Journal: Materials

Article Title: Chlorine Reduction Kinetics and its Mass Balance in Copper Premise Plumbing Systems During Corrosion Events

doi: 10.3390/ma12223676

Figure Lengend Snippet: T-XRF measurements of scales removed from the tested copper pipes. Scales removal was done though simple coupon sonication and sonication-scraping ( a ) Chlorine, ( b ) Copper, ( c ) EDS analysis of the corrosion by-products extracted from the inner-surface of a copper stagnated for 8 hours with synthetic water (DIC = 80 mg CaCO 3 /L; OCl - = 8.0 mg Cl 2 /L).

Article Snippet: A T-XRF (S2 PICOFOX, Bruker, Karlsruhe, Germany) was used to determine the presence of Cu-Cl scales on deeper layers of the film formed on the inner surface of the pipe.

Techniques: Sonication

Journal: Translational Oncology

Article Title: AGEs-RAGE manipulates tumor intrinsic pERK/Sp1/IL6 pathway and reprograms macrophage to promote intrahepatic cholangiocarcinoma progression

doi: 10.1016/j.tranon.2025.102446

Figure Lengend Snippet: AGEs-RAGE axis activates ERK phosphorylation in ICC cells . (A) Stable RAGE knockdown in ICC cells. (B) Western blot analysis of RAGE, p-ERK, E-cadherin, N-cadherin, Vimentin, Sp1 and MMP2 expressions in RBE cells treated with BSA (200 µg/ml) or glucose-AGEs (50,100,200µg/ml) for 24 h. (C) Western blot analysis of p-ERK, E-cadherin, N-cadherin, Vimentin, Sp1 in RBE cells were treated with: BSA (200 µg/ml); glucose-AGEs (200,400µg/ml);or glucose-AGEs(200µg/ml) +U0126(10 µM) for 24 h. (D) RNA-seq analysis of MAPK pathway mRNA expression in shRAGE vs. control cells. ** P < 0.01 compared with control. (E) Western blot analysis of p-ERK, E-cadherin, N-cadherin, Vimentin, Sp1 expressions in shRAGE and control cells. ** P < 0.01 compared with control. (F) Enrichment analysis of downregulated differentially expressed genes (DEGs) in shRAGE vs. control cells.

Article Snippet: Rabbit anti-human RAGE (Abcam, USA) and rabbit anti-human Sp1 (CST, USA) were used as primary antibodies.

Techniques: Phospho-proteomics, Knockdown, Western Blot, RNA Sequencing, Expressing, Control

Journal: Translational Oncology

Article Title: AGEs-RAGE manipulates tumor intrinsic pERK/Sp1/IL6 pathway and reprograms macrophage to promote intrahepatic cholangiocarcinoma progression

doi: 10.1016/j.tranon.2025.102446

Figure Lengend Snippet: S p 1 is a key transcription factor downstream of the AGEs-RAGE axis . (A) Correlation between RAGE and Sp1 gene expression in TCGA data. (B) Representative Sp1 immunohistochemistry in ICC and adjacent liver tissues. (Scale bar, 50µ m). (C) Sp1 mRNA expression (RNA-seq) in shRAGE vs. control cells. ** P < 0.01 compared with control. (D) Western blot analysis of RAGE and Sp1 in RBE cells treated with BSA (200 µg/ml), glucose-AGEs (200µg/ml), both glucose-AGEs(200µg/ml) + RAGE-Ab (5µg/ml) for 24 h. (E) Luciferase activity of IL-6 promoter reporter in RBE cells co-transfected with Sp1 plasmid vs. empty vector for 48 h; * P < 0.05, ** P < 0.01,*** P < 0.001compared with control. (F) IL-6 protein expression (Western blot). RBE cells were transfected with Sp1 gene plasmid or Sp1 siRNA and for 48 h, then treated with: BSA (200µg/ml); glucose-AGEs (200µg/ml); glucose-AGEs (200µg/ml) +RAGE-Ab (5µg/ml) for 24 h.

Article Snippet: Rabbit anti-human RAGE (Abcam, USA) and rabbit anti-human Sp1 (CST, USA) were used as primary antibodies.

