Journal: Journal of Biological Chemistry

Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1

doi: 10.1074/jbc.m808734200

Figure Lengend Snippet: FIGURE 5. In vitro phosphorylation of GST-Sp1 by PKC and Erk1. To investigate whether phosphorylation of Sp1 was regulated directly by PKC or SA-pErk1/2, a human Sp1 fusion construct (GST-Sp1) and its deletion mutants (GST-1, 1–110 residues of Sp1; GST-2, 301–350 residues of Sp1; GST-3, 504–785 residues of Sp1) were prepared as described under “Experimental Procedures,” focusing on the potential phosphorylation sites (A). The recombinant proteins were expressed in E. coli and confirmed by Coomassie Blue stain after SDS-PAGE (B). In vitro phosphorylations of Sp1 by PKC (C) and active Erk1 (D) were evaluated by an in vitro kinase assay. The recombinant proteins were subjected to kinase assay in the presence of 5 Ci of [-32P]ATP and 10 times excess amount of unlabeled ATP. The reaction mixture was separated by SDS-PAGE and then visualized by autoradiography. Note phosphorylated GST-1 and GST-3, but not GST-2, in addition to the GST-Sp1 full sequence by active PKC and active Erk1. To identify phosphorylated residues in the recombinant proteins, immunoblot (IB) analyses were performed with anti-Ser(P) (E) and anti-Thr(P) (F) antibodies. Note the phosphorylations of serine and threonine residues in the GST-1 and GST-3, respectively, by active Erk1.

Article Snippet: Generation of ROS was measured by FACS usingH2-DCFDA, according to themethod described elsewhere (22). siRNA Transfection—Young and mid-old cells were transfected with siRNA against PKC , PKC I, and Sp1 (Santa Cruz) using Oligofectamine (Invitrogen) following the protocol provided by the manufacturer.

Techniques: In Vitro, Phospho-proteomics, Construct, Recombinant, Staining, SDS Page, Kinase Assay, Autoradiography, Sequencing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1

doi: 10.1074/jbc.m808734200

Figure Lengend Snippet: FIGURE 6. In vitro phosphorylation of Sp1 on Ser59 and Thr739 by active Erk1. Based on the above data, serine and threonine residues were suspected as the phosphorylation sites in the GST-1 and GST-3, respec- tively. Therefore, Ser59 to Ala (A) and Thr739 to Ala (C) mutant constructs were prepared by site-directed mutagenesis. Employing kinase analyses, we confirmed that active Erk1 phosphorylated GST-1 and GST-3 proteins as expected, however, it failed in GST-1 and GST-3 proteins with the Ser59 to Ala mutant (B) and the Thr739 to Ala mutant (D), indicating the Ser59 and Thr739 residues as in vitro phosphorylation sites of active Erk1.

Article Snippet: Generation of ROS was measured by FACS usingH2-DCFDA, according to themethod described elsewhere (22). siRNA Transfection—Young and mid-old cells were transfected with siRNA against PKC , PKC I, and Sp1 (Santa Cruz) using Oligofectamine (Invitrogen) following the protocol provided by the manufacturer.

