Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Sp1‐mediated miR ‐193b suppresses atopic dermatitis by regulating HMGB1

doi: 10.1002/kjm2.12693

Figure Lengend Snippet: miR‐193b overexpression promotes cell proliferation and represses inflammatory injury in TNF‐α/IFN‐γ‐treated keratinocytes. (A) Skin tissues were collected from 10 AD patients and 10 control subjects without any history of allergy and used for detecting miR‐193b expression by qRT‐PCR. (B) miR‐193b expression in HaCaT cells after TNF‐α/IFN‐γ treatment was detected using qRT‐PCR. (C) miR‐193b expression was determined using qRT‐PCR after transfecting HaCaT cells with NC or miR‐193b mimics. miR‐193b overexpression plasmid was transfected into the TNF‐α/IFN‐γ‐treated HaCaT cells. (D) The viability of HaCaT cells was analyzed using the MTT assay. (E) The cell cycle was analyzed using flow cytometry. (F) TNF‐α, IL‐1β and IL‐6 levels were analyzed using ELISA. All data were expressed as mean ± SD and were obtained from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The short hairpin RNA of HMGB1 (sh‐HMGB1), overexpression plasmids of HMGB1 (OE‐HMGB1), and Sp1 overexpression (OE‐Sp1), miR‐193b mimics/inhibitor and their negative controls were acquired from GenePharma.

Techniques: Over Expression, Control, Expressing, Quantitative RT-PCR, Plasmid Preparation, Transfection, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Sp1‐mediated miR ‐193b suppresses atopic dermatitis by regulating HMGB1

doi: 10.1002/kjm2.12693

Figure Lengend Snippet: HMGB1 overexpression reversed the effect of miR‐193b overexpression on TNF‐α/IFN‐γ‐induced keratinocyte inflammatory injury. (A), (B) HMGB1 expression in HMGB1‐overexpressing HaCaT cells was examined by qRT‐PCR and Western blot. HMGB1 and miR‐193b overexpression were induced in inflamed HaCaT cells. (C) HaCaT cell viability was determined by MTT assay. (D) The cell cycle was examined using flow cytometry. (E) TNF‐α, IL‐1β and IL‐6 levels were analyzed using ELISA. (F) HMGB1 and p‐p65 NF‐κB levels in cells were detected using Western blot. Data were expressed as mean ± SD and were obtained from three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: The short hairpin RNA of HMGB1 (sh‐HMGB1), overexpression plasmids of HMGB1 (OE‐HMGB1), and Sp1 overexpression (OE‐Sp1), miR‐193b mimics/inhibitor and their negative controls were acquired from GenePharma.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Sp1‐mediated miR ‐193b suppresses atopic dermatitis by regulating HMGB1

doi: 10.1002/kjm2.12693

Figure Lengend Snippet: Sp1 positively regulates miR‐193b expression by directly binding to the miR‐193b promoter. (A) The binding sites between Sp1 and miR‐193b promoter were predicted by TransmiR v2.0 and JASPAR database. (B), (C) The interaction between Sp1 and miR‐193b promoter was analyzed by ChIP and dual luciferase reporter gene assays. (D) Sp1 mRNA levels in TNF‐α/IFN‐γ‐treated HaCaT cells were analyzed using qRT‐PCR. (E), (F) Sp1 expression in HaCaT cells following Sp1 knockdown or Sp1 overexpression was assessed using qRT‐PCR and Western blot. (G) miR‐193b expression in HaCaT cells following Sp1 knockdown or Sp1 overexpression was analyzed using qRT‐PCR. Data were expressed as mean ± SD and were obtained from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The short hairpin RNA of HMGB1 (sh‐HMGB1), overexpression plasmids of HMGB1 (OE‐HMGB1), and Sp1 overexpression (OE‐Sp1), miR‐193b mimics/inhibitor and their negative controls were acquired from GenePharma.

