Journal: Cancer biology & therapy

Article Title: GdX inhibits the occurrence and progression of breast cancer by negatively modulating the activity of STAT3.

doi: 10.1080/15384047.2024.2420383

Figure Lengend Snippet: Figure 6. GdX specifically interacts with STAT3. (a) Dual luciferase assay results indicate that STAT3 is a downstream target gene of GdX; (b) immunofluorescence staining reveals the colocalization of GdX and p-STAT3 in the cell nucleus; (c) Western blot analysis assesses the impact of GdX overexpression on the phosphorylation levels of STAT3. *, p < .05, **, p < .01, ***, p < .001, vs. GdX-nc.

Article Snippet: The membrane was blocked with 5% skimmed milk for 1 h at room temperature, and then incubated with primary antibodies against GdX (Proteintech, 1: 1000), Bcl-XL (Proteintech, 1: 1000), c-Myc (Proteintech, 1: 2000), Cyclin D1 (Proteintech, 1: 5000), STAT3 (Cell Signaling Technology, 1: 1000), phospho-STAT3 (p-STAT3) (Cell Signaling Technology, 1: 2000), and GAPDH (Cell Signaling Technology, 1: 1000) at 4°C overnight.

Techniques: Luciferase, Immunofluorescence, Staining, Western Blot, Over Expression, Phospho-proteomics

Journal: PLoS ONE

Article Title: Nogo-66 Promotes the Differentiation of Neural Progenitors into Astroglial Lineage Cells through mTOR-STAT3 Pathway

doi: 10.1371/journal.pone.0001856

Figure Lengend Snippet: (A) Gel imagine of RT-PCR for NgR mRNA expression in NPCs. (B) Immunocytochemistry for NgR (green) expression in NPCs. Nuclei were stained by PI (shown in red). (C) PI-PLC and NEP1-40 rescued the astroglial induction by Nogo-66. After PI-PLC or NEP1-40 treatment for 2 hours, the proportion of GFAP positive cells induced by Nogo-66 was significantly lower than that induced only by Nogo-66 treatment. ( p <0.01). (D) The statistical result of GFAP positive cells percentage after Y27632 (RhoA-ROCK inhibitor) treatment. The astroglial induction of Nogo-66 was not rescued by Y27632. (E) Rapamycin and AG490 rescued the astroglial induction by Nogo-66. After rapamycin or AG490 treatment for 2 hours, the proportion of GFAP positive cells induced by Nogo-66 was recovered. (F) Nogo-66 activated the phosphorylation of STAT3 at Ser727 and Tyr705 and phosphorylation of mTOR. After starved for 24 hours in serum-free DMEM medium, total cell lysates from NPCs treated with GST (G) or Nogo-66 (N) (100 nM) for the indicated time were immunoblotted and probed with the indicated antibodies. (G) After starved for 24 hours in serum- free DMEM medium, total cell lysates from NPCs not treated (−) or treated (+) with PI-PLC (1 U/ml) or NEP1-40 for 2 hours and then stimulated with Nogo-66 (100 nM) for 30 minutes were immunoblotted and probed with the indicated antibodies. PI-PLC and NEP1-40 could rescue the phosphorylation of STAT3 activated by Nogo-66. (H) Y27632 did not alter the phosphorylation of STAT3 activated by Nogo0-66. After starved for 24 hours in serum-free DMEM medium, total cell lysates from NPCs not treated (−) or treated (+) with Y27632 (10 uM) for 2 hours and then stimulated with Nogo-66 (100 nM) for 30 minutes were immunoblotted and probed with the indicated antibodies. (I) Rapamycin could inhibit the activated phosphorylation of STAT3 induced by Nogo-66. AG490 strongly inhibited phosphorylation of STAT3 at Tyr705. After starved for 24 hours in serum-free DMEM medium, total cell lysates from NPCs not treated (−) or treated (+) with rapamycin (50 uM) or AG 490 (3ug/ml) for 2 hours and then stimulated with Nogo-66 (100 nM) for 30 minutes were immunoblotted and probed with the indicated antibodies. (J) Rapamycin treatment decreased the complex formation between mTOR and Stat3 in NPCs at the presence of Nogo-66. Nondenatured whole cell lysates were immunoprecipitated with an mTOR antibody before western blot analysis using anti-STAT3 and mTOR, respectively. Data are mean±S.E. Error bars indicate SE. * p <0.05 ** p <0.01 (n = 3).

Article Snippet: For immunoblotting, primary antibodies were diluted as follow: NeuN, β III tubulin, α-tubulin, P-STAT3 (Ser727), P-STAT3 (Tyr 705), STAT3 (CST), P-mTOR and mTOR (CST) at 1∶1000; Nogo-A at 1∶300, MBP at 1∶200, GFAP 1∶100.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunocytochemistry, Staining, Phospho-proteomics, Immunoprecipitation, Western Blot