differentiation and NTF3 expression in NT2D1 cells. a POU3F2 expression in POU3F2-knockdown (shPOU3F2) and control (shLuc) NT2D1 cells was determined by Western blot analyses after neuronal induction for 6 h. GAPDH was used as a loading control. The values represent the relative expression compared with that of the non-induced shLuc cells (to which a value of 1 was assigned). b NTF3 mRNA expression of the cells described in a was analyzed by real-time PCR. mRNA levels were calculated relative to that of the non-induced shLuc cells. * p < 0.05; *** p < 0.001. c Neuronal morphology of shLuc and shPOU3F2 cells that were treated with neuronal induction medium for 24 h or left untreated (non). d Quantification of cell numbers of shLuc and shPOU3F2 described in c . All the percentages of the shLuc and shNTF3 cells were compared to that of the non-induction shLuc cells (to which a value of 100% was assigned). e β3-tubulin staining was performed on shLuc and shPOU3F2 cells, which were treated with neuronal induction medium for 0, 6, or 24 h or left untreated, after which neuronal cells were detected. Values represent the mean ± SEM of three independent experiments for b and d

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Knockdown, Control, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of NTF3 silencing and NTF3 recombinant protein treatment on the viability and neuronal differentiation of NT2D1 cells. a NTF3 mRNA levels in NTF3-knockdown (shNTF3) and control (shLuc) NT2D1 cells, which were treated with neuronal induction medium for 0, 24, or 48 h or left untreated (Non), were determined by real-time PCR. mRNA levels were calculated as the relative expression compared with the untreated shLuc cells. *** p < 0.001. b Phase contrast microscopy images of untreated shLuc and shNTF3 cells and those cells 24 h after neuronal induction with concomitant treatment of rNTF3 (5, 20 ng/ml) or vehicle. c Quantification of neuron number of shLuc and shNTF3 cells as described in b . All the percentages of neurons differentiated from shLuc and shNTF3 cells were compared to that of neurons differentiated from the vehicle-treated shLuc cells (to which a value of 100% was assigned). * p < 0.05; ** p < 0.01. d A suggested model of the POU3F2/NTF3 pathway that mediates the process of neuron differentiation. Values are presented as mean ± SEM of at least three independent experiments for a and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Recombinant, Knockdown, Control, Real-time Polymerase Chain Reaction, Expressing, Microscopy

Journal: Nucleic Acids Research

Article Title: Validation of human microRNA target pathways enables evaluation of target prediction tools

doi: 10.1093/nar/gkaa1161

Figure Lengend Snippet: HiTmIR overview and representative selection of miR-34a. ( A ) Combined experimental and computational workflow of HiTmIR. Three computational steps are carried out consecutively before target gene sets are validated by an automated reporter assay. ( B ) Immunocytochemistry of D2R expression in differentiated LUHMES cells. ( C ) Immunocytochemistry of TH expression in differentiated LUHMES cells. (B, C) Expression of dopaminergic markers in differentiated LUHMES cells were analyzed by immunocytochemistry with antibodies against TH and D2R. The nuclei were visualized by DAPI staining. Scale bars are 25 μm. ( D ) Heatmap of the 50 most down-regulated miRNAs in LUHMES cells that were differentiated toward dopaminergic neurons and treated with MPP+ to induce a PD-like phenotype. ( E ) Heatmap of the 50 most up-regulated miRNAs. (D, E) Shown are z-scores of quantile-normalized expression values. ( F ) Validation of microarray results by qRT-PCR of up-regulated and down-regulated miRNAs. Bars present the log 2 fold change between PD-like and controls together with the respective standard deviation. ( G ) Increased expression of miR-34a-5p in the blood of patients, spanning an age range from 20 to 80 years. The orange line shows a smoothed spline with 8 degrees of freedom and the shaded area represents the 95% confidence interval.

Article Snippet: Lund human mesencephalic (LUHMES) cells were purchased from the American Type Culture Collection (ATCC) and transfected for GFP-expression.

Techniques: Selection, Reporter Assay, Immunocytochemistry, Expressing, Staining, Biomarker Discovery, Microarray, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Effects of hypoxia on cell markers, morphology, and migration (A) Expression in breast cancer MCF7 and MDA-MB-231 cells of E-cadherin (epithelial marker), fibronectin (epithelial marker), HIF-1α (hypoxia marker), β-catenin, and GLUT1. Cells were cultured at 37°C in 5% CO 2 atmosphere for normoxic treatment, and in 1% O 2 / 5% CO 2 / 94% N 2 atmosphere for hypoxic treatment. Cells were harvested, lysed in T-PER Reagent, and protein content was determined by BCA assay. Western blotting was performed as described in M&M. (B) Morphological changes under normoxic and hypoxic conditions. Cells (2 × 10 5 per well) were grown in 6-well plates for 24 h under the two conditions. Photos were taken by phase-contrast microscopy at 200× magnification. (C) Cell migration assessed by wound assay. Cell monolayers under the two conditions were scratched with pipette tip. Cells were washed with ice-cold 1× PBS and cultured in serum-free medium. Pictures of wounds were taken at 0 and 24 h by phase-contrast microscopy (100× magnification).