Techniques: Gene Expression, Immunohistochemistry, Expressing, RNA Sequencing, Control, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Translational Oncology

Article Title: AGEs-RAGE manipulates tumor intrinsic pERK/Sp1/IL6 pathway and reprograms macrophage to promote intrahepatic cholangiocarcinoma progression

doi: 10.1016/j.tranon.2025.102446

Figure Lengend Snippet: Glucose-AGEs axis promotes migration and invasion in ICC cells . (A-B) Wound healing assay in RBE cells treated with: BSA (200 µg/mL); glucose-AGEs (200 µg/mL); or glucose-AGEs (200 µg/mL) + RAGE-Ab (5 µg/mL); The wound space was photographed at 0 and 48 h. The wound healing was measured with the following formula: 48-h migration % =(0-h width–48-h width of wound)/(0-h width of wound), *** P < 0.001. (C-D) Transwell migration/invasion assays (RBE cells). Treatments identical to (A-B); All experiments were done in triplicate, and the results are presented as the mean±SD,* P < 0.05, *** P < 0.001. (E) Western blot of RAGE, E-cadherin, N-cadherin, Vimentin, MMP2, and Sp1 in RBE cells. (F-I) RBE cells were subjected to scratch wound-healing assay and transwell assay. RBE cells were transfected with Sp1 siRNA and for 48 h.Then RBE cell were treated with BSA (200µg/ml); glucose-AGEs (200µg/ml), * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Rabbit anti-human RAGE (Abcam, USA) and rabbit anti-human Sp1 (CST, USA) were used as primary antibodies.

Techniques: Migration, Wound Healing Assay, Western Blot, Transwell Assay, Transfection

Journal: Translational Oncology

Article Title: AGEs-RAGE manipulates tumor intrinsic pERK/Sp1/IL6 pathway and reprograms macrophage to promote intrahepatic cholangiocarcinoma progression

doi: 10.1016/j.tranon.2025.102446

Figure Lengend Snippet: RAGE overexpression correlates with poor ICC prognosis . (A) RAGE mRNA expression in ICC (TCGA). (B) Kaplan-Meier survival analysis by RAGE expression (high vs. low). (C) Representative RAGE IHC in ICC vs. adjacent liver (Scale bar: 50 µm). (D) RAGE staining intensity distribution in 153 ICC patients. (E) Confocal imaging showing RAGE expression in normal bile duct vs. ICC tissues (Scale bar: 50 µm). Concurrent upregulation of RAGE and Sp1 in ICC tissues (Western blot).

Article Snippet: Rabbit anti-human RAGE (Abcam, USA) and rabbit anti-human Sp1 (CST, USA) were used as primary antibodies.

Techniques: Over Expression, Expressing, Staining, Imaging, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-17 activates the canonical NF-kappaB signaling pathway in autoimmune B cells of BXD2 mice to upregulate the expression of regulators of G-protein signaling 16.

doi: 10.4049/jimmunol.0903133

Figure Lengend Snippet: FIGURE 3. IL-17 rapidly activates the canonical NF-kB signaling pathway in BXD2 B cells. Purified splenic B cells from BXD2 mice were treated with IL-17 (30 ng/ml). Cells were harvested at the indicated time points. A–F, The levels of the indicated protein in total cell lysates (A), cytoplasmic extract (C), and nuclear extract (E) were analyzed by Western blot analysis with the respective Abs. Quantitation of the indicated protein after normalization to the levels of GAPDH or SP1 transcription factor is graphed as the mean 6 SEM for total cell lysates (B), cytoplasmic extract (D), and nuclear extract (F). The results are representative of three independent experiments. pp , 0.05; ppp , 0.01 compared with unstimulated control.

Article Snippet: Abs against p65 (C22B4), P-p65 (93H1), transcription factor RelB (C1E4), IkBa (44D4), phospho-IkBa (14D4), phospho-p38 MAPK (28B10), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (197G2) rabbit mAb, phospho-C/EBPb (Thr235) (Cell Signaling Technology, Beverly, MA), p50 (H119), p52 (K27), SP1 transcription factor (1C6), C/EBPb (C-19), C/ EBPd (C-22) (Santa Cruz Biotechnology, Santa Cruz, CA), RGS16 (ProSci, Poway, CA), and GAPDH (R&D Systems, Minneapolis, MN) were used to detect their respective Ags.

