Journal: Diabetes

Article Title: Angiotensin II–Induced Ferroptosis in Epithelial Cells Contributes to Kidney Injury via SP1-DPEP1–Mediated SLC3A2 Degradation

doi: 10.2337/db25-0619

Figure Lengend Snippet: AngII-induced renal TECs ferroptosis via SP1-DPEP1–mediated SLC3A2 degradation. In renal TECs, AngII activates the transcription factor SP1, promoting the transcription and translation of the DPEP1 gene. The upregulated DPEP1 facilitates the ubiquitination and degradation of SLC3A2, thereby inhibiting intracellular GSH synthesis. The depletion of GSH, combined with increased iron content and enhanced lipid peroxidation, leads to ferroptosis in renal TECs.

Article Snippet: After SDS-PAGE and transfer, membranes were blocked and incubated with primary antibodies: SP1 (21962-1-AP; Proteintech), DPEP1 (12222-1-AP; Proteintech), ACSL4 (T510198; Abmart), ferritin heavy chain (FTH) (TD6278; Abmart), GPX4 ( T56959 ; Abmart), SLC3A2 (PTM-5485; PTM Bio and A24735; ABclonal), and β-actin (66009-1-lg; Proteintech).

Techniques: Ubiquitin Proteomics

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: Repeated NTG administration induces migraine-like behaviors and elevates SP1, TET1, and KIF1A expression in the SP5C. ( A ) Experimental timelines for NTG-induced migraine modeling, SP1 inhibition (MTM), and Kif1a manipulation (Ad- Kif1a or Ad-mCherry). Behavioral testing was performed as indicated, followed by tissue collection on day 10. ( B – D ) Repeated NTG administration induced sustained nociceptive hypersensitivity, reflected by reduced mechanical allodynia (HML and HMT) and thermal hyperalgesia (PMT) ( n = 8 mice per group). ( E ) Representative WB images showing increased protein levels of TET1, KIF1A, SP1, c-Fos, and CGRP in the SP5C after NTG treatment. ( F ) Quantification of TET1, KIF1A, SP1, c-Fos, and CGRP protein levels normalized to β-actin ( n = 4 mice per group). ( G ) RT-qPCR analysis of Kif1a mRNA expression after NTG treatment the SP5C. ( n = 4mice per group). ( H ) Representative images of IF staining images showing CGRP expression in the SP5C. Nuclei were counterstained with DAPI (blue). Scale bars: 200 μm. ( I ) Quantification of the fluorescence intensity showing the significant upregulation of CGRP in NTG-treated mice ( n = 4 mice per group). Statistical analysis: Two-way ANOVA for ( B - D ); unpaired two-tailed t test for ( F , G , I ). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the VEH group. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Staining, Fluorescence, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: Pharmacological inhibition of SP1 suppresses KIF1A signaling and CGRP expression. ( A ) Representative WB images showing time-dependent suppression of SP1 protein expression following MTM treatment (2, 6, 12, and 24 h) compared with control cells. ( B ) Quantification of SP1 protein levels normalized to β-actin ( n = 4). ( C ) Representative WB images showing reduced protein expression of KIF1A, SP1, c-Fos, and CGRP after MTM treatment compared to DMSO controls. ( D ) Quantitative analysis of CGRP, c-Fos, SP1, and KIF1A protein levels following MTM treatment ( n = 4). ( E ) RT-qPCR analysis showing decreased Kif1a mRNA expression in the MTM-treated group relative to controls ( n = 4). ( F ) Representative IF images showing reduced CGRP expression (green) after MTM treatment. Nuclei were counterstained with DAPI (blue). Scale bar = 100 μm. Statistical significance: One-way ANOVA for ( B ); unpaired two-tailed t-test for ( D , E ). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DMSO group. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Inhibition, Expressing, Control, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: SP1 regulates KIF1A expression through TET1-dependent epigenetic mechanisms. ( A ) Representative WB showing reduced TET1 protein expression following MTM treatment compared to DMSO. ( B ) Quantification of TET1 protein levels normalized to β-actin ( n = 4). ( C ) Screening of Tet1 -targeting sequences in N2a cells. ( D ) Quantification of TET1 protein levels after Tet1 siRNA transfection ( n = 4) showed that sequence 2 produced the strongest knockdown. ( E ) RT-qPCR analysis showing sequence 2 most effectively decreased Tet1 mRNA ( n = 4). (F ) Representative WB showing reduced KIF1A, SP1, c-Fos, and CGRP after Tet1 siRNA transfection. ( G ) Quantification of CGRP, c-Fos, SP1, and KIF1A protein levels following Tet1 siRNA transfection ( n = 4). ( H ) RT-qPCR showing decreased Kif1a mRNA expression in Tet1 siRNA–treated cells ( n = 4). ( I ) IF images showing reduced CGRP expression (green) after Tet1 siRNA transfection. Scale bar = 100 μm. ( J ) Co-IP showing SP1 and TET1 interaction after immunoprecipitation with anti-SP1 antibody. ( K ) Co-IP showing SP1 and TET1 interaction after immunoprecipitation with anti-TET1 antibody. ( L ) Dual-luciferase reporter assay showing increased Kif1a promoter–driven luciferase activity after SP1 overexpression ( n = 3). ( M ) ChIP-qPCR showing SP1 and TET1 enrichment at the Kif1a promoter, reduced after sequential Re-ChIP with TET1 antibodies ( n = 3). Statistical significance: Unpaired two-tailed t-test for ( A , G , H ); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO or MS siRNA. One-way ANOVA for ( D , E ); Two-way ANOVA for ( L ); * p < 0.05, ** p < 0.01, ** p < 0.001 compared the corresponding control groups, as indicated. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, Transfection, Sequencing, Produced, Knockdown, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Over Expression, ChIP-qPCR, Two Tailed Test, Control