Techniques: Expressing, Binding Assay, Luciferase, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Sp1‐mediated miR ‐193b suppresses atopic dermatitis by regulating HMGB1

doi: 10.1002/kjm2.12693

Figure Lengend Snippet: miR‐193b overexpression inhibited AD in vivo. DNFB was applied on the abdomen of BALB/c mice to induce AD before inducing miR‐193b overexpression. (A) Representative photomicrographs of skin sections were stained with HE. (B) The severity score was analyzed. (C) Serum IgE levels were analyzed using ELISA. (D) IL‐6, IL‐13, and IL‐4 expression levels were determined using qRT‐PCR. Data were expressed as mean ± SD. N = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The short hairpin RNA of HMGB1 (sh‐HMGB1), overexpression plasmids of HMGB1 (OE‐HMGB1), and Sp1 overexpression (OE‐Sp1), miR‐193b mimics/inhibitor and their negative controls were acquired from GenePharma.

Techniques: Over Expression, In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Biochimie

Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

doi: 10.1016/j.biochi.2014.12.002

Figure Lengend Snippet: Analysis of the unique bands on SDS-PAGE gels by Q Exactive MS. a

Article Snippet: Sp1 overexpression plasmid was kindly provided by Dr. Gun-tram Suske at Philipps-University Marburg, Germany.

Techniques: Sequencing

Journal: Biochimie

Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

doi: 10.1016/j.biochi.2014.12.002

Figure Lengend Snippet: Analysis of the transcription factors that could bind to hnRNP K promoter. A. In silico analysis showed that at least two Sp1 binding sites in hnRNP K promoter. B. ChIP analysis verified the binding of Sp1 with hnRNP K promoter in the 293T cells. There were two controls, a non-DNA negative control and an input DNA sample. PCR was performed using primers recognizing the K1 and K2 sequences in the hnRNP K promoter and the amplified products were separated in agarose gel and visualized with EB. The sizes of the PCR products were 513 bp (K1) and 229 bp (K2). Similar results were observed in three independent experiments. C. EMSA confirmed the exact binding site of Sp1 in the 37 bp probe. N.E. denotes nuclear extract of 293T cells. Cold means cold competitor and mutant means mutant competitor. D. Promoter luciferase reporter analysis of the sequences with mutant Sp1 binding sites in 293T and HK2 cells. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis. (a) P < 0.05 versus pGL3-basic; (b) P < 0.05 versus pGL3-K1–1.

Article Snippet: Sp1 overexpression plasmid was kindly provided by Dr. Gun-tram Suske at Philipps-University Marburg, Germany.

Techniques: In Silico, Binding Assay, Negative Control, Amplification, Agarose Gel Electrophoresis, Mutagenesis, Luciferase

Journal: Biochimie

Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

doi: 10.1016/j.biochi.2014.12.002

Figure Lengend Snippet: Effect of Sp1 overexpression on hnRNP K and AGT expression in HK2 cells. A. The promoter activity of hnRNP K was significantly enhanced by Sp1. B. Overexpression of Sp1 increased the protein level of hnRNP K. C. Overexpression of Sp1 significantly reduced the mRNA level of AGT. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis.*P < 0.05; **P < 0.01.

Article Snippet: Sp1 overexpression plasmid was kindly provided by Dr. Gun-tram Suske at Philipps-University Marburg, Germany.

Techniques: Over Expression, Expressing, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Curcumol repressed cell proliferation and angiogenesis via SP1/mir-125b-5p/VEGFA axis in non-small cell lung cancer

doi: 10.3389/fphar.2022.1044115

Figure Lengend Snippet: Curcumol decreased VEGFA, SP1, but increased miR-125b-5p Expression. (A) Transcription factors in descending order of score, that may regulate VEGFA transcription. (B) mRNA expression level of VEGFA and SP1 in A549 and H1975, relative to that of BEAS-2B. (C , D) VEGFA, SP1 protein expression levels were detected by Western blot. (E) MicroRNAs with more than seven base binding sites at the 3′UTR of VEGFA mRNA searched on miRmap. (F) Differential expression of miR-125b-5p in human normal tissues and tumor tissues. LUAD, Lung Adenocarcinoma. (G) Expression level of miR-125b-5p in A549 and H1975, relative to that of BEAS-2B. (H) Expression level of miR-125b-5p in A549 and H1975, tested by qRT-PCR. The data are presented from five independent experiments (n = 5) run in triplicate. * p < 0.05, compared with control (Con).