Techniques: In Vitro, Phospho-proteomics, Mutagenesis, Construct

Journal: Journal of Biological Chemistry

Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1

doi: 10.1074/jbc.m808734200

Figure Lengend Snippet: FIGURE 7. In vivo phosphorylation of Sp1 on Ser59 by SA-pErk1/2 and regulation of p21Sdi1 expression. To evaluate in vivo phosphorylation of Sp1 by PKC-, HDF old cells were treated with either GFP-siRNA or PKC-siRNA for 2 days (A), or treated with either vehicle or 10 mM NAC for 12 h (B). The cell lysates (500 g) were incubated with anti-Sp1 antibody and protein G-Sepharose beads for 8 h at 4 °C with continuous agitation. Immunoprecipitated complexes were separated by SDS-PAGE, and subjected to immunoblot analyses with anti-Thr(P), anti-Ser(P), or anti-Sp1 specific antibodies. Note serine phosphorylation of Sp1 in the cells, but not with threonine. Moreover, the degree of Ser(P) in Sp1 was significantly reduced by treatment of the cells with PKC-siRNA and NAC. These findings indicate that Sp1 was in vivo phosphorylated only on the serine residue, but not on threonine. C, at the same time, to evaluate in vivo regulation of Sp1 phosphorylation by SA-pErk1/2, mid-old HDF cells were treated with U0126 for 1 h, and Sp1 protein was extracted by immunoprecipitation. U0126 significantly reduced expressions of pErk1/2 (0.2) and Ser(P) in Sp1 (0.5) than the untreated control. Moreover, threonine phosphorylation in the Sp1 molecule by pErk1/2 was not found. These experiments clearly indicate Sp1 phosphorylation on the serine residue by not only PKC- but also SA-pErk1/2, but not threonine,intheprocessofcellularsenescence.D,toinvestigatewhetherphosphorylationofSp1onSer59regulatesp21Sdi1expression,mid-oldHDFcellswere treated with scrambled-siRNA or Sp1-siRNA for 2 days, and then expressions of Sp1 and p21Sdi1 were determined by immunoblot analyses. Expression of Sp1 was almost completely down-regulated with concurrently reduced p21Sdi1 expression when treated with Sp1-siRNAs. E, to confirm whether Ser(P)59 in Sp1 affects transcription of p21Sdi1 or not, Huh7 cells were transfected with p21Sdi1-luciferase along with either WT-Sp1 or the S59A-Sp1 construct for 24 h, and then serum-starved for 24 h before treatment with EGF for 6 h. As expected, luciferase activity was significantly increased by EGF treatment in the WT-Sp1- transfected cells, as compared with the control (*, p 0.01). However, the effect of EGF was not found in the S59A-Sp1-transfected cells, indicating the role of Ser(P)59-Sp1 in regulation of p21Sdi1 transcription after EGF treatment.

Article Snippet: Generation of ROS was measured by FACS usingH2-DCFDA, according to themethod described elsewhere (22). siRNA Transfection—Young and mid-old cells were transfected with siRNA against PKC , PKC I, and Sp1 (Santa Cruz) using Oligofectamine (Invitrogen) following the protocol provided by the manufacturer.

Techniques: In Vivo, Phospho-proteomics, Expressing, Incubation, Immunoprecipitation, SDS Page, Western Blot, Residue, Control, Transfection, Luciferase, Construct, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1

doi: 10.1074/jbc.m808734200

Figure Lengend Snippet: FIGURE 8. Phosphorylation of Sp1 on Ser59 by SA-pErk1/2 downstream of PKC, and induction of p21Sdi1 expression. ROS and PKC concurrently stimulated each other during cellular senescence. Accumulated ROS stimu- lates the activity of PKC, and the active PKC in turn stimulates ROS gener- ation during cellular senescence, demonstrated by treatment of the old cells with PKC-siRNA. Moreover, activated PKC regulates the expression of senescence-associated pErk1/2 in the cytoplasm (SA-pErk1/2), which in turn increases transcription of p21Sdi1 via in vivo phosphorylation of Sp1 on Ser59. Elevatedp21Sdi1inducescell-cyclearrestoftheactivelygrowingcellsatG1phase, leading to growth arrest of senescent cells. On the other hand, down-regulation of PKC expression by treatment of old cells with PKC-siRNA significantly reducesSA-pErk1/2inbothnuclearandcytoplasmicfractionsoftheoldcells,and inhibits Sp1 phosphorylation on Ser59, but not Thr739, and transcription of p21Sdi1, resulting in the release of senescent cells from G1 arrest. In summary, in vivo regulation of p21Sdi1 expression occurs via Sp1 phosphorylation on Ser59 by pErk1/2 downstream of PKC in the process of cellular senescence.

