sta21  (Santa Cruz Biotechnology)

Journal: Neural regeneration research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury.

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

Article Snippet: To assess the role of STAT3 signaling, astrocytes were pretreated with 10 μM STA21 (an inhibitor of STAT3, Selleck, Shanghai, China, Cat# S7951) for 72 hours before coculture.

Techniques: In Vitro, Staining, Western Blot, Incubation, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-β1

doi: 10.1073/pnas.2023230118

Figure Lengend Snippet: Transcriptional factor ICER promotes ADAM9 expression in Th17 cells. (A and B) ICER/CREM-deficient or -sufficient naïve CD4+ T cells cultured under Th17-polarizing conditions for 3 d. (A) Relative ADAM9 mRNA expressions were assessed by qRT-PCR. Cumulative data are shown (mean ± SEM, n = 12). (B) Pro-ADAM9, ADAM9 (active), and β-actin expressions on day 3 were determined by Western blotting. A representative (of six) blot is shown (Left) and cumulative densitometric readings of ADAM9 (active) and β-actin are shown (Right) (mean ± SEM, n = 6). (C) Naïve CD4+ T cells from ICER/CREM-deficient or -sufficient IL-17GFP mice were polarized under Th17 conditions for 3 d. ADAM9 mRNA expression of FACS-sorted GFP+ cells (IL-17A–producing cells) was assessed by qRT-PCR (mean ± SEM, n = 7). (D) Th17-polarized naive CD4+ T cells were cultured in the presence of a STAT3 inhibitor (STA21, 10 μM) or dimethyl sulfoxide (DMSO) for 3 d. Pro-ADAM9, ADAM9 (active), and β-actin expressions were determined by Western blotting. A representative (of five) blot is shown (Left) and cumulative densitometric readings of ADAM9 (active) and β-actin are shown (Right) (mean ± SEM, n = 5). (E) Naive CD4+ T cells from ICER/CREM-deficient IL-17GFP mice were cultured under Th17-polarizing conditions and empty vector (empty) or ADAM9 overexpression (ADAM9 O.E.) plasmids were transfected into cultured T cells on day 1. Representative flow plots on day 3 (Left) and cumulative data (Right) are shown (mean ± SEM, n = 5). (F–H) ICERγ binds to the ADAM9 promoter directly and increases its activity. ICER/CREM-deficient or -sufficient naïve CD4+ T cells were cultured under Th17-polarizing conditions. (F) Schematic representation of the used reporter constructs. Numbers represent the position from the transcription start site of the murine ADAM9 gene. (G) The full-length ADAM9 promoter region (full) or a version containing a mutated CRE binding site (Δ-134/-130) were transfected into Th17-polarized T cells on day 1. Cells were harvested and lysed on day 2. Cumulative results of four independent experiments are shown (mean ± SEM). (H) The FLAG-tagged ICERγ overexpression vector was transfected into ICER/CREM-deficient CD4+ T cells on day one. Cells were harvested and lysed on day 3, and binding of FLAG/ICERγ to the CRE was assessed by ChIP assay. CRE at the intron1 of the ADAM9 gene and CRE at the intron 3 of the adjacent gene (Tm2d2) were used as negative controls for ChIP enrichment. Representative blots from three experiments are shown. *P < 0.05, **P < 0.01.

Article Snippet: For signal transduction studies, STA21 (STAT3 inhibitor, Santa Cruz Biotechnology) was added to cultures on day 0.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Over Expression, Transfection, Activity Assay, Construct, Binding Assay