Article Snippet: A549 cells were seeded on slides (SPL life sciences, South Korea) then transfected with SP1 overexpression vectors (pcDNA3.1-SP1) and pcDNA3.1 negative control vectors (GeneCopoeia, United States), followed by treating with Curcumol for 24 h. MiR-125b-5p mimics, inhibitors and NC were generated by GenePharma (Shanghai, China).

Techniques: Expressing, Western Blot, Binding Assay, Quantitative Proteomics, Quantitative RT-PCR, Control

Journal: Frontiers in Pharmacology

Article Title: Curcumol repressed cell proliferation and angiogenesis via SP1/mir-125b-5p/VEGFA axis in non-small cell lung cancer

doi: 10.3389/fphar.2022.1044115

Figure Lengend Snippet: Overexpressed SP1 impacted Curcumol-mediated cell viability, VEGFA and miR-125b-5p. (A) A549 and H1975 were transfected with SP1 overexpression plasmid (pcDNA3.1-SP1 0.5 μg/ml) or control plasmid (pcDNA3.1) for 24 h, followed by added with Curcumol for another 24 h. Cell viability was calculated by OD (optical density) number of each well. (B , C) Protein expression level of SP1, VEGFA were detected by Western blot, with β-Tubulin as internal control. (D , E) The expression of VEGFA protein in A549 cells was detected by cellular immunofluorescence, and the localization and semi quantification were performed. DAPI was used to display nuclei. Scale bar, 20 μm. (F) NSCLC cells were transfected with vectors, followed by treating with Curcumol (final concentration 400 μM) for 24 h. Expression of miR-125b-5p were detected by qTR-PCR, with U6 as internal reference. The data are presented from five independent experiments (n = 5) run in triplicate. * p < 0.05, compared with control (Con). # p < 0.05, compared with pcDNA3.1 + Curcumol group.

Article Snippet: A549 cells were seeded on slides (SPL life sciences, South Korea) then transfected with SP1 overexpression vectors (pcDNA3.1-SP1) and pcDNA3.1 negative control vectors (GeneCopoeia, United States), followed by treating with Curcumol for 24 h. MiR-125b-5p mimics, inhibitors and NC were generated by GenePharma (Shanghai, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Expressing, Western Blot, Immunofluorescence, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Curcumol repressed cell proliferation and angiogenesis via SP1/mir-125b-5p/VEGFA axis in non-small cell lung cancer

doi: 10.3389/fphar.2022.1044115

Figure Lengend Snippet: MiR-125b-5p mimics or inhititor modulated Curcumol-mediated cell viability, VEGFA and SP1 Expression. (A , B) Quantification of miR-125b-5p was shown after addition of mimics or inhibitor for 24 h. Subsequently, cell viability of A549 and H1975 were measured by MTT assay. (C) The luciferase reporter constructed with wild type or mutant VEGFA sequences were shown. 293T cells were transfected with VEGFA 3′UTR-WT or VEGFA 3′UTR-Mut vectors (0.5 μg/ml each well) for 24 h, then transfected with miR-125b-5p mimics (50 nM) or NC for another 24 h. The luciferase activity was detected on fluorescence microplate reader. VEGFA protein was displayed by (D,E) , Western blot analysis as well as (F) cellular immunofluorescence assay after transfection of miR-125b-5p inhibitor (50 nM) and addition of Curcumol (400 μM). Scale bar, 20 μm. (G , H) The bands of SP1 by Western blot analysis. β-Tubulin was used as internal control. (I) Dual-Luciferase Reporter Assay was carried out to detect SP1 promoter luciferase activity. The data are presented from five independent experiments (n = 5) run in triplicate. * p < 0.05, compared with control (Con). # p < 0.05, compared with NC + Curcumol group.

