Journal: Journal of Nanobiotechnology

Article Title: Apoptotic tumor cell-derived microparticles loading Napabucasin inhibit CSCs and synergistic immune therapy

doi: 10.1186/s12951-023-01792-8

Figure Lengend Snippet: NAP inhibited colon cancer by reducing STAT1 expression. A Western blot and RT-qPCR analysis of CD44 and BMI1 in NAP-treated CT26 cells. B Western Blot and RT-qPCR analyses of STAT1, STAT2, and STAT3 in the NAP-treated CT26 cells. C The correlations of STAT1, STAT2, and STAT3 with CD44 in human colon cancer tissues. D The heatmap of STAT1, STAT2, and STAT3 with CD44 in human colon cancer tissues. E Overexpression of STAT1 increased the protein levels of CD44. F Knockdown of STAT1 decreased the protein levels of CD44. G The overexpression of STAT1 reversed the effects of NAP on CD44 inhibition. H A STAT1 binding peak in the promoter region of CD44. I A consensus binding motif for STAT1. J The presence of STAT1 binding sites. K STAT1 bound to the promoter region of CD44

Article Snippet: The antibodies used in this experiment are as follows; STAT1 antibody (#10144-2-AP, 1:2000), STAT2 antibody (#66485–1-Ig, 1:4000), STAT3 antibody (#10253-2-AP, 1:2000), CD44 antibody (#15675-1-AP, 1:2000), BMI1 antibody (#10832-1-AP, 1:2000), TBK1 antibody(#28397-1-AP, 1:2500) and IRF3 antibody(#11312-1-AP,1:5000) were purchased from Proteintech.

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

Journal: Journal of Nanobiotechnology

Article Title: Apoptotic tumor cell-derived microparticles loading Napabucasin inhibit CSCs and synergistic immune therapy

doi: 10.1186/s12951-023-01792-8

Figure Lengend Snippet: In vivo antitumor effect and in vivo toxicity. A The therapeutic schedule. B Tumor growth curves in each group at given time points. C Average tumor weights in each group in the end. D Representative images of CT26 tumor tissues in the end. E Immunohistochemical staining of Ki67 in tumor tissues. F Western blot analysis of CD44 and STAT1 in CT26 tumor-bearing mice after treatment. G Immunohistochemical staining of CD44 in CT26 tumor tissues. H Body weight change curves. I – K Liver function markers (ALT, AST, and ALP) and kidney function markers (BUN and CRE) after different treatments

Article Snippet: The antibodies used in this experiment are as follows; STAT1 antibody (#10144-2-AP, 1:2000), STAT2 antibody (#66485–1-Ig, 1:4000), STAT3 antibody (#10253-2-AP, 1:2000), CD44 antibody (#15675-1-AP, 1:2000), BMI1 antibody (#10832-1-AP, 1:2000), TBK1 antibody(#28397-1-AP, 1:2500) and IRF3 antibody(#11312-1-AP,1:5000) were purchased from Proteintech.

Techniques: In Vivo, Immunohistochemical staining, Staining, Western Blot

Journal: Molecular Medicine Reports

Article Title: Role of SUMOylation of STAT1 in tubular epithelial-mesenchymal transition induced by high glucose

doi: 10.3892/mmr.2023.12929

Figure Lengend Snippet: STAT1 activation inhibits phenotypic transition of HK-2 cells. The activation of STAT1 (A) was detected using western blotting and (B) was semi-quantified using densitometry. High glucose increased the relative expression levels of p-STAT1 (Tyr701). The data were analyzed by two-way ANOVA and Bonferroni post hoc test. The efficiency of STAT1 knockdown (C) was analyzed by western blotting and (D) was semi-quantified using densitometry. The data were analyzed by one-way ANOVA and Tukey post hoc test. (E) Efficiency of plasmids transfection. The expression of p-STAT1, E-cadherin and α-SMA post-transfection with STAT1 siRNA (F) was detected using western blotting and (G) was semi-quantified using densitometry. The reduction in p-STAT1 aggravated the effects of high glucose on the expression levels of E-cadherin and α-SMA. The data were analyzed by one-way ANOVA and Tukey post hoc test. The expression of p-STAT1, E-cadherin, vimentin and α-SMA post-transfection with STAT1-WT or STAT1-YE plasmid (H) was detected using western blotting and (I) was semi-quantified using densitometry. Upregulation of STAT1 phosphorylation alleviated the high glucose-induced expression levels of E-cadherin, α-SMA and vimentin. The data were analyzed by one-way ANOVA and Tukey post hoc test. n=3. *P<0.05; # P<0.05 vs. 0 h; ▲ P<0.05 vs. normal glucose group. p-, phosphorylated; siRNA, small interfering RNA; WT, wild type.