Techniques: Western Blot, Quantitation Assay, Control

Journal: The Journal of biological chemistry

Article Title: Activation of Nur77 by selected 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes induces apoptosis through nuclear pathways.

doi: 10.1074/jbc.M500107200

Figure Lengend Snippet: FIG. 4. Nuclear localization of Nur77. A, immunostaining. Panc-28 cells were treated with Me2SO or 10 M Nur77 agonists for 6 h, and cells were immuno- stained for Nur77 as described under “Materials and Methods.” Nur77 staining was not observed in cells treated with nonspecific IgG. B, nuclear localization in subcellular fractions. Panc-28 cells were treated with the various compounds for 12 h and Nur77 protein expression in cy- tosolic, and nuclear extracts were deter- mined by Western blot analysis. Sp1 pro- tein and a nonspecific (NS) band serve as loading controls, and Sp1, a nuclear pro- tein, also serves as a control for separa- tion of nuclear and cytosolic extracts.

Article Snippet: Antibodies for PARP (sc8007), Sp1 (sc-59), and TRAIL (sc7877) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Nur77 (IMG-528) from Imgenex (San Diego, CA).

Techniques: Immunostaining, Staining, Expressing, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

doi: 10.1038/s41419-025-08143-5

Figure Lengend Snippet: MDA-MB-231 cells were transfected with oligonucleotides targeting NR4A1 ( A ), NR4A2 ( B ), Sp1 ( C ), Sp4 ( D ) and Sp3 ( E ), and after 72 h, whole cell lysates were analyzed by Western blots and quantified as outlined in the “Methods”. F Effects of Sp1 overexpression on CD71 were obtained by transfection of the p CVM-Sp1 expression plasmid and subsequent western blot analysis of whole cell lysates and quantified as outlined in the “Methods”. Cells were treated with 10 µM DIM-3,5-CI 2 ( G ) and DIM-3-CI-5-CF 3 ( H ) for up to 24 h, and effects on CD71, Sp1 and Sp4 (separate blot) were determined by Western blots of whole cell lysates and quantified as outlined in the “Methods”.

Article Snippet: The plasmid containing Sp1 coding sequence (Addgene plasmid # 12097) and empty vector as negative control (Addgene plasmid # 20783) using LipofectamineTM 3000 reagent (Invitrogen, # L3000008) according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Over Expression, Expressing, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Orphan nuclear receptor 4A1 (NR4A1) and NR4A2 are endogenous regulators of CD71 and their ligands induce ferroptosis in breast cancer

doi: 10.1038/s41419-025-08143-5

Figure Lengend Snippet: Interactions of NR4A1 ( A ), NR4A2 ( B ), Sp1 ( C ) and Sp4 ( D ) with the CD71 gene promoter containing the Sp binding region was determined after treatment with DIM-3,5 compounds in a ChIP assay (in triplicate) as outlined in the “Methods section”. E Summary of the CD71 gene promoter, Sp binding sites (−1576 to −1566) and the primers used for detecting protein interactions in this region of the promoter, along with the overall mechanism of ferroptosis induction. Results are expressed as means ± SD for replicate (3) determinations for each treatment group ( A – D ) and significant ( p < 0.05) induction is indicated (*).

Article Snippet: The plasmid containing Sp1 coding sequence (Addgene plasmid # 12097) and empty vector as negative control (Addgene plasmid # 20783) using LipofectamineTM 3000 reagent (Invitrogen, # L3000008) according to the manufacturer’s instructions.

Techniques: Binding Assay

Journal: The FASEB Journal

Article Title: SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells

doi: 10.1096/fj.202201274rr

Figure Lengend Snippet: FIGURE 1 The fertility was prolonged in GC-Sp1−/− mice. (A) Localization and expression of SP1 protein (green) in the primordial follicle, primary follicle, secondary follicle, and antral follicle. SP1 protein (green) is localized in the nuclei of both GCs and oocytes. Hoe (blue): Hoechst 33342. Scale bar: 80 μm. (B) The expression pattern of SP1 protein in ovaries of mice at 1 dpp, 3 dpp, 5 dpp, 7 dpp, 14 dpp, and 21 dpp. (C) Quantification of ratio of SP1 to β-actin in (B). n = 3 biologically independent repeats. (D) A schematic strategy in producing conditional knockout of Sp1 in GCs. (E) Western blotting analysis of the expression levels of SP1 protein in granulosa cells in PD56 Sp1fl/fl and GC-Sp1−/− mice after PMSG priming. (F) Quantification of ratio of SP1 to β-actin in (E). n = 3 biologically independent repeats. (G) Fertility check showed a better fertility phenotype in the GC-Sp1−/− females with significantly increased litter size and number (n = 23) compared to that in Sp1fl/fl mice (n = 30) during 60 weeks of mating. (H and I) The levels of SP1 in the ovaries of PD14 and PD56 Sp1fl/fl and GC-Sp1−/− mice. The proteins were co-stained with FOXL2 and SP1 antibodies. White arrows in 1H indicate the primordial follicle. Scale bar: 100 μm. Results were presented as mean ± SEM and analyzed by a Student's t-test, two unpaired. **p < .01.

