Journal: Cell Death & Disease

Article Title: Origin recognition complex 6 overexpression promotes growth of glioma cells

doi: 10.1038/s41419-024-06764-w

Figure Lengend Snippet: Multiple databases predicted the common potential transcription factors of ORC6 ( A ). P1 glioma cells were transfected with the specified siRNAs (at 200 nM) targeting the described transcription factors or a scramble non-sense siRNA (siC, at 200 nM) for 48 h, the expression of ORC6 mRNA was assessed ( B ). P1 glioma cells were genetically engineered to stably express the lentiviral RBPJ shRNA (shRBPJ-Sq1 or shRBPJ-Sq2) ( C and D ), scramble control shRNA (“shC”) ( C , D ), the lentiviral RBPJ-expressing construct (oeRBPJ) ( E , F ), or an empty vector (“Vec”) ( E , F ); The expression of the listed mRNAs and proteins was assessed ( C – F ). Chromatin Immunoprecipitation (ChIP) assay results showed the relative levels of RBPJ-bound ORC6 promoter region in specified glioma tissues (“T”) and matched adjacent normal brain tissues (“N”) ( G ) as well as in listed glioma cells and human astrocytes ( H ). RBPJ protein expression in primary human astrocytes (“Astrocytes1/2”), immortalized (A172) or primary (“P1,” “P2,” “P3”) glioma cells was presented ( I ). Values were mean ± standard deviation (SD). * P < 0.05 versus “siC” ( B ). * P < 0.05 versus “shC”/ “Vec” cells ( C – F ). * P < 0.05 versus “N” tissues or “Astryocytes1” ( G , H ). “n. s.” indicates non-statistical difference ( P > 0.05). The experiments were conducted five times (biological repeats), yielding consistent results.

Article Snippet: Genechem (Shanghai, China) provided validated siRNAs targeting specific transcription factors (STAT5A, RBPJ, NFYA, NFATC1, WT1, SP1 and STAT1).

Techniques: Transfection, Expressing, Stable Transfection, shRNA, Control, Construct, Plasmid Preparation, Chromatin Immunoprecipitation, Standard Deviation

Journal: Antioxidants

Article Title: Specificity Protein 1-Mediated Promotion of CXCL12 Advances Endothelial Cell Metabolism and Proliferation in Pulmonary Hypertension

doi: 10.3390/antiox12010071

Figure Lengend Snippet: NOX1 activates the transcription factor specificity protein 1 (Sp1), Sp1 consensus DNA binding, and CXCL12 gene promotion. ( A ) Western blot analysis of human lung lysates for p-Sp1/β-actin (n = 6). ( B ) Sp1 consensus DNA binding activity in human lung lysates measured by Active Motif TransAM assay (n = 6). ( C ) hPAECs transfected with scrambled or NOX1-targeted siRNA for 24 h were exposed to 1% O 2 and analyzed by Western blot for Sp1 activation, p-Sp1/β-actin (n = 3–6). ( D ) hPAECs transfected with scrambled or NOX1-targeted siRNA for 24 h were exposed to 1% O 2 and analyzed by Active Motif Trans AM assay. ( E ) Sp1 transcriptional activation was tested in HUVECs transfected with SpRE luciferase plasmid in addition to scrambled or NOX1-targeted siRNA. Cells were placed in normoxia or 1% O 2 for 48 h and luciferase activity was measured by plate reader luminometer (n = 3–6). ( F ) hPAECs transfected with scrambled or Sp1-targeted siRNA for 24 h were exposed to 1% O 2 48 h. CXCL12 expression was measured by flow cytometry (n = 3–9). Representative flow cytometry histograms depict mean fluorescent intensity of FITC along the x axis, with a rightward shift of the peak indicating increased CXCL12 expression. ( G ) HUVECs transfected with luciferase reporter construct containing 1 kb of the human CXCL12 promoter were transfected with scrambled or Sp1-targeted siRNA. Cells were then exposed to 48 h hypoxia (1% O2) and Sp1′s ability to promote luciferase activity measured by plate reader luminometer (n = 3–4). Results were analyzed by Student t-test ( A , B ), one-way ANOVA followed by the Holm–Sidak test ( C – G ) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TransAM ® Transcription Factor Activation Assay for Sp1 was purchased from Active Motif (Carlsbad, CA, USA).

