Journal: Mechanisms of development

Article Title: Developmental cooperation of leukemia inhibitory factor and insulin-like growth factor I in mice is tissue-specific and essential for lung maturation involving the transcription factors Sp3 and TTF-1.

doi: 10.1016/s0925-4773(02)00449-5

Figure Lengend Snippet: Fig. 4. Sp3 expression in neonatal bone. Representative H&E stained cross- sections at a proximal level of the humerus of wild type (A) and double null (B) neonates. Both images were taken with the same objective (20 £ ) but the double null bone is smaller and the bone area fits completely in the field shown. Sections at similar levels were immunostained for Sp3 and equivalent areas (* in A) are shown at a higher magnification (C–F). Note the lower number of Sp3 expressing cells in the double null mice humerus (F) compared to the rest of the genotypes. Scale bar, 120 mm in (A,B) and 60 mm in (C–F).

Article Snippet: For immunocytochemistry, paraffin sections were dewaxed, rehydrated, washed with PBS, incubated with 7.4% H2O2 for 10 min and exposed to a solution of 0.1% Triton X-100 and 10% goat serum in PBS for 1 h. They were incubated overnight at 4 8C with the polyclonal anti Sp3 antibody (D-20, does not recognize Sp1, 2 and 4, from Santa Cruz Biotechnology, Santa Cruz, CA), at 1:200.

Techniques: Expressing, Staining

Journal: Mechanisms of development

Article Title: Developmental cooperation of leukemia inhibitory factor and insulin-like growth factor I in mice is tissue-specific and essential for lung maturation involving the transcription factors Sp3 and TTF-1.

doi: 10.1016/s0925-4773(02)00449-5

Figure Lengend Snippet: Fig. 8. Sp3 protein and mRNA expression in neonatal lung. Representative sections at similar levels were immunostained for Sp3 (A–D) or underwent in situ hybridization (E–H). Note the widespread protein expression in control (A) and LIF-null (B) lungs, a moderate decrease in the IGF-I null (C), and a very marked decrease in Sp3 staining in the non-aerated double null embryo lung (D); staining, probably non-specific, remains in bronchial structures (arrow). Hybridization with radiolabeled oligodeoxynucleotides in comparable trunk sections of mice from a different litter (E–H). The autoradiographic signal of the film exposure was converted to a pseudocolor scale to show expression levels from blue (lowest) to red (highest). Note the lower Sp3 mRNA expression in the double null mice lung (asterisk), as well as in ribs (arrows) compared to the rest of the genotypes. Scale bar, 60 mm in (A–D) and 500 mm in (E–H).

Article Snippet: For immunocytochemistry, paraffin sections were dewaxed, rehydrated, washed with PBS, incubated with 7.4% H2O2 for 10 min and exposed to a solution of 0.1% Triton X-100 and 10% goat serum in PBS for 1 h. They were incubated overnight at 4 8C with the polyclonal anti Sp3 antibody (D-20, does not recognize Sp1, 2 and 4, from Santa Cruz Biotechnology, Santa Cruz, CA), at 1:200.