Article Snippet: A549 cells were seeded on slides (SPL life sciences, South Korea) then transfected with SP1 overexpression vectors (pcDNA3.1-SP1) and pcDNA3.1 negative control vectors (GeneCopoeia, United States), followed by treating with Curcumol for 24 h. MiR-125b-5p mimics, inhibitors and NC were generated by GenePharma (Shanghai, China).

Techniques: Expressing, MTT Assay, Luciferase, Construct, Mutagenesis, Transfection, Activity Assay, Fluorescence, Western Blot, Immunofluorescence, Control, Reporter Assay

Journal: Frontiers in Pharmacology

Article Title: Curcumol repressed cell proliferation and angiogenesis via SP1/mir-125b-5p/VEGFA axis in non-small cell lung cancer

doi: 10.3389/fphar.2022.1044115

Figure Lengend Snippet: Curcumol reduced tumor burden and MVD of nude mice. (A , B) Mice (n = 6 per group) were divided into four groups (Con [saline], Axitinib [25 mg/kg], Curcumol [40 mg/kg], and Curcumol [80 mg/kg]). Tumor tissue were harvested and photographed after 25 days intragastric administration. Tumor volume and tumor weight were measured. (C) Tumor growth degree was evaluated by measuring bioluminescence signals in IVIS imaging system on 1st day and 25th day. (D) Body weight of mice in each group recorded every 5 days. (E – G) Immunohistochemistry (IHC) images of PCNA, VEGFA, SP1, CD31. Scale bar, 20 μm. ImageJ software was used to count positive cells. Microvessel density (MVD) was calculated through CD31 antibody staining. (H , I) VEGFA and SP1 protein expression level in mice fresh tumor tissue were detected by Western blot. Axi, Axitinib 25 mg/kg. Cur-L, Curcumol 40 mg/kg. Cur-H, Curcumol 80 mg/kg. (J) MiR-125b-5p expression level were detected by qTR-PCR, with U6 as internal control. (K) The diagram showed that Curcumol inhibited NSCLC cell growth and angiogenesis through suppressing SP1 expression, increasing miR-125b-5p expression, followed by downregulation of VEGFA. There is feedback regulatory axis and complex interaction network among these targets that together contribute to the inhibitory effect of Curcumol on NSCLC cells. * p < 0.05, compared with control (Con).

Article Snippet: A549 cells were seeded on slides (SPL life sciences, South Korea) then transfected with SP1 overexpression vectors (pcDNA3.1-SP1) and pcDNA3.1 negative control vectors (GeneCopoeia, United States), followed by treating with Curcumol for 24 h. MiR-125b-5p mimics, inhibitors and NC were generated by GenePharma (Shanghai, China).

Techniques: Saline, Imaging, Immunohistochemistry, Software, Staining, Expressing, Western Blot, Control