Techniques: Binding Assay, Western Blot, Activity Assay, Transfection, Activation Assay, Luciferase, Plasmid Preparation, Expressing, Flow Cytometry, Construct

Journal: Antioxidants

Article Title: Specificity Protein 1-Mediated Promotion of CXCL12 Advances Endothelial Cell Metabolism and Proliferation in Pulmonary Hypertension

doi: 10.3390/antiox12010071

Figure Lengend Snippet: Hypoxia-induced proliferation and migration of pulmonary endothelial cells are NOX1-, Sp1- and CXCL12-dependent. ( A , B ) Normoxic vs. hypoxic hPAECs (1% O 2 ) were transfected with either scrambled or Sp1-targeted siRNA and analyzed by ( A ) Crystal Violet and ( B ). Wound closure assay; insets are wound images taken at 24 h. ( C ) hPAECs were subjected to normoxic vs. hypoxic (1% O 2 ) conditions following treatment with vehicle (DMSO) or Sp1 inhibitor mithramycin; proliferation was analyzed by Click-It TM EdU incorporation and flow cytometry. ( D , E ) hPAECs transfected with scrambled or CXCL12-targeted siRNA analyzed by ( D ). Crystal Violet and ( E ). wound closure assay. ( F ) hPAECs were treated with scrambled or Nox1-selective inhibitory peptide NoxA1ds, in addition to recombinant CXCL12 or vehicle control (PBS),and were subjected to hypoxia (1% O 2 ) or normoxia. Proliferation was analyzed by Click-It EdU incorporation and flow cytometry. Representative flow cytometry histograms depict populations of EdU-positive and -negative cells in a sample along the x axis, with right hand peaks indicating proliferating cells taken as percentage to the total number of cells in the sample (see for gating). Results were analyzed by one-way ANOVA and Holm–Sidak post hoc analysis ( A – F ). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TransAM ® Transcription Factor Activation Assay for Sp1 was purchased from Active Motif (Carlsbad, CA, USA).

Techniques: Migration, Transfection, Wound Closure Assay, Flow Cytometry, Recombinant, Control

Journal: Antioxidants

Article Title: Specificity Protein 1-Mediated Promotion of CXCL12 Advances Endothelial Cell Metabolism and Proliferation in Pulmonary Hypertension

doi: 10.3390/antiox12010071

Figure Lengend Snippet: Working model of NOX1-mediated SP1 regulation of CXCL12 in hypoxia and PAH . Working model depicting NOX1 (alongside its critical subunits p22 phox , NOXO1, NOXA1, and Rac1/2) induction of CXCL12 via Sp1 following hypoxia. Resultant CXCL12 is postulated to be secreted from cells, leading to downstream promotion of vascular remodeling and PAH symptoms via pro-proliferative metabolic alteration and glucose uptake.

Article Snippet: TransAM ® Transcription Factor Activation Assay for Sp1 was purchased from Active Motif (Carlsbad, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

doi: 10.1371/journal.pone.0029527

Figure Lengend Snippet: Plasmids generated for luciferase reporter assays.

Article Snippet: Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG (Cell Signaling Technologies).

Techniques: Generated, Luciferase

Journal: PLoS ONE

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

doi: 10.1371/journal.pone.0029527

Figure Lengend Snippet: Mutations in Sp1 and CREB binding sites in the promoter, and HNF-1 and NF-Y in the enhancer of human NAGS .

Article Snippet: Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG (Cell Signaling Technologies).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

doi: 10.1371/journal.pone.0029527

Figure Lengend Snippet: DNA sequence of the promoter (A) and enhancer (B) regions were aligned using CLUSTALW alignment software. CLOVER analysis was used to identify transcription factor binding motifs. Binding sites for C/EBP (green), Sp1 (red), CREB/ATF (pink), AP-2 (purple), HNF-1 (blue), NF-Y (olive), and SMAD 3 (cyan) were highly conserved.

Article Snippet: Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG (Cell Signaling Technologies).

Techniques: Sequencing, Software, Binding Assay

Journal: PLoS ONE

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

doi: 10.1371/journal.pone.0029527

Figure Lengend Snippet: Two probes for the promoter (A) and one probe for the enhancer (B) encompass the highly conserved transcription factor binding motifs of NAGS . The motif colors reflect the colors used in . Assays followed by immunoblot confirmed binding of Sp1 and CREB, but not C/EBP within the promoter (C) and HNF-1 and NF-Y, but not SMAD3 or AP-2 within the enhancer regions (D). Lanes 1–4 represent precipitated proteins from mouse liver nuclear extract bound to biotinylated probes of the regions of interest (Lane 1), non-biotinylated probes of the regions of interest (Lane 2), biotinylated probes of non-specific regions (Lane 3), and no probe (Lane 4). Lanes 5–8 represent supernatant fluid from overnight incubation of biotinylated probes of the region of interest (Lane 5), non-biotinylated probes of the region of interest (Lane 6), biotinylated probes of the non-specific regions (Lane 7), or no probe (Lane 8). Immunoblots are representative of at least three replicate experiments.