Article Snippet: Generation of ROS was measured by FACS usingH2-DCFDA, according to themethod described elsewhere (22). siRNA Transfection—Young and mid-old cells were transfected with siRNA against PKC , PKC I, and Sp1 (Santa Cruz) using Oligofectamine (Invitrogen) following the protocol provided by the manufacturer.

Techniques: Phospho-proteomics, Expressing, Activity Assay, In Vivo

Journal: PLoS ONE

Article Title: Oestrogens Downregulate Tissue Factor Pathway Inhibitor through Oestrogen Response Elements in the 5’-Flanking Region

doi: 10.1371/journal.pone.0152114

Figure Lengend Snippet: MCF7 cells were transfected with siRNA for Sp1 (A) or c-JUN (B). 24 hours after transfection, the cells were starved in phenol red-free medium for another 24 hours and thereafter treated with ± 10 nM EE2 for 16 hours before harvesting. mRNA levels were determined using qRT-PCR. Relative mRNA expression levels were calculated with the 2 -ΔΔCt method. The bars represent the mean relative mRNA expression levels after adjusting for the PMM1 endogenous control gene levels. The error bars represent standard deviation from three (A) or two (B) independent experiments with three biological parallels (*** ≤0.0001 relative to the non-transfected (NTC) cells).

Article Snippet: Bovine serum albumin (BSA) was purchased from New England BioLabs (Ipswich, MA, USA). siRNAs against c-JUN (ID 3725 Trilencer-27 siRNA SR302499) and Sp1 (ID 6667 Trilencer-27 siRNA SR304537) were purchased from ORIGENE, as was the Universal Scrambled Negative Control siRNA SR30004.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Control, Standard Deviation

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Prediction of transcription factor binding sites and mutation analysis of the mSTING minimal promoter. ( A ) A schematic representation of the putative binding sites for DNA-binding proteins in the proximal promoter of the mSTING gene. The 129 nt DNA sequence (from −77 to +52) was analyzed with TFSEARCH softwares. The putative transcription factor binding sites were outlined with black box, and the names of the transcription were annotated. The transcription start site (TSS) was marked by black arrow. ( B ) Conserved base sequence of GATA1, IK2, Sp1/Sp3 or STAT binding site. The base size presented the binding affinity coefficient of transcription factor and promoter. ( C ) Mutation analysis of the mSTING minimal promoter. The left: Binding sites for GATA1, IK2, Sp1/Sp3 and STAT were indicated with open different shapes. Mutations were shown in bold above the histogram. The right: The relative luciferase activities derived from mutational pSTING-254. The site-special mutagenized plasmids were cotransfected with pRL-TK into NIH3T3 cells and lucifarase assays were performed. The level of firefly luciferase activities was normalized to the Renilla luciferase activity. Each bar represented the mean ± SD of three independent experiments. (n = 3, * p < 0.05 vs. pSTING-254).

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Binding Assay, Mutagenesis, DNA Binding Assay, Sequencing, Luciferase, Derivative Assay, Activity Assay

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Effect of GATA1 on promoter activity of the mSTING gene. ( A ) Expression of the exogenous GATA1 increased the luciferase reporter gene activity of STING and knockdown of the endogenous GATA1 decreased the promoter activity. NIH3T3 cells were cotransfected with plasmids GATA1 or pcDNA 3.1(+) (200 ng), GATA1 siRNA or negative control siRNA (50 nM) and pSTING-254 (100 ng), pRL-TK (3 ng). Luciferase assays were performed after 24 h. ( B – D ) The mRNA and protein levels of mSTING were significantly increased after GATA1 overexpression. qRT-PCR and western blot were performed after GATA1 plasmid or pcDNA 3.1(+) were transiently cotransfected into NIH3T3 cells. The protein expression was quantified to GAPDH. Each bar represented the mean ± SD of three independent experiments (n = 3, * P < 0.05 vs. Control). ( E – G ) Knockdown of GATA1 by siRNA (50 nM) reduced the mRNA and protein level of mSTING. The protein expression was quantified to GAPDH. siRNA negative control was set as 1. Each bar represented the mean ± SD of three independent experiments (n = 3, * P < 0.05 vs. NC).