Article Snippet: The antibodies included SP1 antibody (Novus Biologicals, NBP2- 20460) (1:100), FDX1 antibody (Proteintech, 12592- 1- AP) (1:100), CYP11A1 antibody (Cell Signaling Technology, 14217S) (1:200), FOXO3A antibody (Cell Signaling Technology, 12829S) (1:200), and CleavedCapased3 antibody (Abcam, ab2302) (1:200).

Techniques: Expressing, Knock-Out, Western Blot, Staining

Journal: The FASEB Journal

Article Title: SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells

doi: 10.1096/fj.202201274rr

Figure Lengend Snippet: FIGURE 2 Primordial follicle depletion slowed down in GC-Sp1−/− mice. (A) The structures of mouse ovaries recovered from PD7, PD14, and PD21, 3 months, 8 months, and 12 months of the Sp1fl/fl and GC-Sp1−/− mice. Scale bar: 200 μm. (B) Analysis of the available numbers of primordial follicles (PmF) with one layer of flat GCs, primary follicles (PF) with one layer of cuboidal GCs, secondary follicles (SF) with two or more layers of GCs), and antral follicles (AF) in ovaries of mice. n = 3 biologically independent for GC-Sp1−/− and Sp1fl/fl mice. Results were presented as mean ± SEM and analyzed by a Student's t-test, two unpaired. *p < .05; **p < .01; ns ≥ 0.05, no difference.

Article Snippet: The antibodies included SP1 antibody (Novus Biologicals, NBP2- 20460) (1:100), FDX1 antibody (Proteintech, 12592- 1- AP) (1:100), CYP11A1 antibody (Cell Signaling Technology, 14217S) (1:200), FOXO3A antibody (Cell Signaling Technology, 12829S) (1:200), and CleavedCapased3 antibody (Abcam, ab2302) (1:200).

Techniques:

Journal: The FASEB Journal

Article Title: SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells

doi: 10.1096/fj.202201274rr

Figure Lengend Snippet: FIGURE 5 SP1 directly binds to the Fdx1 promoter. (A) A schematic representation of the SP1 binding site on the Fdx1 promoter predicted by JASPAR, in which the region of −1931/−1922 is the potential binding site of SP1. (B) ChIP-qPCR analysis of SP1 binding to the Fdx1 promoter in GCs of PD7 mice ovary. n = 3 biologically independent repeats. (C) EMSA analysis of the combined capability between nucleoprotein (SP1) and Fdx1 promoter. n = 3 biologically independent repeats. (D) Hematoxylin staining of Control, Sp1-KD, Fdx1-OE, and Sp1-KD; Fdx1-OE group. Black arrows indicate the primordial follicles, red arrows indicate the primordial to primary follicles transition, yellow arrows indicate primary follicles, and green arrows indicate secondary follicles. Scale bar: 80 μm. (E) Analysis of the numbers of PmF, PPT, and PF. n = 5 biologically independent. (F) Western blotting analysis of the expression of SP1, CYP11A1, PCNA, and FDX1. (G) Quantification of ratio of SP1, CYP11A1, PCNA, and FDX1 to β-Actin in (F). n = 3 biologically independent repeats. Results are expressed as mean ± SD, and the student's t test (two unpaired) was used to analyze the significance of the data. *p < .05; **p < .01.

Article Snippet: The antibodies included SP1 antibody (Novus Biologicals, NBP2- 20460) (1:100), FDX1 antibody (Proteintech, 12592- 1- AP) (1:100), CYP11A1 antibody (Cell Signaling Technology, 14217S) (1:200), FOXO3A antibody (Cell Signaling Technology, 12829S) (1:200), and CleavedCapased3 antibody (Abcam, ab2302) (1:200).