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, Expressing, Marker, Cell Culture, BIA-KA, Western Blot, Microscopy, Transferring

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MALDI-TOF-MS spectra of N-glycans from MCF7 cells MCF7 cells were cultured in 10-cm dishes under normoxic and hypoxic conditions, and N-glycans were separated and desalted as described in M&M. Lyophilized N-glycans were dissolved in MW, and an aliquot of mixture with DHB solution was spotted on MTP AnchorChip sample target and air-dried. MALTI-TOF-MS was performed in positive-ion mode. Experiments were performed in biological triplicate, and representative N-glycan spectra are shown. Peaks (signal-to-noise ratio > 5) were selected for relative proportion analysis. Detailed structures were analyzed using the GlycoWorkbench program. Proposed structures are indicated by m/z value.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Culture, Glycoproteomics

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Relative proportions of various types of N-glycans in MCF7 and MDA-MB-231 cells under normoxia and hypoxia.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques:

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Variation of fine glycan structures detected by lectin microarray analysis (A) Variation of levels of glycans from MCF7 (upper) and MDA-MB-231 (lower) cells, detected by 37 lectins, is presented as a heatmap. Lectin microarray analysis was performed as described as M&M. Red: fluorescence signal activation. Green: signal inhibition. Black: missing data. (B) Altered glycan levels evaluated by lectin histochemistry. Four lectins (Con A, MAL-I, LCA, PHA-E) were applied, and lectin histochemistry was performed as described in M&M. Signals are shown from merge images of Cy3-conjugated lectins and DAPI staining of nuclei in MCF7 (left) and MDA-MB-231 (right) under normoxic and hypoxic conditions (60× magnification). (C) Expression in MCF7 and MDA-MB-231 cells of HIF-1α, MGAT3, and tubulin.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Glycoproteomics, Microarray, Fluorescence, Activation Assay, Inhibition, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: Differential glycopatterns in normoxia- vs. hypoxia-treated MCF7 cells revealed by lectin microarray analysis.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Microarray

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 overexpression suppresses hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-transfected MCF7 cells. Cells were stably transduced with a GFP-marked lentivirus carrying mock gene or MGAT3 gene, harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M using anti-MGAT3 and anti-GFP antibody. (B) Levels of bisecting GlcNAc structures in mock- and MGAT3-transfectants. Whole cell lysates of the two transfectants were subjected to PHA-E lectin blotting as described in M&M. (C) Proliferation of transfectant cells. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (E) Colony formation ability. The two transfectants (2500 cells each) were cultured in 6-cm dishes for 1–2 week, fixed, stained with crystal violet solution, and photographed. Acetic acid was added to dissolve crystal violet, and OD 595 was determined (D) . * p < 0.05; *** p < 0.001. (F) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (H) . * p < 0.05. (G) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Over Expression, Expressing, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Staining, Migration, BIA-KA

Journal: Frontiers in Physiology

Article Title: Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells

doi: 10.3389/fphys.2018.00210

Figure Lengend Snippet: MGAT3 knockdown promotes hypoxia-induced EMT in MCF7 cells (A) MGAT3 expression in mock- and MGAT3-shRNA-transfected MCF7 cells. Cells were stably transduced with lentivirus carrying anti-MGAT3 shRNAs (MCF7/shMGAT3-1/2) or shNC (MCF7/mock), harvested, and lysed in T-PER Reagent. Western blotting was performed as described in M&M. (B) Cell proliferation. The two transfectants were cultured for 24, 36, 48, 60, and 72 h, and proliferation was assessed by MTS assay. (C) Cell migration. Migration assays of the two transfectants under normoxic and hypoxic conditions were performed as described in M&M, and relative migration rate was shown (E) . * p < 0.05; ** p < 0.01. (D) Expression of HIF-1α, MGAT3, AKT, p-AKT, E-cadherin, fibronectin, β-catenin, and tubulin in the two transfectants under normoxic and hypoxic conditions. Cells were cultured as described in Figure , harvested, and lysed in T-PER Reagent. Protein content was determined by BCA assay. Western blotting was performed as described in M&M.

Article Snippet: Human breast cancer MCF7 and MDA-MB-231 cell lines were from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Expressing, shRNA, Transfection, Stable Transfection, Transduction, Western Blot, Cell Culture, MTS Assay, Migration, BIA-KA