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Activity Assay, Expressing, Luciferase, Knockdown, Negative Control, Over Expression, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: ChIP assay of the mSTING proximal promoter was performed in NIH3T3 cells. The immunoprecipitated chromatin fragments were analyzed by semi-quantitative PCR using primer pairs spanning the putative GATA1 and Sp1 binding site (the target locus) or exon 6 of the mSTING (a non-target locus). ( A ) GATA1 bound to the mSTING promoter in vivo . A band of 199 nt containing the GATA1 binding site in the mSTING promoter region was amplified. Lane M: DNA marker 1000. Lane INPUT: PCR product derived by ChIP-GATA1 primers from direct input DNA template without immunoprecipitation. Lane IgG: PCR product derived by ChIP-GATA1 primers from DNA template immunoprecipitated by normal IgG as a negative control. Lane GATA1: PCR product derived by ChIP-GATA1 primers from DNA template immunoprecipitated by anti-GATA1 antibody. Lane STING: PCR product derived by primers flanking exon 6 of the mSTING from DNA template immunoprecipitated by anti-GATA1 antibody. ( B ) Sp3 bound to the mSTING promoter in vivo . A band of 107 nt containing Sp3 binding site in the mSTING promoter region was amplified. Lane M: DNA marker 1000. Lane INPUT: PCR product derived by ChIP-Sp3 primers from direct input DNA template without immunoprecipitation. Lane IgG: PCR product derived by ChIP-Sp3 primers from DNA template immunoprecipitated by normal IgG as a negative control. Lane Sp3: PCR product derived by ChIP-Sp3 primers from DNA template immunoprecipitated by anti-Sp3 antibody. Lane STING: PCR product derived by primers flanking exon 6 of them STING from DNA template immunoprecipitated by anti-Sp3 antibody.

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, In Vivo, Amplification, Marker, Derivative Assay, Negative Control

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Sequences of oligonucleotides used to clone the mSTING gene promoters and site-directed mutagenesis.

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques: Mutagenesis, Sequencing

Journal: Scientific Reports

Article Title: Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

doi: 10.1038/s41598-017-02242-w

Figure Lengend Snippet: Sequences of oligonucleotides used in RT-PCR and ChIP assay.

Article Snippet: GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company (Shanghai, China).

Techniques:

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: A. The expression of eIF4E in normal colon tissues around cancer. B. The negative expression of eIF4E in colon cancerous tissues. C. The positive expression of eIF4E in colon cancerous tissues. D. The expression of VEGF-C in normal colon tissues around cancer. E. The negative expression of VEGF-C in colon cancerous tissues. F. The positive expression of VEGF-C in colon cancerous tissues. G. The expression of E-cadherin (E-cad) in normal colon tissues around cancer. H. The negative expression of E-cad in colon cancerous tissues. I. The positive expression of E-cad in colon cancerous tissues. J. The expression of MMP-2 in normal colon tissues around cancer. K. The negative expression of MMP-2 in colon cancerous tissues. L. The positive expression of MMP-2 in colon cancerous tissues. Bar = 50 μM.

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: Relationship between the expression of eIF4E, VEGF-C, E-cadherin and MMP-2 as well as clinical pathological parameters

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: Log rank test χ2= 75.0054. p< 0.0001. Fragments of human colon tumors were implanted subcutaneously (s.c.) into nude mice, and mice with tumors that reached 4–5 mm in diameter (n=5 per group) were applied to survival statistics. A. Kaplan–Meier survival curves of mice bearing eIF4E negative and positive tumors. B. Kaplan–Meier survival curves of mice bearing VEGF-C negative and positive tumors. C. Kaplan–Meier survival curves of mice bearing MMP-2 negative and positive tumors. D. Kaplan–Meier survival curves of mice bearing E-cadherin negative and positive tumors.