Article Snippet: Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG (Cell Signaling Technologies).

Techniques: Binding Assay, Western Blot, Incubation

Journal: PLoS ONE

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

doi: 10.1371/journal.pone.0029527

Figure Lengend Snippet: ChIP with transcription factor antibodies was compared to negative control IgG antibody. Real-Time PCR using promoter or enhancer specific primers was compared to primers for the negative locus MIP-2. The results confirmed that Sp1 and CREB but not C/EBP bind within the promoter (A) and HNF-1 and NF-Y but not AP-2 or SMAD2/3 bind within the enhancer region (B) of NAGS . Calculated error was from three replicate experiments and reported as ± SEM. One asterisk (*) indicates p<0.05 and two asterisks (**) indicate p<0.005.

Article Snippet: Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG (Cell Signaling Technologies).

Techniques: Negative Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Transcriptional Regulation of N-Acetylglutamate Synthase

doi: 10.1371/journal.pone.0029527

Figure Lengend Snippet: Mutagenesis of the putative transcription factor binding sites significantly decreases transcription by the promoter (A) and the enhancer with TATA promoter (B) in liver derived cells when compared to non-mutated sites. Addition of Sp1 with the promoter (A) and HNF-1 with the enhancer (B) increases transcription driven by non-mutated constructs. Calculated results are an average of three independent experiments that were each carried out in triplicate, normalized to Rluc expression, and expressed relative to the promoter for each experiment with error reported as ±SEM. Lowercase letters indicate statistically significant differences.

Article Snippet: Immunoprecipitation was conducted using antibodies to transcription factors Sp1 (Millipore), CREB (Santa Cruz Biotech), C/EBP (Santa Cruz Biotech), HNF-1 (Santa Cruz Biotech), NF-Y (Santa Cruz Biotech), SMAD2 (Santa Cruz Biotech) and AP-2 (Santa Cruz Biotech) and control antibodies to histone H3 and non-specific rabbit IgG (Cell Signaling Technologies).

Techniques: Mutagenesis, Binding Assay, Derivative Assay, Construct, Expressing

Journal: Scientific reports

Article Title: Syndecan-2 regulates PAD2 to exert antifibrotic effects on RA-ILD fibroblasts.

doi: 10.1038/s41598-022-06678-7

Figure Lengend Snippet: Figure 3. Syndecan-2 (SDC2) inhibits α-SMA and PAD2 expression by regulating CD148/PI3K/Akt signaling in RA-ILD fibroblasts. Control (n = 4) and RA-ILD (n = 4) fibroblasts were transfected with scramble (scr) or shCD148 via lentivirus as described in the Methods. Stably transfected cells were treated with syndecan-2 (10 μg/ml) for 24 h. After treatment, cells were subjected to (A) Western blot to measure phospho-Akt, α-SMA, and PAD2 expression, (B) RT-PCR to measure mRNA levels of PADI2 (PAD2), or (C) a Sp1 transcriptional activity assay as described in the Methods. (D) RA-ILD fibroblasts were transfected via lentivirus with empty vector or pLenti-GIII-CMV-C-term-HA plasmid carrying the PADI2 (PAD2-over). Stably transfected cells were treated with syndecan-2 (10 μM) for 24 h. After treatment, cells were subjected to RT-PCR to measure mRNA levels of COL1A1 and PADI2. Data are mean ± SEM. P < 0.05; significant comparisons by one-way ANOVA: * vs. control, † vs. RA-ILD fibroblasts + Scr, # vs. RA-ILD fibroblasts + Scr + SDC2. Abbreviations: α-SMA = alpha- smooth muscle actin, ILD = interstitial lung disease, p-Akt = phosphorylated Akt, PAD2 = peptidylarginine deiminase 2, PI3K = phosphoinositide 3-kinase, RA = rheumatoid arthritis, scr = scrambled short hairpin RNA, SDC2 = syndecan-2, shCD148 = CD148 short hairpin RNA, shSp1 = Sp1 short hairpin RNA.

Article Snippet: Sp1 transcriptional activity was measured using a commercially available kit from RayBiotech (Peachtree Corners, GA) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Transfection, Stable Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Plasmid Preparation, shRNA