Journal: eLife

Article Title: HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

doi: 10.7554/eLife.61531

Figure Lengend Snippet: ( A ) Nissl staining of the coronal session of Trrap-FBΔ brain at postnatal day 0 (P0) and 10 (P10). Ctx: cortex; str: striatum; v: ventricle. ( B ) The Venn diagram depicts the overlap between the differentially expressed genes (DEGs) measured by RNA-seq in the cortex and striatum. The numbers refer to the DEGs in the indicated data sets. ( C ) Log 2 of the fold changes of the 2695 common DEGs in Trrap-FBΔ cortex and striatum. ( D ) Top50 GO terms of the 2695 overlapping hits identified in the RNA-seq data set of the cortex and striatum. Note that microtubule dynamics related processes are highlighted in red. ( E ) Transcription factor binding site (TFBS) enrichment analysis of the 1261 common DEGs in aNSCs, the cortex, and the striatum identified by RNA-seq. ( F ) Luciferase assays using a Sp1-responsive construct. The graph shows the luciferase activity normalized by Bradford assay. N: the number of cell lines analyzed; Mock: empty vector, Sp1: overexpression; luc: luciferase. Co.: control; aNSCsΔ: Trrap-aNSCsΔ. n: the number of cell lines analyzed. Mean ± standard error of the mean is shown. Unpaired t-test was performed for statistical analysis. n.s.: not significant; *p≤0.05, **p≤0.01.

Article Snippet: Recombinant DNA reagent , pN3-Sp1FL , Addgene , Cat#: 24543 , Sp1 overexpression reporter; H. sapiens.

Techniques: Staining, RNA Sequencing, Binding Assay, Luciferase, Construct, Activity Assay, Bradford Assay, Plasmid Preparation, Over Expression, Control

Journal: eLife

Article Title: HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

doi: 10.7554/eLife.61531

Figure Lengend Snippet: ( A ) Western blot analysis of the Trrap deletion and expression of STMNs in the forebrain of indicated genotype at postnatal day 10 (P10). β-actin is a loading control. Co: control; FBΔ: Trrap-FBΔ. ( B ) The quantification of the expression of the indicated proteins in mutant forebrains measured by western blots are related value to adjacent controls after normalization to β-actin. N: the number of mice analyzed. The error bar presents the standard error. Paired t-test was used for statistical analysis. *p≤0.05, **p≤0.01. ( C and D ) Sagittal sections of 4-month-old Trrap-PCΔ mice were stained against STMN3 (green, C ) and STMN4 (green, D ), the Purkinje cell marker Calbindin (red) and counterstained with DAPI (blue). The right panel shows the average intensity of STMN3 or STMN4 in Purkinje cells normalized by the intensity in the neighboring cells (not Trrap-deleted). n: the number of cells analyzed; N = 4 mice analyzed. ( E ) ChIP analysis on the STMN3 and STMN4 promoters in control striata using antibodies against Sp1, AcH4, and IgG. qPCR analysis was performed to quantify the binding of the indicated factors to the promoter. The binding enrichment was calculated as fold enrichment over IgG. N = 3 mice analyzed. The primers that contain the Sp1 site for ChIP assays are marked in . ( F and G ) ChIP analysis on the proximity of STMN3 ( F ) and STMN4 ( G ) promoters in control and TrrapΔ aNSCs. Protein binding value is presented in percentage of input. The large error bars in Oct4 ChIP are due to an inclusion of a high value from one pair of samples. N = 3–4 mice analyzed. ( H ) Western blot analysis of STMN3 and STMN4 expression after siRNA-mediated knockdown of Sp1 in aNSCs. Oct4 is an Sp1 independent transcription factor control and β-actin controls loading. ( C–H ) Mean ± standard error of the mean is shown. Unpaired t-test was performed for statistical analysis. *p≤0.05, **p≤0.01.

Article Snippet: Recombinant DNA reagent , pN3-Sp1FL , Addgene , Cat#: 24543 , Sp1 overexpression reporter; H. sapiens.