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: Effects of MTM treatment on TET1, KIF1A, SP1, and CGRP expression in the NTG-induced migraine model. ( A – C ) NTG induced mechanical allodynia (decreased HML and HMT) and thermal hyperalgesia (decreased PMT). MTM treatment significantly attenuated NTG-induced nociception compared with NTG + DMSO ( n = 8 mice per group). ( D ) Representative WB images showing protein expression levels of TET1, KIF1A, SP1, c-Fos, and CGRP in the VEH and NTG groups treated with DMSO or MTM. ( E ) Quantification of TET1, KIF1A, SP1, c-Fos, and CGRP protein levels normalized to β-actin ( n = 4 mice per group). ( F ) Quantification of Kif1a mRNA expression in the VEH and NTG groups treated with DMSO or MTM ( n = 4 mice per group). ( G ) Representative IF images showing CGRP expression (green) in the VEH and NTG groups treated with DMSO or MTM. Nuclei were counterstained with DAPI (blue). Scale bar = 200 μm. ( H ) Quantification of CGRP fluorescence intensity across groups ( n = 4 mice per group). ( I ) Methylation analysis of the Kif1a promoter in the VEH and NTG groups treated with DMSO or MTM, showing alterations in DNA methylation patterns. (L) Quantitative analysis of Kif1a promoter DNA methylation ( n = 3 mice per group). Statistical significance: Two-way ANOVA for ( A – C ); One-way ANOVA for ( E , F , H , J ); * p < 0.05, ** p < 0.01, *** p < 0.001 compared the VEH+DMSO groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the NTG+DMSO groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, Fluorescence, Methylation, DNA Methylation Assay