Article Snippet: Anti-E-cadherin (cat. no. 20874-1-AP), protein inhibitor of activated STAT (PIAS)1 (cat. no. 23395-1-AP), PIAS3 (cat. no. 13486-1-AP), GFP (cat. no. 66002-1-lg) and anti-β-actin (cat. no. 66009-1-lg) antibodies were purchased from Proteintech Group, Ltd. Anti-SUMO1 antibody (cat. no. 4930) was purchased from Cell Signaling Technology, Inc. Rabbit anti-STAT1 antibody (cat. no. 10144-2-AP) was purchased from Proteintech Group, Ltd. and mouse anti-STAT1 antibody (cat. no. sc-464) was purchased from Santa Cruz Biotechnology, Inc. Anti-PIAS4 antibody (cat. no. AF5329) was purchased from Affinity Biosciences, Ltd.

Techniques: Activation Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Role of SUMOylation of STAT1 in tubular epithelial-mesenchymal transition induced by high glucose

doi: 10.3892/mmr.2023.12929

Figure Lengend Snippet: High glucose upregulates STAT1 SUMOylation in HK-2 cells. (A) Co-IP suggested that the binding of STAT1 and UBC9 was upregulated under high-glucose conditions. The data were analyzed by Student's t-test. (B) Efficiency of UBC9 knockdown was analyzed by western blotting and was semi-quantified using densitometry. The data were analyzed by one-way ANOVA and Tukey post hoc test. (C and D) Co-IP suggested that the SUMOylation of STAT1 was enhanced under high-glucose conditions. The data were analyzed by one-way ANOVA and Tukey post hoc test. (E) Binding of STAT1 and PIASs was analyzed. Results suggested that PIAS4 mediated the upregulation of STAT1 SUMOylation. The data were analyzed by Student's t-test. (F) Efficiency of PIAS4 knockdown was analyzed by western blotting and was semi-quantified using densitometry. The data were analyzed by one-way ANOVA and LSD post hoc test. The effects of PIAS4 siRNA transfection on STAT1 SUMOylation (G) were analyzed by co-IP and (H) were semi-quantified using densitometry. The results suggested that PIAS4 knockdown inhibited STAT1 SUMOylation. The data were analyzed by one-way ANOVA and Tukey post hoc test. IP:STAT1 means the cell lysate was incubated with STAT1 antibody. Input was used as a positive control to detect the presence of target proteins. n=3. *P<0.05. Co-IP, co-immunoprecipitation; PIAS, protein inhibitor of activated STAT; siRNA, small interfering RNA; SUMO, small ubiquitin-related modifier.

Article Snippet: Anti-E-cadherin (cat. no. 20874-1-AP), protein inhibitor of activated STAT (PIAS)1 (cat. no. 23395-1-AP), PIAS3 (cat. no. 13486-1-AP), GFP (cat. no. 66002-1-lg) and anti-β-actin (cat. no. 66009-1-lg) antibodies were purchased from Proteintech Group, Ltd. Anti-SUMO1 antibody (cat. no. 4930) was purchased from Cell Signaling Technology, Inc. Rabbit anti-STAT1 antibody (cat. no. 10144-2-AP) was purchased from Proteintech Group, Ltd. and mouse anti-STAT1 antibody (cat. no. sc-464) was purchased from Santa Cruz Biotechnology, Inc. Anti-PIAS4 antibody (cat. no. AF5329) was purchased from Affinity Biosciences, Ltd.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Transfection, Incubation, Positive Control, Immunoprecipitation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Role of SUMOylation of STAT1 in tubular epithelial-mesenchymal transition induced by high glucose

doi: 10.3892/mmr.2023.12929

Figure Lengend Snippet: Inhibition of STAT1 SUMOylation upregulates STAT1 activity induced by high glucose. (A) Transfection efficiency of STAT1-WT and STAT1-YE plasmids was detected by western blotting. The expression level of p-STAT1 (B) was detected using western blotting and (C) was semi-quantified using densitometry. Compared with in the STAT1-WT plasmid group, the level of p-STAT1 was higher in the STAT1-KR plasmid group. (D) Dual luciferase reporter assay suggested that STAT1 activity was increased in STAT1-KR plasmid group. All of the data were analyzed by one-way ANOVA and Tukey post hoc test. n=3. p-, phosphorylated; WT, wild type; Exo, exogenous STAT1; End, endogenous STAT1