Techniques: Western Blot, Expressing, Control, Mutagenesis, Staining, Marker, Binding Assay, Protein Binding, Knockdown

Journal: eLife

Article Title: HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

doi: 10.7554/eLife.61531

Figure Lengend Snippet: ( a ) The scheme depicts the overlap between the common differentially expressed genes (DEGs) measured in the RNA-seq analyses of the cortex and striatum of Trrap-FBΔ mice and acetylated histone depleted peaks in aNSCs. The numbers refer to common genes identified in the indicated data sets. ( b ) Level of histone acetylation across SP1-motif in the whole genome. Box plot shows the distribution of lower quartile 25% and upper quartile 75%. RPKM: Fragment per kb pair region per million sequence reads. Whiskers present minimal to maximal value. Unpaired t-test was performed for statistical analysis. *p≤0.05. ( c ) Level of histone acetylation across SP1-motif in the genome at Trrap DEGs. Box plot shows the distribution of lower quartile 25% and upper quartile 75%. Whiskers present minimal to maximal value. ‘Up’ indicates the acetylated histone peak among genes that are upregulated after Trrap-deletion, ‘down’ indicates those which are downregulated. Unpaired t-test was performed for statistical analysis. *p≤0.05, **p≤0.01. ( d ) Genes overlapping in RNA-seq from cortices and striatum and AcH3 ChIP-seq in aNSCs. The log 2 of the fold changes in RNA-seq of cortices and AcH3 ChIP-seq of the 282 genes where decreased AcH3 was observed concomitant with changes in RNA levels. STMN3 and STMN4 are targets analyzed in this study and related to neuronal homeostasis. ( e ) Genes overlapping in RNA-seq from cortices and striatum and AcH4 ChIP-seq in aNSCs. Log 2 of the fold changes in RNA-seq of cortices and AcH4 ChIP-seq of the 136 genes where decreased AcH4 was observed concomitant with changes in RNA levels. STMN4 is a target analyzed in this study and related to neuronal homeostasis. ( f ) Quantitative PCR (q-PCR) analysis of Sp1 target genes in Trrap-FBΔ cortex and striatum. N = 4, the number of mice analyzed. Unpaired t-test was performed for statistical analysis. *p≤0.05; ***p≤0.001; n.s.: not significant. ( g ) Genomic view of the Stmn3 and Stmn4 gene showing the levels of histone H3 and H4 acetylation in control and mutant aNSCs from ChIP-seq data sets . The primers used for ChIP assays are located in the promoter proximity of respective genes. Lower panels depict the sequence of both ChIP probes, in which Sp1 consensus is shown.

Article Snippet: Recombinant DNA reagent , pN3-Sp1FL , Addgene , Cat#: 24543 , Sp1 overexpression reporter; H. sapiens.

Techniques: RNA Sequencing, Sequencing, ChIP-sequencing, Real-time Polymerase Chain Reaction, Control, Mutagenesis

Journal: eLife

Article Title: HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

doi: 10.7554/eLife.61531

Figure Lengend Snippet: ( a ) The experimental workflow of the Trrap deletion in aNSCs in culture by 4-OHT followed by experiments at indicated day post 4-OHT treatment (DPO). ( b ) Western blot analysis of the Trrap deletion in aNSC at 5DPO. GAPDH is a loading control. Quantification of the Trrap protein levels from three animals (N = 3) after normalization to GAPDH is shown on the left. Co.: control; aNSCΔ: Trrap-aNSCΔ. ( c ) The Venn diagram depicts the overlap between the differentially expressed genes (DEGs) measured by RNA-seq in the cortex, striatum, and aNSCs. The numbers refer to DEGs in the indicated data sets. ( d ) Log 2 of the fold changes of the 2120 common DEGs in Trrap-FBΔ cortex and Trrap-aNSCΔ aNSCs. ( e ) Transcription factor binding site (TFBS) enrichment analysis of the 1261 common DEGs in aNSCs, cortex, and striatum identified by RNA-seq. ( f ) A list of the Sp1 targets based on published ChIP-seq results was obtained from the Harmonizome database and compared with our RNA-seq results to determine the amount of Sp1 targets in each data set. ( g ) Top50 GO terms of the processes regulated by Sp1. Note that microtubule dynamics related processes are highlighted in red.

Article Snippet: Recombinant DNA reagent , pN3-Sp1FL , Addgene , Cat#: 24543 , Sp1 overexpression reporter; H. sapiens.