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: SP1 inhibition suppresses synaptic protein expression in vitro and in vivo. ( A ) Representative WB images showing reduced expression of the synaptic proteins PSD-95, Syt1, and SYP following MTM treatment compared with DMSO in N2a cells. ( B ) Quantification of PSD-95, Syt1, and SYP protein levels normalized to β-actin ( n = 4). ( C – E ) Representative IF images showing decreased expression of SYP ( C ), Syt1 ( D ), and PSD-95 ( E ) (green) after MTM treatment. Nuclei were counterstained with DAPI (blue). ( F ) Representative WB images showing PSD-95, Syt1, and SYP protein expression in the SP5C of VEH and NTG mice treated with DMSO or MTM. ( G ) Quantification of PSD-95, Syt1, and SYP protein levels normalized to β-actin ( n = 4 mice per group). ( H ) Representative IF images showing Syt1 expression (green) in the SP5C of VEH and NTG mice treated with DMSO or MTM. Scale bar = 100 μm. ( I ) Quantification of Syt1 fluorescence intensity across groups ( n = 4 mice per group). ( J ) Representative IF images showing PSD-95 expression (green) in the SP5C of VEH and NTG mice treated with DMSO or MTM. Scale bar = 100 μm. ( K ) Quantification of PSD-95 fluorescence intensity across groups ( n = 4 mice per group). Statistical significance: Unpaired two-tailed t-test for (B); * p < 0.05, ** p < 0.01, ** p < 0.001 vs. DMSO; One-way ANOVA for ( G , I , K ), * p < 0.05, ** p < 0.01, ** p < 0.001 compared the VEH+DMSO groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the NTG+DMSO groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Inhibition, Expressing, In Vitro, In Vivo, Fluorescence, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: TET1 mediates SP1-dependent regulation of synaptic protein expression and CGRP signaling in vitro. ( A ) Representative WB images showing reduced expression of the synaptic proteins PSD-95, Syt1, and SYP following Tet1 siRNA transfection compared with MS siRNA controls. ( B ) Quantification of PSD-95, Syt1, and SYP protein levels normalized to β-actin ( n = 4). ( C – E ) Representative IF images showing decreased expression of SYP ( C ), Syt1 ( D ), and PSD-95 ( E ) (green) after Tet1 siRNA transfection. ( F ) Representative WB images showing expression of KIF1A, PSD-95, Syt1, SYP, and CGRP in cells transfected with pcDNA3.1- Tet1 or control plasmid, with or without MTM treatment. ( G ) Quantification of CGRP, SYP, Syt1, PSD-95, and KIF1A protein levels normalized to β-actin ( n = 4). ( H ) RT-qPCR analysis showing Kif1a mRNA expression following pcDNA3.1- Tet1 transfection in the presence or absence of MTM ( n = 4). ( I ) Representative IF images showing CGRP expression (green) under the indicated conditions. Scale bar = 100 μm. Statistical significance: Unpaired two-tailed t-test for ( B ); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO; One-way ANOVA for (G, H), * p < 0.05, ** p < 0.01, ** p < 0.001 compared the control groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the MTM groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Expressing, In Vitro, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Headache and Pain

Article Title: SP1 recruits TET1 to mediate Kif1a demethylation and synaptic remodeling in a mouse model of chronic migraine

doi: 10.1186/s10194-026-02296-0

Figure Lengend Snippet: KIF1A mediates SP1 inhibition–induced synaptic ultrastructural and dendritic spine remodeling in the SP5C. ( A ) Representative TEM images of synaptic ultrastructure in the SP5C from the VEH, NTG, NTG + MTM, NTG + MTM + Ad- Kif1a , and NTG + MTM + Ad-mCherry groups. Dashed boxes indicate synaptic regions shown at higher magnification (Zoom). Scale bars = 500 nm. ( B ) Quantification of synaptic cleft width. NTG reduced synaptic cleft width, which was reversed by MTM and restored by Ad- Kif1a . ( C ) Quantification of synaptic interface curvature. NTG increased synaptic interface curvature; this increase was attenuated by MTM and restored by Ad- Kif1a . ( D ) Quantification of PSD thickness. NTG increased PSD thickness, which was reduced by MTM and re-elevated by Ad- Kif1a but not by Ad-mCherry. ( E ) Representative Golgi-Cox–stained images of dendritic morphology and spines in SP5C neurons. Enlarged images (Zoom) show dendritic segments used for spine analysis. Scale bars = 50 μm. ( F ) Quantification of dendritic spine density, expressed as the number of spines per 20 μm of dendritic length. NTG increased spine density, which was partially reversed by MTM and restored by Ad- Kif1a . Statistical significance: One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 compared the VEH groups, # p < 0.05, ## p < 0.01, ### p < 0.001 compared the NTG groups; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 compared the NTG+MTM groups. All the data are presented as the means ± SEMs

Article Snippet: For the first immunoprecipitation, chromatin was incubated overnight at 4 °C with a rabbit anti-SP1 antibody (Proteintech, China), with normal rabbit IgG serving as a negative control.

Techniques: Inhibition, Staining