flag stat3  (Sino Biological)

Journal: Advanced Science

Article Title: Detachment‐Induced FAK‐STAT3‐NNMT Inhibits CTCs Anoikis to Promote Breast Cancer Metastasis by Enhancing Fatty Acid Oxidation

doi: 10.1002/advs.202522837

Figure Lengend Snippet: Detachment‐induced NNMT is upregulated by the FAK‐STAT3 axis. (A) Representative image of the positive correlation between FAK and STAT3 in breast cancer and pan‐cancer according to the GEPIA 2 database analysis. (B) Representative results of STAT3, P‐STAT3, FAK, and P‐FAK proteins by WB in the detached BT‐549, MCF‐7, and MDA‐MB‐231 cells. (C,D) The protein and mRNA levels of NNMT and P‐STAT3 after treating detached cells with FAK inhibitor PF‐562271 (10 µ m ) and P‐STAT3 inhibitor C188‐9 (10 µ m ) for 48 h by WB and QPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (E,F) The protein and mRNA levels of NNMT and P‐STAT3 after treating detached cells with FAK inhibitor PF‐562271 (10 µ m for 48 h) and P‐STAT3 activator (1 µ m for 24 h) by WB and QPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (G,H) ChIP assay using P‐STAT3 antibodies in MDA‐MB‐231 cells cultured under suspension conditions was performed, followed by PCR and qPCR, which showed P‐STAT3 enrichment at the NNMT promoter vs. IgG control. ** p < 0.01. I: Dual‐luciferase reporter assays showed that co‐transfection with the full‐length NNMT promoter and a STAT3‐expressing plasmid significantly enhanced luciferase activity, whereas this enhancement was abolished when a truncated NNMT promoter was used. Data were presented as mean ± SEM. **** p < 0.0001.

Article Snippet: All plasmids used in this assay, including the firefly luciferase reporter vectors (e.g., pGL4‐basic containing the wild‐type or truncated NNMT promoter), the STAT3 expression plasmid, and the Renilla luciferase internal control plasmid (pRL‐TK), were custom‐synthesized by GeneChem Co., Ltd. (Shanghai, China).

Techniques: Cell Culture, Suspension, Control, Luciferase, Cotransfection, Expressing, Plasmid Preparation, Activity Assay

Journal: Advanced Science

Article Title: ECSIT‐X4 is Required for Preventing Pressure Overload‐Induced Cardiac Hypertrophy via Regulating Mitochondrial STAT3

doi: 10.1002/advs.202414358

Figure Lengend Snippet: ECSIT‐X4 interacts with STAT3 and enhanced its serine 727 phosphorylation in the mitochondria of hypertrophic cardiomyocytes. A) NMCMs were isolated from Ecsit f/f mice transfected with Adv‐Flag‐Ecsit‐X4. Mitochondria isolated from the NMCMs were lysed and immunoprecipitated with either anti‐IgG or anti‐Flag antibody. Flag‐labeled immunoprecipitates were visualized using Coomassie blue staining and subsequently subjected to LC‐MS/MS analysis to identify the target protein (STAT3), a mitochondrial complex I‐related protein that interacts with ECSIT‐X4. B) LC‐MS/MS fragment analysis of the specific peptide segment (AILSTKPPGTFLLR) of the STAT3 protein. The X‐axis represents the mass‐to‐charge ratio (m/z), while the Y‐axis represents signal intensity. Red line: b ions; blue lines: y ions; gray line: fragment ions. n = 1 independent experiments. C) The structure of ECSIT‐X4 protein was build using AlphaFold2, and the interaction between ECSIT‐X4 (blue) and STAT3 (yellow) was predicted by ZDOCK (version 3.0.2). Score = 1408.166. D) Interaction between ECSIT‐X4 and STAT3 was demonstrated by GST‐pull down. n = 3 independent experiments. E) HEK293T cells were transfected with expression plasmid encoding Flag‐ECSIT‐X4 for 24 h. Immunofluorescence staining with anti‐Flag antibody (green), mitochondria marker VDAC1 (red), and DAPI (blue). Scale bar = 20 µm. n = 3 independent experiments. F,G) HEK293T cells were co‐transduced with expression plasmid encoding FLAG‐ECSIT‐X4 and HA‐STAT3 for 36 h, and interaction between ECSIT‐X4 and STAT3 in mitochondria was determined by immunoprecipitation. n = 3 independent experiments. H) HEK293T cells were co‐transduced with expression plasmid encoding His‐STAT3 and FLAG‐ECSIT‐X4 or FLAG‐ΔECSIT‐X4 (mutant with 110–310 amino acid) for 36 h, and interaction between STAT3 and ECSIT‐X4 or ΔECSIT‐X4 in mitochondria was determined by immunoprecipitation. n = 3 independent experiments. I) Interaction between FLAG‐ECSIT‐X4 and endogenous STAT3 in mitochondria of H9c2 cells was determined by immunoprecipitation with anti‐Flag antibody followed by immunoblot with anti‐STAT3 antibody. n = 3 independent experiments. J) Representative Western blots and statistical results of total STAT3 and p (S727)‐STAT3 were shown. n = 3 independent experiments. K) Interaction between FLAG‐ECSIT‐X4 and endogenous STAT3 in the mitochondria of NMCM‐Ecsit cKO is illustrated, along with representative Western blots and statistical results for total STAT3 and p (S727)‐STAT3. n = 4 independent experiments. Data were presented as mean ± SD, p ‐values were determined by one‐way ANOVA corrected by the post hoc Turkey's test. p < 0.05 was considered statistically significant.