Techniques: Western Blot, Control, RNA Sequencing, Binding Assay, ChIP-sequencing

Journal: eLife

Article Title: HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

doi: 10.7554/eLife.61531

Figure Lengend Snippet: ( A ) Immunofluorescent images of primary neurons isolated from E16.5 forebrains at 6 days post co-transfection of siRNA (siScramble, siTrrap-4) with GFP, or with the GFP-STMN3, or with GFP-STMN4 expressing vector. ( B ) The neurite length after the Trrap knockdown and rescue by the STMN3 or STMN4 overexpression was analyzed at 6 days post co-transfection of the indicated siRNA with the GFP-, STMN3-, or STMN4-expressing vector. The neurite length is measured with NeuronJ (ImageJ plug-in). Only GFP-positive neurons (indicative of transfection) were analyzed. Each bar represents the data from four to six mouse embryos; the experiments were repeated more than three times. Unpaired t-test was performed for statistical analysis. **p≤0.01, ***p≤0.001, n.s., not significant. ( C ) The neurite branching after the Trrap knockdown and rescue by STMN3 or STMN4 overexpression was analyzed at 6 days post co-transfection of the indicated siRNA with the GFP, STMN3-, or STMN4-expressing vector. The neurite length is measured with NeuronJ (ImageJ software). Only GFP-positive neurons were scored and are shown. Each bar represents the data from four to six mouse embryos; the experiments were repeated more than three times. Unpaired t-test was performed for statistical analysis. *p≤0.05, **p≤0.01, ***p≤0.001, n.s., not significant. ( D ) Working model of Trrap-HAT-Sp1 in brain homeostasis and neurodegeneration. The Trrap deletion compromises HAT to acetylate histones resulting in insufficient binding of Sp1 and the subsequent downregulation of target genes involved in microtubule dynamics (STMNs). The dysregulation of STMNs provokes the axonal swelling, declines of neurite lengths and branching of postmitotic neurons, ultimately, leading to defective neuronal homeostasis and neurodegeneration.

Article Snippet: Recombinant DNA reagent , pN3-Sp1FL , Addgene , Cat#: 24543 , Sp1 overexpression reporter; H. sapiens.

Techniques: Isolation, Cotransfection, Expressing, Plasmid Preparation, Knockdown, Over Expression, Transfection, Software, Binding Assay

Journal: eLife

Article Title: HAT cofactor TRRAP modulates microtubule dynamics via SP1 signaling to prevent neurodegeneration

doi: 10.7554/eLife.61531

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pN3-Sp1FL , Addgene , Cat#: 24543 , Sp1 overexpression reporter; H. sapiens.

Techniques: Control, Plasmid Preparation, Transfection, Cell Culture, Isolation, Construct, Recombinant, Over Expression, Expressing, Activity Assay, Sequencing, Real-time Polymerase Chain Reaction, Luciferase, Purification, Sample Prep, Electron Microscopy, Protease Inhibitor, Magnetic Beads, SYBR Green Assay, Software, Staining, Sonication, Microscopy, Imaging

Journal: Science translational medicine

Article Title: Immune Checkpoint B7-H3 is a Therapeutic Vulnerability in Prostate Cancer Harboring PTEN and TP53 Deficiencies

