Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 1. Characterization of the BMI1 promoter. (A) Schematic diagram of the BMI1 promoter fragments spanning from 2063 to 783 and from 783 to +375. The 2063/783 and 783/+375 regions were cloned upstream of the firefly luciferase reporter gene in the pGL3- basic vector. (B) Transcriptional activity analysis of the potential BMI1 promoter fragments in CNE2 and HNE1 cells. (C) Schematic diagram of the 5′-truncated and 3′-truncated fragments spanning from 783 to +375 of BMI1. (D) Promoter activity analysis of the 5′-truncated and 3′-truncated fragments spanning from 783 to +375. For (B) and (D), CNE2 and HNE1 cells were transfected with either the pGL3-basic vector or the pGL3-basic vector containing the indicated fragments of the potential BMI1 promoter. pRL-SV40, which expresses Renilla luciferase, was cotransfected to calibrate the differences in both transfection and harvesting efficiency. Luciferase activity was detected 24 h post-transfection, and presented as relative luciferase units (RLU) (firefly luciferase/Renilla luciferase). All data are presented as the mean SEM from triplicate measurements. A representative result of three independent experiments is shown. For (A), (C) and (E), the numbers on the schematic diagram indicate the location of the fragments relative to the TSS, which was assigned as +1. ***P < 0.001. (E) The fragment from +167 to +232 in the 5′-flanking region of the BMI1 is highly conserved in humans and mice. Sequence alignment of the human BMI1 promoter region (from +167 to +232) with that of the mouse is shown. The sequence of the human BMI1 promoter region (from +167 to +232) was BLAST-searched (NCBI database).

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Sequencing

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 3. Essential roles of the Sp1 binding sites and the E-box element in BMI1 transcription. (A) Schematic of potential binding sites for the transcription factors within the region from 783 to +375. The E-box element (181) and the binding sites for E2F1 (122) and E2F1 (319) were subjected to site-directed mutagenesis, whereas the ETF-Egr binding site cluster (+126/+179) and Sp1 binding site cluster (+164/+241) were deleted. The mutation and deletion are labeled as ‘m’ and ‘D’, respectively. The indicated fragments with mutation or deletion were cloned upstream of the firefly luciferase reporter gene in the pGL3-basic vector. (B) Promoter activity analysis of the potential transcription factor binding sites with point mutations or partial deletions in CNE2 cells. (C) Schematic of the E-box elements and Sp1 binding sites within the fragment from 783 to +232. The E-box elements (181) or Sp1 binding sites (+181/+214) were subjected to site- directed mutagenesis. The indicated fragments with a single mutation or double mutations were cloned upstream of the firefly luciferase reporter gene in the pGL3-basic vector. (D) Promoter activity analysis of the potential transcription factor binding sites with the indicated mutations in CNE2 and HNE1 cells. For (B) and (D), NPC cells were transfected with either the pGL3-basic vector or pGL3-basic vector containing the indicated fragments of the potential BMI1 promoter. All data are presented as the mean SEM from triplicate measurements. A representative result of three independent experiments is shown. **P < 0.01; ***P < 0.001. RLU, relative luciferase units; WT, wild type.

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: Binding Assay, Mutagenesis, Labeling, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 4. Sp1 interacts with the BMI1 promoter in vitro and in vivo. (A) EMSA verified Sp1 as a potential nuclear protein interacting with the BMI1 promoter. The analysis was performed in the presence (+, lanes 1–4) of CNE2 nuclear extracts. Shift bands were present in the binding reaction that included a c-32P-labeled wild- type (wt) probe comprising the Sp1 binding site (+181/+214) (lanes 1–3), but were absent in the reaction that included the labeled mutant (mut) probe comprising the mutant Sp1 binding site (lane 4). A supershifted band was present when the antibody against Sp1 was added to the reaction (lane 3), but was absent when the antibody against FLAG was added (lane 2). A representative result of three experiments is shown. (B) Scheme of the location of the ChIP amplicons in the E-box and the binding sites for Sp1. (C) ChIP assay showed that Sp1 directly interacted with the Sp1 binding sites, but not with the 239/87 E-box element within the BMI1 promoter. Formaldehyde-crosslinked chromatin isolated from CNE2 cells was precipitated with antibody against Sp1 or FLAG (negative control), respectively. The isolated chromatin complexes were subsequently amplified by PCR with 29 GC Hotstar Taq buffer with primer sets that encompassed the E-box region (from 239 to 87) or the predicted Sp1 binding sites (from +90 to +324). A specific strong band of the expected size was detected in the input DNA (lane 3). The fragment containing the Sp1 binding sites was detected (lane 2, top) whereas the fragment containing the E-box region was significantly faint in DNA precipitated by antibody against Sp1 (lane = 2, bottom). No band was detected in the chromatin complex precipitated by antibody against FLAG (lane 1).

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: In Vitro, In Vivo, Binding Assay, Labeling, Mutagenesis, Isolation, Negative Control

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 5. MITA treatment suppressed the promoter activity and expression of Bmi1. (A) MITA treatment repressed the promoter activity of the fragment from 783 to +232 within the BMI1 promoter. CNE2 and HNE1 cells were cotransfected with both pGL3-basic containing the fragment (783/+232) and pRL-SV40 for 16 h, exposed to 125 nM MITA for 48 h, and then subjected to luciferase assays. ***P < 0.001. (B) MITA treatment downregulated the expression of Bmi1. CNE2 and HNE1 cells (4 9 105) were placed in six-well plates for 16 h, and then treated with 125 nM MITA for 72 h before western blotting. GAPDH served as an internal control. RLU, relative luciferase units.