Techniques: Expressing, In Situ Hybridization, Control, Staining, Hybridization

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 is high expressed in normal tissues and was blocked in the spleen, overexpression of Sp3 attenuates the clinical symptoms and inflammation in the CNS of EAE mice. a The expression of SP3 in brain from 42 health and 37 MS patients in GEO ( GSE131282 ). b The expression of SP3 from 92 normal tissues or compartments of human in genecards ( www.genecards.org ). c The expression of Sp3 was checked by real-time PCR in the spleen from normal wild type C57BL/6 and EAE mice. Changes in mRNA levels were quantified using the 2-ΔΔCT method with mGapdh mRNA as a control. Data were from three independent experiments (mean ± SD). * p < 0.05; ** p < 0.01, as determined using the Student’s t test. d The protein levels of Sp3 was tested by immunoblotting. β-actin was used as the loading control. EAE was induced by MOG 35-55 in female C57BL/6 mice (n = 6).The clinical scores of all the EAE mice were assessed daily according to the same criteria for 24 continuous days. Incidence ( e ), disease onset ( f ), daily clinical scores ( g ), cumulative disease scores ( h ), and peak disease scores ( i ) were monitored. e The incidence of EAE (mice with a clinical score ≥ 1 for 2 continuous days). The disease incidence is represented by the percentage of mice suffering from EAE. f The line graph depicts the survival rate of EAE between WT and LV-Sp3 mice. g The clinical scores of all the mice for 24 continuous days. h The mean disease cumulative score. i The mean maximum clinical score. j Hematoxylin and eosin (H&E) staining of representative spinal cord sections from LV-Sp3 mice and WT mice showing the infiltration of inflammatory cells in the white matter. k , m Showing the infiltration of inflammatory cells or demyelination in the white matter. l Luxol fast blue staining of intact myelin (blue) and demyelination (pink). The data are representative of at least two experiments with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Overexpression of Sp3 suppressed Th1 and Th17cells in vivo and in vitro . Flow cytometry analysis of activated signature markers of Th1 and Th17 (IFN-γ and IL-17A) cells on the range of CD4 + cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-Sp3-treated EAE mice (n = 6 mice per group) detected on the day 18 after the induction of EAE. Data are shown in a representative plot of the Th1 and Th17 cell subpopulations in three types of tissues ( a - c ). The percentage of respective subpopulations in CD4 + cells summarized by histograms. Treatment of cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-Sp3-treated EAE mice was performed according to the description in the “Intracellular cytokine staining” section. Cells from the spleen ( d ) and lymph nodes ( e ) of LV-Ctrl and LV-Sp3-treated EAE mice were collected, and total RNA was extracted based on the manufacturer’s instructions using TRIzol reagent. The quantity of mRNA was recorded using real-time PCR. Gene expression was normalized based on the mGapdh content. f Naïve CD4 + T cells were separated from the spleen of female C57BL/6 mice, and then Th1 and Th17 cells were induced under various conditions according to the “Naïve CD4 + T cell preparation and differentiation” section. The cells were evaluated per the description in the “Intracellular cytokine staining” section. g , h ELISA was used to assess the expression of IFN-γ and IL-17A in the supernatants of Th1 and Th17 cells. Data were from three independent experiments (mean ± SEM). * p < 0.05; ** p < 0.01, as determined using the Student’s t test.

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Over Expression, In Vivo, In Vitro, Flow Cytometry, Isolation, Staining, Real-time Polymerase Chain Reaction, Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 as a direct target of miR-223. a Potential target genes of miR-223 as predicted by miRanda, TargetRank and TargetScan. b The miR-223 target sequence in the 3′UTR of Sp3 and Hsp72 in mouse. c EL4 cells were transfected with LV-Ctrl or LV-anti-miR-223. Quantitative PCR (real-time PCR) analysis of Hsp72, Sp3 and Sp1 mRNA levels. d The protein level of Sp3 and Hsp72 was tested by immunoblotting. H3 and β-actin were used as the nuclear and cytoplasm loading controls. e A schematic representing the luciferase constructs with the wild-type (CN) or mutant 3′UTR miR-223 MRE sequences of Sp3 and Hsp72. Mutations are marked in lowercase letters and underlined. f Normalized luciferase activity in lysates from 293 T cells co-transfected with wild-type or mutant Sp3-luciferase constructs and miR-223 or control mimics (mean ± SEM, n = 3, * p < 0.05, ** p < 0.01). g - i EL4 cells were transfected with LV-Ctrl or LV-anti-miR-223 and stimulated with PMA/I for 0 min, 10 min, 30 min, and 60 min. Quantitative PCR analysis of IFN-γ, IL-17A and IL-17F mRNA levels (g). h , i The protein levels of Smad2/3, phospho-Smad2/3, and Stat3 were tested by immunoblotting. β-actin was used as the loading control. The data are expressed as the mean ± SEM; experiments were performed in triplicate (* p < 0.05, ** p < 0.01).