doi: 10.1126/scitranslmed.adf6724

Figure Lengend Snippet: (A) Western blot analysis of B7-H3 in LNCaP cells (PTEN-null) with or without TP53 knockout. (B) Dual luciferase assay of vector (Vec) or pGL4 plasmid containing CD276 promoter (CD276-reporter) in LNCaP cells with or without TP53 knockout (n=3 per group). (C) Western blot analysis of B7-H3 in DU145 cells (TP53-deficient) with or without PTEN knockout. (D) Dual luciferase assay of vector or CD276-reporter in DU145 cells with or without PTEN knockout (n=4 per group). (E) Binding of Sp1 protein to the promoter region of the CD276 gene was determined using ChIP-qPCR (n=4 per group). Locations of primers in the CD276 gene promoter region are shown. TSS: transcription start site. (F,G) PTEN/p53-deficient LNCaP cells (PTEN-null; TP53 KO) were transfected with siRNA targeting SP1 or scrambled control, followed by dual luciferase assay of CD276-reporter (F) and qPCR of CD276 (G). Relative luciferase activities were normalized to vector control samples (n=4 per group). (H) Western blot analysis of B7-H3 and Sp1 in LNCaP cells treated with different doses of AKT inhibitor MK2206. (I) Western blot analysis of Sp1 and B7-H3 in LNCaP cells upon TP53 deletion. (J) Schematics of the mechanism by which PTEN-AKT and p53 pathways regulate B7-H3 expression. Data represent the mean ± SD. One-way ANOVA with Tukey’s post hoc tests (B, D, F, and G) and student’s t-tests (E) were performed using GraphPad Prism version 9.2.0.. ∗P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: Human SP1 overexpression plasmid was purchased from Genscript (OHu18205D).

Techniques: Western Blot, Knock-Out, Luciferase, Plasmid Preparation, Binding Assay, ChIP-qPCR, Transfection, Control, Expressing

Journal: Biochimie

Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

doi: 10.1016/j.biochi.2014.12.002

Figure Lengend Snippet: Analysis of the unique bands on SDS-PAGE gels by Q Exactive MS. a

Article Snippet: Transient transfection Sp1 overexpression plasmid was kindly provided by Dr. Gun-tram Suske at Philipps-University Marburg, Germany.

Techniques: Sequencing

Journal: Biochimie

Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

doi: 10.1016/j.biochi.2014.12.002

Figure Lengend Snippet: Analysis of the transcription factors that could bind to hnRNP K promoter. A. In silico analysis showed that at least two Sp1 binding sites in hnRNP K promoter. B. ChIP analysis verified the binding of Sp1 with hnRNP K promoter in the 293T cells. There were two controls, a non-DNA negative control and an input DNA sample. PCR was performed using primers recognizing the K1 and K2 sequences in the hnRNP K promoter and the amplified products were separated in agarose gel and visualized with EB. The sizes of the PCR products were 513 bp (K1) and 229 bp (K2). Similar results were observed in three independent experiments. C. EMSA confirmed the exact binding site of Sp1 in the 37 bp probe. N.E. denotes nuclear extract of 293T cells. Cold means cold competitor and mutant means mutant competitor. D. Promoter luciferase reporter analysis of the sequences with mutant Sp1 binding sites in 293T and HK2 cells. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis. (a) P < 0.05 versus pGL3-basic; (b) P < 0.05 versus pGL3-K1–1.

Article Snippet: Transient transfection Sp1 overexpression plasmid was kindly provided by Dr. Gun-tram Suske at Philipps-University Marburg, Germany.

Techniques: In Silico, Binding Assay, Negative Control, Amplification, Agarose Gel Electrophoresis, Mutagenesis, Luciferase

Journal: Biochimie

Article Title: Transcriptional regulation of heterogeneous nuclear ribonucleoprotein K gene expression

doi: 10.1016/j.biochi.2014.12.002

Figure Lengend Snippet: Effect of Sp1 overexpression on hnRNP K and AGT expression in HK2 cells. A. The promoter activity of hnRNP K was significantly enhanced by Sp1. B. Overexpression of Sp1 increased the protein level of hnRNP K. C. Overexpression of Sp1 significantly reduced the mRNA level of AGT. Values represent the mean ± SD of three independent experiments. T-test was used for statistical analysis.*P < 0.05; **P < 0.01.

Article Snippet: Transient transfection Sp1 overexpression plasmid was kindly provided by Dr. Gun-tram Suske at Philipps-University Marburg, Germany.

Techniques: Over Expression, Expressing, Activity Assay