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: Activity Assay, Expressing, Luciferase, Western Blot, Control

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 6. Ectopic expression of Sp1 elevated BMI1 promoter activity. (A) Sp1 was ectopically expressed in CNE2 and HNE1 cells. (B) Sp1 overexpression induced transcriptional activity of the BMI1 promoter. CNE2 and HNE1 cells were cotransfected with 10 ng of pRL-SV40, 0.1 lg of the firefly luciferase reporter plasmids and 1.5 lg of either the pCDNA6-myc-HisB vector or the pCDNA6-myc- HisB-SP1 plasmid in each reaction. Cell lysates were harvested 48 h post-transfection, and subjected to western blotting (A) and luciferase assay (B). A representative result of three experiments is shown. ***P < 0.001. RLU, relative luciferase units.

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: Expressing, Activity Assay, Over Expression, Luciferase, Plasmid Preparation, Transfection, Western Blot

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 7. Effect of Sp1 overexpression or knockdown on the Bmi1 level in NPC cells. (A, B) Sp1 overexpression enhanced the protein (A) and mRNA (B) levels of Bmi1 in HNE1 cells. Seventy-two hours post-transfection, HNE1 cells that were successfully transfected with pEGFP-N1 and either the empty vector (pCDNA6-myc-HisB) or the SP1 overexpression plasmid (pCDNA6-myc-HisB-SP1) were sorted on the basis of enhanced green fluorescent protein expression with fluorescence-activated cell sorting. The sorted cells were harvested and subjected to western blotting (A) and qRT-PCR (B). (C, D) Knockdown of Sp1 reduced the endogenous Bmi1 level. CNE2 and HNE1 cells were transfected with the indicated siRNA duplex for 72 h, and this was followed by western blotting (C) and qRT-PCR (D). GAPDH served as an internal control. A representative result of three experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

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

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 8. Expression of Bmi1 and expression of Sp1 are positively correlated in NPECs, NPC cells, and NPC tumor specimens. (A–C) Bmi1 and Sp1 showed a similar expression trend in the mRNA (A, B) and protein levels (C) among eight NPECs/NPC cells. The arrow indicates Sp1 protein, and the asterisks indicate the possible isoforms or proteolytic degradation products of Spl. (D) A significant positive correlation was found between the mRNA levels of BMI1 and SP1 in NPC tissues. The mRNA levels of BMI and SP1 in 69 NPC tissue specimens were examined by qRT-PCR. The correlation was analyzed with Spearman’s correlation coefficient. (E) The protein expression levels of Bmi1 and Sp1 in NPC tumors were statistically correlated. The expression levels of Bmi1 and Sp1 in 22 NPC tumors were analyzed by immunohistochemical staining. The overall staining intensity and percentage were quantified with the Nuance multispectral imaging system. A value designated as the immunohistochemical score (H-score) was derived by summing percentages of cells stained at each intensity multiplied by the weighted intensity of staining. An H-score of 1 was considered to be low, and an H-score of > 1 was considered to be high. (F) Representative immunohistochemical staining of Bmi1 in consecutive NPC tumor sections with low (biopsy 1) and high (biopsy 2) Sp1 expression.

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Imaging, Derivative Assay

Journal: The FEBS journal

Article Title: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma.

doi: 10.1111/febs.12299

Figure Lengend Snippet: Fig. 9. Effect of c-Myc on the expression of Bmi1. (A) A scheme of the location of the ChIP amplicons for the E-box and the adjacent region is shown. (B) ChIP assay showed that c-Myc directly interacted with the 239/177 region where the E-box region located, but not with the adjacent 118/26 region. Formaldehyde-crosslinked chromatin isolated from CNE2 and C666-1 cells was precipitated with antibody against c-Myc or FLAG, respectively. The isolated chromatin complexes were subsequently amplified by qRT-PCR with primer sets that encompassed the E-box region (from 239 to 177) or the adjacent region (from 118 to 26). Relative enrichment was calculated by normalizing the threshold cycle (CT) value of DNA coprecipitated by antibody against Myc against the CT value of DNA coprecipitated by antibody against FLAG control (IgG). (C, D) Ectopic expression of c-Myc augmented the expression and promoter activity of Bmi1 in HNE1 cells. HNE1 cells were cotransfected with 10 ng of pRL-SV40, 0.1 lg of the firefly luciferase reporter plasmids and 1.5 lg of either the pCDNA6-myc-HisB vector or the pCDNA6-myc-HisB-MYC plasmid in each reaction. Cell lysates were harvested 48 h post-transfection, and subjected to western blotting (C) and luciferase assay (D). (E, F) Knockdown of c-Myc reduced Bmi1 expression in CNE2 cells. After transfection with either siMyc or NC for 72 h, the expression levels of Bmi1 or c-Myc in CNE2 cells were analyzed by western blotting (E) and qRT-PCR (F). (G) There was a positive correlation between the mRNA levels of BMI1 and MYC in NPC tissues. The mRNA levels of BMI1 and MYC in 69 NPC tissue specimens were examined by qRT-PCR. The correlation was analyzed with Spearman’s correlation coefficient. GAPDH served as an internal control. A representative result of three experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001. RLU, relative luciferase units.

Article Snippet: The blots were probed with rabbit polyclonal antibodies against Bmi1 (Proteintech Group, Wuhan, China), Sp1 (#07-645; Millipore, Temecula, CA, USA), Sp3 (A302-484A; Bethyl Laboratories, Montgomery, TX, USA), and c-Myc (#04-216; Millipore).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Western Blot, Knockdown