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques:

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: A. The negative expression of eIF4E in colon cancerous tissues. B. The positive expression of eIF4E in colon cancerous tissues. C. The negative expression of VEGF-C in colon cancerous tissues. D. The positive expression of VEGF-C in colon cancerous tissues. E. The negative expression of E-cadherin in colon cancerous tissues. F. The positive expression of E-cadherin in colon cancerous tissues. G. The negative expression of MMP-2 in colon cancerous tissues. H. The positive expression of MMP-2 in colon cancerous tissues. Bar = 25 μM.

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques: Expressing

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: A. Western blotting showed that the stable cell lines including HCT-15/Rluc/E-cadherin, HCT-15/Rluc/VEGF-C, HCT-15/Rluc/MMP-2, and HCT-15/Rluc/eIF4E were constructed successfully. B. The stable colon cancer cell lines with overexpression of E-cadherin, MMP-2, VEGF-c and eIF4E as well as parental HCT-15/Rluc cell line showed different growth rates.

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques: Western Blot, Stable Transfection, Construct, Over Expression

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: A. The HCT-15/Rluc/eIF4E cells (1.0 × 10 6 ) and corresponding HCT-15-Rluc were injected via tail-vein 3 days later. The bioluminescence seen represents the thorax region of the mouse where HCT-15/Rluc and HCT-15/Rluc/eIF4E cells are trapped in the lungs. B. The HCT-15/Rluc/VEGF-C cells (1.0 × 10 6 ) and corresponding HCT-15/Rluc were injected via tail-vein 3 days later. The bioluminescence seen represents the thorax region of the mouse where HCT-15/Rluc and HCT-15/Rluc/VEGF-C cells are trapped in the lungs. C. The HCT-15/Rluc/MMP-2 cells (1.0 × 10 6 ) and corresponding HCT-15/Rluc were injected via tail-vein 3 days later. The bioluminescence seen represents the thorax region of the mouse where HCT-15/Rluc and HCT-15/Rluc/MMP-2 cells are trapped in the lungs. D. The HCT/15-Rluc/E-cadherin cells (1.0 × 10 6 ) and corresponding HCT-15-Rluc were injected via tail-vein 3 days later. The bioluminescence seen represents the thorax region of the mouse where HCT-15/Rluc and HCT-15/Rluc/E-cadherin cells are trapped in the lungs.

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques: Injection

Journal: Oncotarget

Article Title: Expression analysis and clinical significance of eIF4E, VEGF-C, E-cadherin and MMP-2 in colorectal adenocarcinoma

doi: 10.18632/oncotarget.13453

Figure Lengend Snippet: A. Western blotting showed the expression of eIF4E, VEGF-C, MMP-2, and E-cadherin in the corresponding stable SW 480 cell lines with the overexpression and knockdown of eIF4E. Control, Control stable SW480 cell lines by use of lentiviral infection packaged with control empty vectors. Overexpression, the stable SW480 cell line with the overexpression of eIF4E. sheIF4E, the stable SW480 cell lines with knockdown of eIF4E. The cells were lysed for loading on SDS-PAGE. The blotting were performed by use of indicated antibodies. B. Q-PCR showed the expression of eIF4E, VEGF-C, MMP-2, and E-cadherin in the stable SW 480 cell lines with the overexpression of eIF4E. C. Q-PCR showed the expression of eIF4E, VEGF-C, MMP-2, and E-cadherin in the stable SW 480 cell lines with the knockdown of eIF4E. Control, Control stable SW480 cell lines by use of lentiviral infection packaged with control empty vectors. Overexpression of eIF4E, the stable SW480 cell line with the overexpression of eIF4E. sheIF4E, the stable SW480 cell lines with knockdown of eIF4E. *, p<0.05. D. Relative number of migrating cells ( y -axis) in a transwell migration assay is shown for Control, eIF4E-overexpression, and eIF4E-knockdown SW 480 cells. *, p<0.05. E. Number of mammospheres ( y -axis) formed by Control, eIF4E-overexpression and eIF4E-knockdown SW480 cells. *, p<0.05.