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Sequencing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Construct, Mutagenesis, Activity Assay, Control

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 regulated the Smad2/3 and Stat3 signaling pathways during Th17 cell differentiation through Socs3. a , b The protein levels of Smad2/3, phospho-Smad2, Stat3 and phospho-Stat3 were tested by immunoblotting in the spleen and CNS from LV-Ctrl, LV-Sp3 or LV-anti-Sp3-treated EAE mice. c RORγt, a key transcription factor in Th17 cells, was tested in CNS by immunoblotting. d EL4 cells were transfected with LV-Ctrl or LV-Sp3 and stimulated with PMA/I for 0 min, 10 min, 30 min, and 60 min. The protein levels of Smad2/3, phospho-Smad2/3, and Stat3 were assessed by immunoblotting. e Analysis SP3 binding genes by TRRUST, FIMO_JASPAR, GSE131282 database. f Analysis SOCS3 gene expression in MS patients in GEO ( GSE131282 ). g JASPAR database predicts that transcription factor SP3 binds to the upstream 1740–1750 bp position of the SOCS3 gene. h The protein level of Socs3 was tested by immunoblotting in EL4 cells. i The protein level of Sp3 was tested by immunoblotting in EL4 cells and 293 T cells. β-actin was used as the loading control. Densitometric ratios were quantified using ImageJ software. The data are expressed as the mean ± SEM (* p < 0.05, ** p < 0.01).

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Protein-Protein interactions, Cell Differentiation, Western Blot, Transfection, Binding Assay, Gene Expression, Control, Software

Journal: Journal of Advanced Research

Article Title: Sp3 ameliorated experimental autoimmune encephalomyelitis by triggering Socs3 in Th17 cells

doi: 10.1016/j.jare.2025.01.051

Figure Lengend Snippet: Sp3 in CD4 T cells attenuated or aggravated the clinical symptoms of Sp3 +/+CD4Cre or Sp3 -/-CD4Cre EAE mice. The clinical scores of all the EAE mice were assessed daily according to the same criteria for 24 continuous days. Incidence ( a ), Daily clinical scores ( b ), peak disease scores ( c ), and cumulative disease scores ( d ) were monitored Sp3 fl/fl and Sp3 +/+CD4Cre EAE mice. The data are representative of at least two experiments with similar results. Flow cytometry analysis of CD4 + cells isolated from the spleen and lymph node of Sp3 fl/fl and Sp3 +/+CD4Cre EAE mice (n = 6 mice per group) at day 18 after the induction of EAE. Data are shown in a representative plot of the CD4 + cell subpopulations in spleen and lymph node ( e, f, g ). Knockout of Sp3 in CD4 T cells of Sp3 -/-CD4Cre EAE mice. Incidence ( h ), Daily clinical scores ( i ), peak disease scores ( j ), and cumulative disease scores ( k ) were monitored. The data are representative of at least two experiments with similar results. Flow cytometry analysis of CD4 + cells isolated from the spleen of Sp3 fl/fl and Sp3 -/-CD4Cre EAE mice at day 15 after the induction of EAE. Data are shown in a representative plot of the CD4 + cell subpopulations in spleen ( l ). m Naïve CD4 + T cells were separated from the spleen of female Sp3 fl/fl and Sp3 -/-CD4Cre mice and then induced to differentiate into Th17 cells. Data were from three independent experiments (mean ± SEM). * p < 0.05; ** p < 0.01, as determined using the Student’s t test. n Schematic of the molecular mechanism by which Sp3 regulates Th17 cell differentiation. As the target of miR-223, Sp3 positively regulates Socs3, which regulates Sp3 via a feedback mechanism. Socs3 negatively regulates the IL-6/JAK2/Stat3 and TGF-β/Smad2/3 signal transduction pathways to control Th17 cell differentiation. Thus, RORγt contributes to Th17 cell differentiation and EAE induction.

Article Snippet: Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China).

Techniques: Flow Cytometry, Isolation, Knock-Out, Cell Differentiation, Transduction, Control