Journal: Oncotarget

Article Title: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

doi:

Figure Lengend Snippet: (A) qPCR confirming the overexpression of HOXA9 in hTERT/E6/E7-HOXA9 and U87MG-HOXA9 cells and HOXA9 -silencing in U251-shHOXA9 and GBML18-shHOXA9 cells, comparing to their respective control counterparts. (B) Venn diagram summarizing the number of differentially expressed transcripts in the microarray data in all cell lines. The numbers in each area represent the total number of transcripts within each intersection. (C–F) DAVID was used to query the HOXA9-transcriptome from each cell line (C, hTERT/E6/E7; D, U87MG; E, U251; F, GBML18), in order to identify enriched biological terms on the differentially expressed genes extracted from the microarray data. Statistically significant enriched GO terms are shown for each cell line.

Article Snippet: For HOXA9 silencing , GBML18 and U251 cells were transfected with pGFP-V-RS plasmid containing HOXA9-specific shRNAs or non-effective shRNAs sequences (Origene).

Techniques: Over Expression, Microarray

Journal: Oncotarget

Article Title: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

doi:

Figure Lengend Snippet: (A–D) GSEA reveals that the HOXA9 transcriptomes in hTERT/E6/E7 (A) and U251 (C) cells are associated with transcriptional signatures of glioma stem-like cells (Enrichment Score, ES = −0.54, False Discovery Rate, FDR = 0.19; and ES = 0.50, FDR = 0.11, respectively); in U87MG cells (B) , the HOXA9 transcriptome is positively associated with genes that are upregulated in embryonic stem cells (ES = 0.50, FDR < 0.0001); in GBML18 cells (D) , HOXA9 transcriptome is inversely associated with genes upregulated during the neuronal differentiation (ES = 0.75, FDR = 0.21). (E) Representative phase contrast photographs of hTERT/E6/E7 and U87MG neurospheres are shown. (F) Quantification of neurospheres number and size for each cell line ( n = 3; * p < 0.05; *** p < 0.001). (G) Immunofluorescence showing increased Nestin staining in HOXA9-positive cells.

Article Snippet: For HOXA9 silencing , GBML18 and U251 cells were transfected with pGFP-V-RS plasmid containing HOXA9-specific shRNAs or non-effective shRNAs sequences (Origene).

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

doi:

Figure Lengend Snippet: (A and B) Determination of the half inhibitory concentration (IC 50 ) values after 6 days of temozolomide (TMZ) treatment in HOXA9 -positive or HOXA9 -negative hTERT/E6/E7 and U87MG (A), and HOXA9 -silenced or control U251 and GBML18 (B) cell lines. (C–F) Cell viability trypan blue assays in HOXA9 -negative/low or HOXA9 –positive/high hTERT/E6/E7 (C), U87MG (D), U251 (E) and GBML18 (F) cells, exposed to temozolomide or vehicle. (G) Cell death was evaluated by annexin V staining followed by flow cytometry in HOXA9 -positive/high and HOXA9 -negative/low hTERT/E6/E7, U87MG, U251 and GBML18 cell lines, both in basal conditions and after exposure to temozolomide (TMZ). HOXA9 expression decreases cell death of all GBM cell models, both in basal conditions and after TMZ treatment, except in basal conditions for U251 cell line. (H) Cell invasion in the same cell lines, both in basal conditions and after exposure to TMZ. HOXA9 increases the invasion of hTERT/E6/E7, U87MG, and GBML18 cells. U251 cells did not show significant differences in invasion profiles due to HOXA9 levels or TMZ treatment. Results are representative of at least three independent experiments, performed in triplicates (data points represent mean ± SEM). Statistical differences were calculated by Student's t -test (panels A, B, G, H) and two-way ANOVA (panels C–F) (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: For HOXA9 silencing , GBML18 and U251 cells were transfected with pGFP-V-RS plasmid containing HOXA9-specific shRNAs or non-effective shRNAs sequences (Origene).

Techniques: Concentration Assay, Staining, Flow Cytometry, Expressing

Journal: Oncotarget

Article Title: miR-137 acts as a tumor suppressor via inhibiting CXCL12 in human glioblastoma

doi: 10.18632/oncotarget.20589

Figure Lengend Snippet: (A-B) RT-PCR analysis of miR-137 and CXCL12 expression in glioblastoma U87 and U251 cell lines. Quantification analysis was defined as the relative density of miR-137 and CXCL12 mRNA to U6 and GAPDH respectively. U6 or GAPDH was used as an internal control. Results shown are the mean ± SD of repeated independent experiments. * p <0.01, compared with NHA cells, one-way ANOVA. (C) The expression of CXCL12 protein was examined in GBM cell lines U87 and U251 using western blot. The CXCL12 expression was normalized to β-actin expression. Results shown are the mean ± SD of 3 repeated independent experiments. * p <0.01, compared with NHA cells, one-way ANOVA.