Article Snippet: eIF4E cDNA was inserted into the eukaryotic retroviral expression vector pBabe. eIF4E and control siRNAs were purchased from Ribobio (Cat No Q000001977-1-B, Guangzhou, People's Republic of China).

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Infection, SDS Page, Transwell Migration Assay

Journal:

Article Title: Lung Kr?ppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A 2 ? promoter

doi: 10.1042/BJ20041458

Figure Lengend Snippet: (A) Individual clones of RL-65 cells, stably transfected with plasmid encoding an siRNA directed against LKLF (LKLF-1 and -2, lanes 4 and 5), with control empty vector (pU6, lanes 1 and 2), or with a scrambled LKLF siRNA (SCR, lane 3) were harvested and protein lysates prepared in RIPA buffer [50 mM Tris/HCl (pH 7.0), 150 mM NaCl, 1% Nonidet P40, 1% sodium deoxycholate, 0.1% SDS, 50 mM sodium fluoride, 2 mM EDTA and 200 μM Na3VO4]. Equal amount of lysate protein was immunoblotted for expression of LKLF (upper panel) or cPLA2 (lower panel). Steady-state cPLA2 expression was decreased in both clones expressing the LKLF siRNA compared with controls or cells transfected with scrambled siRNA. Loading control for the LKLF blot is shown on the right. (B) Extracts were prepared from pools of RL-65 cells, stably transfected with empty vector (pU6) or siRNA against LKLF after Dounce homogenization and ultracentrifugation as described previously in [16]. Enzyme activity was assayed using [14C]arachidonoyl-phosphatidylcholine in the presence of 5 mM Ca2+. (C) One of the control clones (pU6) and both clones expressing LKLF-siRNA (LKLF-1 and -2) were transiently transfected with the cPLA2 promoter construct with or without an H-Ras expression plasmid. Promoter activity was determined 48 h after transfection. Both siRNA clones showed a decrease in H-Ras-induced promoter activity. (D) H2122 cells were transiently transfected with the cPLA2 promoter construct along with the plasmids encoding siRNA for LKLF, Sp1 or both constructs together. Control cells were transfected with empty vector (pU6). Promoter activity normalized to β-galactosidase was measured after 48 h incubation. Both siRNA constructs significantly decreased steady-state promoter activity. *P<0.05 versus pU6.

Article Snippet: Plasmids encoding siRNAs for LKLF and Sp1 were obtained from GenScript Corporation (Edison, NJ, U.S.A.).

Techniques: Clone Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Homogenization, Activity Assay, Construct, Incubation

Journal:

Article Title: Lung Kr?ppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A 2 ? promoter

doi: 10.1042/BJ20041458

Figure Lengend Snippet: (A) Drosophila SL-2 cells were transfected with expression plasmids in the pACT2 vector encoding LKLF, Sp1, Sp3 or combinations of these factors, along with the cPLA2 promoter construct. Cells were harvested after 72 h, and promoter activity normalized to protein was determined. (B) SL-2 cells were transfected with empty vector or with the expression plasmids encoding LKLF, Sp1 or Sp3, along with either the wild-type cPLA2 promoter construct (WT), construct encoding mutations at −37/−30 (DE mut), or mutations at −21/−8 (F mut). Promoter activity normalized to protein was determined as in (A).

Article Snippet: Plasmids encoding siRNAs for LKLF and Sp1 were obtained from GenScript Corporation (Edison, NJ, U.S.A.).