Article Snippet: Mouse expression plasmids encoding HA‐tagged Stat3 was purchased from Genechem (Shanghai, China).

Techniques: Phospho-proteomics, Isolation, Transfection, Immunoprecipitation, Labeling, Staining, Liquid Chromatography with Mass Spectroscopy, Expressing, Plasmid Preparation, Immunofluorescence, Marker, Transduction, Mutagenesis, Western Blot

Journal: Advanced Science

Article Title: ECSIT‐X4 is Required for Preventing Pressure Overload‐Induced Cardiac Hypertrophy via Regulating Mitochondrial STAT3

doi: 10.1002/advs.202414358

Figure Lengend Snippet: Overexpression of Ecsit‐X4 enhances mitochondrial OXPHOS function via STAT3 in hypertrophic cardiomyocytes. A) H9c2 cells were transfected with Adv‐Flag‐Ecsit‐X4 or Adv‐GFP for 24 h, and then were treated with 5 µ m Stattic or dmso for 4 h before Saline or Ang II stimulation for 48 h. B) Representative Western blots and statistical results of p (S727)‐STAT3, t‐STAT3, NDUFAF1 and NDUFB8 in mitochondria were presented. n = 3 independent experiments. C) ATP content in H9c2 cells. n = 3 independent experiments. D) H9c2 cells incubated with MitoSOX. Fluorescence measurement was performed to detect mROS, and statistical result of relative MitoSOX staining was shown. Scale bar = 40 µm. n = 3 independent experiments. E) NMCMs were transfected with Si‐Scramble or Si‐Stat3 for 24 h, and then were transfected with Adv‐Flag‐Ecsit‐X4 or Adv‐GFP for 24 h before Saline or Ang II stimulation for 48 h. F) Representative Western blots and statistical results of p (S727)‐STAT3, t‐STAT3 and ETC complex components in mitochondria were presented. n = 4 independent experiments. G) ATP content in NMCMs. n = 4 independent experiments. H,I) NMCMs incubated with MitoSox, and mROS was detected by fluorescence measurement. Statistical result of relative MitoSox staining was shown. Scale bar = 40 µm. n = 4 independent experiments. Data were presented as mean ± SD, p ‐values were determined by one‐way ANOVA corrected by the post hoc Turkey's test. p < 0.05 was considered statistically significant.

Article Snippet: Mouse expression plasmids encoding HA‐tagged Stat3 was purchased from Genechem (Shanghai, China).

Techniques: Over Expression, Transfection, Saline, Western Blot, Incubation, Fluorescence, Staining

Journal: Advanced Science

Article Title: ECSIT‐X4 is Required for Preventing Pressure Overload‐Induced Cardiac Hypertrophy via Regulating Mitochondrial STAT3

doi: 10.1002/advs.202414358

Figure Lengend Snippet: ECSIT‐X4 alleviates cardiac hypertrophy and enhances mitochondrial OXPHOS function through STAT3 in hypertrophic Ecsit cKO mice. A) Treatment regimen. B) Representative gross appearance of whole hearts. Scale bar = 1 mm. C,D) The ratios of HW/BW and LW/BW. E) WGA and H&E staining were performed to detect cardiomyocyte cross‐sectional area in mouse hearts. Scale bar = 40µm. Statistical result of cell size was shown. F) The mRNA expression of ANP, BNP, and β‐MHC. All were normalized to HPRT. G–J) Representative Western blots and statistical results of STAT3, ECSIT‐X4, and ETC complex components in mitochondria were presented. n = 6 mice per group. Data were presented as mean ± SD, p ‐values were determined by one‐way ANOVA corrected by the post hoc Turkey's test. p < 0.05 was considered statistically significant.

Article Snippet: Mouse expression plasmids encoding HA‐tagged Stat3 was purchased from Genechem (Shanghai, China).

Techniques: Staining, Expressing, Western Blot