Techniques: Binding Assay, ChIP-qPCR, Staining, Control, Western Blot, Expressing

Journal: The FASEB Journal

Article Title: SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells

doi: 10.1096/fj.202201274rr

Figure Lengend Snippet: FIGURE 6 The PPT was accelerated in mice ovary by supplemented cholesterol. (A) 10 μM cholesterol cultured newborn ovaries for 4 days. Hematoxylin staining of the control and the cholesterol group. Black arrows indicate the primordial follicles, red arrows indicate the primordial to primary follicles transition, and yellow arrows indicate primary follicles. Scale bar: 80 μm. (B) Images of the control and the cholesterol group were co-stained with Foxo3a antibody (red) and BrdU (green), Hoechst33342 (blue). Yellow arrows indicate the dormant primordial follicle, white arrows indicate the activated primordial follicle. Scale bar: 80 μm. (C) Analysis of the numbers of PmF, PPT, and PF. n = 6 biologically independent repeats. (D) The proportion of oocytes with CL-FOXO3a (%): Oocytes with cytoplasmic localization of FOXO3a/oocytes with nuclear and cytoplasmic localization of FOXO3a. n = 5 biologically independent repeats. (E) Analysis of average fluorescence intensity of EdU per section by ImageJ. (F) Western blotting analysis of the expression of SP1, CYP11A1, PCNA, FDX1, p-mTOR, mTOR, p-RPS6, and RPS6. (G) Quantification of ratio of SP1, CYP11A1, PCNA, FDX1, p-mTOR, mTOR, p-RPS6, and RPS6 to β- Actin in (F). n = 3 biologically independent repeats. (H) The ovarian oxygen consumption rate (OCR) in the ovary of the control (n = 5) and the cholesterol group (n = 5). (I) AUC (area under the curve) of (H) Data were presented as mean ± SEM and analyzed by a Student's t test, two unpaired. *p < .05; **p < .01; ns ≥ 0.05, no difference.

Article Snippet: The antibodies included SP1 antibody (Novus Biologicals, NBP2- 20460) (1:100), FDX1 antibody (Proteintech, 12592- 1- AP) (1:100), CYP11A1 antibody (Cell Signaling Technology, 14217S) (1:200), FOXO3A antibody (Cell Signaling Technology, 12829S) (1:200), and CleavedCapased3 antibody (Abcam, ab2302) (1:200).

Techniques: Cell Culture, Staining, Control, Fluorescence, Western Blot, Expressing

Journal: The FASEB Journal

Article Title: SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells

doi: 10.1096/fj.202201274rr

Figure Lengend Snippet: FIGURE 7 The proposed molecular mechanisms of SP1-mediated PPT. (A) The process of the primordial to primary follicle transition. (B) Molecular mechanism of SP1-mediated PPT: under physiological conditions, SP1 in GCs participates in maintaining the activity of CYP11A1 and promoting cholesterol metabolism by regulating the transcription of Fdx1. And the expression of SP1 is up-regulated with PPT. Meanwhile, SP1 regulates the mTOR signal pathways, which attribute to improving the process of PPT.

Article Snippet: The antibodies included SP1 antibody (Novus Biologicals, NBP2- 20460) (1:100), FDX1 antibody (Proteintech, 12592- 1- AP) (1:100), CYP11A1 antibody (Cell Signaling Technology, 14217S) (1:200), FOXO3A antibody (Cell Signaling Technology, 12829S) (1:200), and CleavedCapased3 antibody (Abcam, ab2302) (1:200).

Techniques: Activity Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Regulation of the Human MAT2A Gene Encoding the Catalytic α2 Subunit of Methionine Adenosyltransferase, MAT II

doi: 10.1074/jbc.m002347200

Figure Lengend Snippet: FIG. 6. Competitive inhibition of complexes with various oli- gonucleotides and probes. Probe D was incubated with nuclear extracts from Jurkat T cells in the absence or presence of various competitors. Competition was conducted as detailed under “Materials and Methods.” The competition pattern indicates that the major com- plexes are formed on the region between 276 and 254, which contains the Sp1-3 site (a and b). However, an Sp1 competitor reduced but did not abolish complex formation (c).

Article Snippet: An Sp1-specific competitor was purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA (catalogue no. SC2502).

Techniques: Inhibition, Incubation