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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis

doi: 10.1007/s00262-008-0645-8

Figure Lengend Snippet: Sodium butyrate enhances the expression of Hsp70 and transcription factor Sp1 in HeLa cells. a, b HeLa cells were starved in serum-free medium, then stimulated (a) with 2 mM sodium butyrate for the indicated times or (b) with the indicated dose of sodium butyrate for 24 h. The mRNA expression of Hsp70 and transcription factors, Sp1 and HSF1, was measured using RT-PCR. Relative band intensities, normalized to β-actin, are shown at right. c Cells were stimulated as above, and protein levels of Hsp70, Sp1 and HSF1 were measured by western blotting. Relative band intensities, normalized to β-actin, are shown below. *P < 0.01 compared to unstimulated cells. Representative results from three independent experiments are shown

Article Snippet: siRNAs targeting Sp1 (si-Sp1) and the nontargeting siRNA (si-control) were purchased from Ribobio (Guangzhou, China).

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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis

doi: 10.1007/s00262-008-0645-8

Figure Lengend Snippet: Chromatin immunoprecipitation analyses of Sp1 or HSF1 binding to the MICA or MICB promoter in vivo. HeLa cells were starved in serum-free medium and then treated with or without 2 mM sodium butyrate or sodium valproate, as indicated (VPA, SB). After formaldehyde crosslinking, soluble chromatin from cell lysates was immunoprecipitated with the indicated antibodies (anti-Sp1, anti-HSF1, or IgG as a control). The total extracted DNA (input) prior to immunoprecipitation and the immunoprecipitated DNA were PCR-amplified using primers specific to MICA promoter fragment (a) or MICB promoter fragment (b). ChIP PCR products were separated on 1% agarose gels and stained with ethidium bromide (top of each panel). Relative band intensities (bottom of each panel) are presented as a percentage of the corresponding input. Data represent mean ± SD from three independent experiments. *P < 0.01 compared to untreated control

Article Snippet: siRNAs targeting Sp1 (si-Sp1) and the nontargeting siRNA (si-control) were purchased from Ribobio (Guangzhou, China).

Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Immunoprecipitation, Control, Amplification, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis

doi: 10.1007/s00262-008-0645-8

Figure Lengend Snippet: siRNA targeting Sp1 diminished the enhancement of MICA expression by sodium butyrate. HeLa and HepG2 cells were starved in serum-free medium and treated with or without 2 mM sodium butyrate for 24 h. The cells were then transfected with lipofectamine 2000 alone (mock) or si-Sp1, or si-control complexed with lipofectamine 2000 at concentration of 100 nM and cultured for 24 h. The silence effect of si-Sp1 and the mRNA expression of MICA were tested by RT- PCR. Band intensities are normalized to β-actin. Error bars represent SD of three independent experiments

Article Snippet: siRNAs targeting Sp1 (si-Sp1) and the nontargeting siRNA (si-control) were purchased from Ribobio (Guangzhou, China).

Techniques: Expressing, Transfection, Control, Concentration Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Hereditas

Article Title: Activation of TRAF1 induced by USP7/SP1 exacerbates the severity of infantile pneumonia

doi: 10.1186/s41065-025-00410-x

Figure Lengend Snippet: SP1 transcriptionally activated TRAF1 in WI-38 cells. ( A ) The schematic illustration showed the binding sites of SP1 in the promoter region of the TRAF1 mRNA. ( B ) ChIP analysis of the association of SP1 with TRAF1. ( C ) The efficiency of SP1 knockdown was analyzed. ( D ) Luciferase reporter analysis of the association of SP1 with TRAF1. ( E ) SP1 mRNA expression in the serum of IP patients and healthy controls. ( F ) The correlation of SP1 with TRAF1 in the serum of IP patients. ( G ) The effect of SP1 knockdown on TRAF1 protein expression. * P < 0.05, ns: not significant

Article Snippet: Small interfering RNAs (siRNAs) of TRAF1 (si-TRAF1, si-TRAF1#1, and si-TRAF1#2), USP7 (si-USP7, si-USP7#1, and si-USP7#2) and SP1 (si-SP1, si-SP1#1, and si-SP1#2), USP7 overexpression plasmid (OE-USP7), TRAF1 overexpression plasmid (OE-TRAF1), and the matched controls (si-NC and OE-control) were provided by GenePharma (Shanghai, China).

Techniques: Binding Assay, Knockdown, Luciferase, Expressing