Article Snippet: Cell lines U87, and U251 were obtained from the KeyGEN Company (China) and cultured in DMEM, 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere under 5% CO2.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Oncotarget

Article Title: miR-137 acts as a tumor suppressor via inhibiting CXCL12 in human glioblastoma

doi: 10.18632/oncotarget.20589

Figure Lengend Snippet: (A) Cells were transfected with miR-137 mimics and identified by RT-PCR. Cell proliferation was measured using a CCK-8 assay. U87 and U251 cells were transfected with miR-137 mimics or scramble control. (B) Relative EGFR, Bcl-2 and Bax expression in U87 and U251 cells was measured after the cells were transfected with miR-137 mimics or NC miRNA using western blot. Results shown are the mean ± SD of 3 repeated independent experiments. * p <0.01, compared with miR-NC, one-way ANOVA.

Article Snippet: Cell lines U87, and U251 were obtained from the KeyGEN Company (China) and cultured in DMEM, 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere under 5% CO2.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Control, Expressing, Western Blot

Journal: Oncotarget

Article Title: miR-137 acts as a tumor suppressor via inhibiting CXCL12 in human glioblastoma

doi: 10.18632/oncotarget.20589

Figure Lengend Snippet: (A) Wound healing assay performed with U87 and U251 cells over 48 h. Cells and wounds were pretreated as described above. Wound healing within the scrape line was recorded every day. Representative scrape lines are shown at day 3; dashed line indicates the margin of the scratch at day 1. (B) Representative fields (×10 magnification) showing invasive cells after 24 hour culture in Matrigel invasion chambers. Panels show U87 and U251 cell invasion after transfection with miR-137 mimics or miR-NC. Quantitative analysis of cell invasion experiments demonstrates that miR-137 decreases U87 and U251 cell invasion compared to miR-NC ( * p<0.01, vs. miR-NC control). (C) Relative MMP2/9 expressions in U87 and U251 cells were measured after the cells were transfected with miR-137 mimics or miR-NC using western blot. Results shown are the mean ± SD of 3 repeated independent experiments. Bar=20 μm, * p <0.01, compared with miR-NC, one-way ANOVA.

Article Snippet: Cell lines U87, and U251 were obtained from the KeyGEN Company (China) and cultured in DMEM, 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere under 5% CO2.

Techniques: Wound Healing Assay, Transfection, Control, Western Blot

Journal: Oncotarget

Article Title: miR-137 acts as a tumor suppressor via inhibiting CXCL12 in human glioblastoma

doi: 10.18632/oncotarget.20589

Figure Lengend Snippet: (A) The WT and Mut of 3′UTR of CXCL12 mRNA contains the binding sequences of miR-137. The miR-137 mimic inhibited the luciferase activity controlled by wild-type CXCL12-3’-UTR (B) but did not affect the luciferase activity controlled by mutant CXCL12-3’-UTR (C) in U87 and U251 cells. Results shown are the mean ± SD of 3 repeated independent experiments. * p <0.01, compared with miR-NC, one-way ANOVA.

Article Snippet: Cell lines U87, and U251 were obtained from the KeyGEN Company (China) and cultured in DMEM, 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere under 5% CO2.

Techniques: Binding Assay, Luciferase, Activity Assay, Mutagenesis

Journal: Oncotarget

Article Title: miR-137 acts as a tumor suppressor via inhibiting CXCL12 in human glioblastoma

doi: 10.18632/oncotarget.20589

Figure Lengend Snippet: (A) The proliferation capacity of miR-137-overexpressing U87 and U251 cells was partially improved when cells were transfected with CXCL12 plasmids in comparison with miR-NC. (B) The invasion capacity of miR-137-overexpressing U87 and U251 cells were effectively improved when cells were transfected with CXCL12 plasmids. * p <0.01, vs. control. (C) The proliferation capacity of miR-137-overexpressing U87 and U251 cells was partially inhibited when cells were transfected with si-CXCL12 compared with si-control. (D) The invasion capacity of miR-137-overexpressing U87 and U251 cells were effectively improved when cells were transfected with si-CXCL12. Bar=20 μm, * p <0. 01, vs. si-control.

Article Snippet: Cell lines U87, and U251 were obtained from the KeyGEN Company (China) and cultured in DMEM, 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere under 5% CO2.

Techniques: Transfection, Comparison, Control

Journal: PLoS ONE

Article Title: Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF

doi: 10.1371/journal.pone.0211501

Figure Lengend Snippet: (A) U251 cells treated with 50 ng/ml GDNF for 0.5, 24 and 48 h and the control group were stained and imaged to detect the GA. Golgi-ID green staining showing the GA (green), nucleus (blue) and merged images (bar = 50 μm). (B) Quantitative analysis of the area of the GA is presented as the mean ± standard deviation of three independent experiments. Compared with the control group, the average area of the GA body was increased by GDNF treatment at 24 and 48 h (*P<0.05). GA, Golgi apparatus; GDNF, glial cell line-derived neurotrophic factor.