Article Snippet: Anti-E-cadherin (cat. no. 20874-1-AP), protein inhibitor of activated STAT (PIAS)1 (cat. no. 23395-1-AP), PIAS3 (cat. no. 13486-1-AP), GFP (cat. no. 66002-1-lg) and anti-β-actin (cat. no. 66009-1-lg) antibodies were purchased from Proteintech Group, Ltd. Anti-SUMO1 antibody (cat. no. 4930) was purchased from Cell Signaling Technology, Inc. Rabbit anti-STAT1 antibody (cat. no. 10144-2-AP) was purchased from Proteintech Group, Ltd. and mouse anti-STAT1 antibody (cat. no. sc-464) was purchased from Santa Cruz Biotechnology, Inc. Anti-PIAS4 antibody (cat. no. AF5329) was purchased from Affinity Biosciences, Ltd.

Techniques: Inhibition, Activity Assay, Transfection, Western Blot, Expressing, Plasmid Preparation, Luciferase, Reporter Assay

Journal: Molecular Medicine Reports

Article Title: Role of SUMOylation of STAT1 in tubular epithelial-mesenchymal transition induced by high glucose

doi: 10.3892/mmr.2023.12929

Figure Lengend Snippet: Inhibition of STAT1 SUMOylation improves the phenotypic transition of HK-2 cells induced by high glucose. Expression levels of vimentin, α-SMA and E-cadherin were detected using western blotting and were semi-quantified using densitometry. The results suggested that activated STAT1 suppresses the high glucose-induced phenotypic transition of HK-2 cells. The data were analyzed by one-way ANOVA and Tukey post hoc test. n=3. *P<0.05. WT, wild type.

Article Snippet: Anti-E-cadherin (cat. no. 20874-1-AP), protein inhibitor of activated STAT (PIAS)1 (cat. no. 23395-1-AP), PIAS3 (cat. no. 13486-1-AP), GFP (cat. no. 66002-1-lg) and anti-β-actin (cat. no. 66009-1-lg) antibodies were purchased from Proteintech Group, Ltd. Anti-SUMO1 antibody (cat. no. 4930) was purchased from Cell Signaling Technology, Inc. Rabbit anti-STAT1 antibody (cat. no. 10144-2-AP) was purchased from Proteintech Group, Ltd. and mouse anti-STAT1 antibody (cat. no. sc-464) was purchased from Santa Cruz Biotechnology, Inc. Anti-PIAS4 antibody (cat. no. AF5329) was purchased from Affinity Biosciences, Ltd.

Techniques: Inhibition, Expressing, Western Blot

Journal: Parasites & Vectors

Article Title: Effect of B7-H4 downregulation induced by Toxoplasma gondii infection on dysfunction of decidual macrophages contributes to adverse pregnancy outcomes

doi: 10.1186/s13071-022-05560-9

Figure Lengend Snippet: Downregulation of B7-H4 by T. gondii infection resulted in changes in dMφ function by affecting the JAK2/STAT1 signaling pathways. a , b Representative western blot and histogram analysis of B7-H4, JAK2, p-JAK2, STAT1 and p-STAT1 levels of dMφ in uninfected, infected and B7-H4 neutralized infected groups. c , d Representative western blot and histograms analysis of B7-H4, STAT1, p-STAT1, iNOS and TNF-α of dMφ in the uninfected, infected, infected + STAT1 inhibitor (Infected + Flu), B7-H4 neutralized infected and B7-H4 neutralized infected + STAT1 inhibitor (Neutralized infected + Flu) groups. The data in all panels are representative of at least three independent experiments. Data are presented as the means ± SD, with the unpaired t-test. Asterisks indicate significant differences between groups at * P < 0.05, ** P < 0.01. Flu, Fludarabine; JAK2, Janus kinase 2; p-JAK2, phosphorylated JAK2; p-STAT1, phosphorylated signal transducer and activator of transcription 1; STAT1, signal transducer and activator of transcription 1

Article Snippet: The membranes were blocked for 2.5 h in 5% non-fat dry milk in Tris-buffered saline with Tween-20 at room temperature (20–25 °C), and then incubated overnight at 4 °C with the primary antibodies for B7-H4 (1:2000; Abcam, Cambridge, UK), CD80 (1:1000; Proteintech, Wuhan, China), Arg-1 (1:1000; Proteintech), iNOS (1:1000; Abcam), JAK2 (1:1000; Abcam), phosphorylated JAK2 (p-JAK2; 1:1000; Abcam), STAT1 (1:2000; Proteintech) and phosphorylated STAT1 (p-STAT1; 1:2000; Proteintech), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:40,000; Proteintech).

Techniques: Infection, Western Blot