Article Snippet: Human U251 glioma cells [Obio Technology (Shanghai) Corp., Ltd, Shanghai, China] were cultured in a 37°C, 5% CO2 humidified incubator using Dulbecco’s modified Eagle’s medium (DMEM, SH30022.01, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, SV30087.02, GE Healthcare Life Sciences), 100U/ml penicillin and 100U/ml streptomycin.

Techniques: Staining, Standard Deviation, Derivative Assay

Journal: PLoS ONE

Article Title: Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF

doi: 10.1371/journal.pone.0211501

Figure Lengend Snippet: (A and B) Western blotting detected the expression of golgin-160 and GMAP-210 in U251 cells treated by 0, 25, 50 and 75 ng/ml GDNF for 48 h. The expression of golgin-160 and GMAP-210 was increased most significantly following treatment 50 ng/ml GDNF (***P<0.001). (C and D) Immunofluorescence analyzed the stack area of the GA in U251 cells treated with 0, 25, 50 and 75ng/ml GDNF for 48 h. The GA area in U251 cells was increased following GDNF treatment, and it was significant following treatment with 50 ng/ml (*P<0.05; bar = 50 μm). GMAP-210, Golgi microtubule-associated protein 210; GA, Golgi apparatus; GDNF, glial cell line-derived neurotrophic factor.

Article Snippet: Human U251 glioma cells [Obio Technology (Shanghai) Corp., Ltd, Shanghai, China] were cultured in a 37°C, 5% CO2 humidified incubator using Dulbecco’s modified Eagle’s medium (DMEM, SH30022.01, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, SV30087.02, GE Healthcare Life Sciences), 100U/ml penicillin and 100U/ml streptomycin.

Techniques: Western Blot, Expressing, Immunofluorescence, Derivative Assay

Journal: PLoS ONE

Article Title: Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF

doi: 10.1371/journal.pone.0211501

Figure Lengend Snippet: (A) The effect of GDNF on U251 cell migration was assessed by wound healing assay (*P<0.05, **P<0.01, bar = 100μm). (B) The effect of GDNF on U251 cell invasion was assessed by Transwell invasion assay (***P<0.001; bar = 100 μm). GDNF, glial cell line-derived neurotrophic factor.

Article Snippet: Human U251 glioma cells [Obio Technology (Shanghai) Corp., Ltd, Shanghai, China] were cultured in a 37°C, 5% CO2 humidified incubator using Dulbecco’s modified Eagle’s medium (DMEM, SH30022.01, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, SV30087.02, GE Healthcare Life Sciences), 100U/ml penicillin and 100U/ml streptomycin.

Techniques: Migration, Wound Healing Assay, Transwell Invasion Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF

doi: 10.1371/journal.pone.0211501

Figure Lengend Snippet: (A) Golgin-160 was knocked down by lentivirus, and its expression was assessed by western blotting and RT-qPCR (***P<0.001). The effect of golgin-160 knockdown on U251 cell (B and C) migration and (D and E) invasion was assessed by wounding healing and cell invasion assays. Cell migration and invasion were inhibited by the depletion of golgin-160, and GDNF could improve it (##P<0.05 and **P<0.01; bar = 100 μm). RT-qPCR, reverse transcription quantitative polymerase chain reaction; GDNF, glial cell line-derived neurotrophic factor.

Article Snippet: Human U251 glioma cells [Obio Technology (Shanghai) Corp., Ltd, Shanghai, China] were cultured in a 37°C, 5% CO2 humidified incubator using Dulbecco’s modified Eagle’s medium (DMEM, SH30022.01, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, SV30087.02, GE Healthcare Life Sciences), 100U/ml penicillin and 100U/ml streptomycin.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Migration, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: PLoS ONE

Article Title: Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF

doi: 10.1371/journal.pone.0211501

Figure Lengend Snippet: (A) GMAP-210 was knocked down by lentivirus and its expression was assessed by western blotting and RT-qPCR (***P<0.001). The effect of GMAP-210 knockdown on U251 cell (B and C) migration and (D and E) invasion was assessed by wounding healing and cell invasion assays. Cell migration and invasion were inhibited by the depletion of GMAP-210, and GDNF could improve it (#P<0.05 and **P<0.01; bar = 100 μm). GMAP-210, Golgi microtubule-associated protein 210; GDNF, glial cell line-derived neurotrophic factor; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

Article Snippet: Human U251 glioma cells [Obio Technology (Shanghai) Corp., Ltd, Shanghai, China] were cultured in a 37°C, 5% CO2 humidified incubator using Dulbecco’s modified Eagle’s medium (DMEM, SH30022.01, Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, SV30087.02, GE Healthcare Life Sciences), 100U/ml penicillin and 100U/ml streptomycin.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Migration, Derivative Assay, Real-time Polymerase Chain Reaction