Journal: British Journal of Pharmacology

Article Title: Ursolic acid promotes apoptosis and mediates transcriptional suppression of CT45A2 gene expression in non‐small‐cell lung carcinoma harbouring EGFR T790M mutations

doi: 10.1111/bph.14793

Figure Lengend Snippet: Ursolic acid (UA) inhibited β‐catenin/TCF4 signalling pathway in H1975 cells. (A) Left: H1975 cells, with or without transfection with siRNA against TCF4 (siTCF4) or a negative control siRNA (siNC), were incubated with PBS or UA (25 μmol L‐1) for 8 hr. The indicated gene was detected by PCR. Right: TCF4 and CT45A2 mRNA levels in Fig. ​Fig.6A,6A, as analyzed by Image J software. (B) H1975 cells, with or without transfection with siRNA against TCF4 (siTCF4) or a negative control siRNA (siNC), were incubated with PBS or UA for 24 hr, cell viability was evaluated using by MTT assay. (C) H1975 cells, with or without transfection with siRNA against TCF4 (siTCF4) or a negative control siRNA (siNC), were incubated with PBS or UA for 24 hr, and then stained with AnnexinV‐FITC and propidium iodide for detecting apoptosis by flow cytometry. The graph shows the mean values from FACS assays. (D) Left: Protein expression of β‐catenin, GSK‐3β and their phosphorylated forms, were determined by western blot analysis. Phosphorylation of β‐catenin and GSK‐3β was decreased after UA treatment. Erlotinib was used as the negative control. Right: The graph shows the mean values from western blotting. Protein levels were quantified using gray value analyses by Image J software. (E) Left: UA inhibited β‐catenin into the nucleus. H1975 cells were treated with UA stimuli for 24 hr, the distribution of β‐catenin (green) was detected by immunofluorescence. The nucleus was visualized by Hoechst33342 staining. White arrows indicate the nucleus. Red arrows indicate the cytoplasm. Scale bar=100 μm. Right: Quantification of β‐catenin nuclear/cytoplasmic ratio in Fig.​Fig.6E.6E. Mean of nuclear/cytoplasmic rations were calculated from 30 H1975 cells. Mean pixel intensity was calculated using GNU Image Manipulator. Data shown are means ± SD from five independent experiments *P < 0.05, #P < 0.05, significantly different as indicated; ns, non‐significant effects

Article Snippet: Phosphorylated GSK‐3β (Ser 9 ; AF2016) and phosphorylated β‐catenin (Ser 33/37 /Thr 41 ; DF2989) were purchased from Affinity Biosciences (Shanghai, China).

Techniques: Transfection, Negative Control, Incubation, Software, MTT Assay, Staining, Flow Cytometry, Expressing, Western Blot, Phospho-proteomics, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: Ursolic acid promotes apoptosis and mediates transcriptional suppression of CT45A2 gene expression in non‐small‐cell lung carcinoma harbouring EGFR T790M mutations

doi: 10.1111/bph.14793

Figure Lengend Snippet: Ursolic acid (UA) inhibited CT45A2 mRNA expression and β‐catenin/TCF4 signalling pathway in vivo. (A) Left: UA inhibited CT45A2 mRNA expression in tumour tissues. Tumour tissues from groups treated with vehicle, UA (25 mg kg‐1) or erlotinib (20 mg kg‐1), were harvested for total RNA extraction and mRNA levels were measured with PCR. GAPDH is used as a control. Right: CT45A2 mRNA levels in Fig. ​Fig.7A,7A, as analyzed by Image J software. (B) Left: UA inhibited β‐catenin/TCF4 signalling protein expression in tumour tissues. Tumour tissues from groups treated with vehicle, UA (25 mg kg‐1) or erlotinib (20 mg kg‐1), were immunoblotted with antibodies to detect β‐catenin, GSK‐3β, p‐β‐catenin(Ser33/37/Thr41), p‐GSK‐3β(Ser9), TCF4 and GAPDH. Right: Analysis of western blotting results in Fig. ​Fig.7B.7B. Protein levels were quantified using gray value analyses by Image J software. (C) Left: Immunohistochemical analysis of the cleaved‐caspase3 expression in xenograft tumours formed by H1975 cells in mice treated with vehicle, UA, or erlotinib. Representative images are shown. Scale bar=200 μm. Right: quantitative analysis of the immunohistochemical staining by using Image J software. Data shown are means ± SD from five independent experiments for vehicle group and UA group; three independent experiments for erlotinib group. *P < 0.05, significantly different as indicated; ns, non‐significant effects

Article Snippet: Phosphorylated GSK‐3β (Ser 9 ; AF2016) and phosphorylated β‐catenin (Ser 33/37 /Thr 41 ; DF2989) were purchased from Affinity Biosciences (Shanghai, China).

Techniques: Expressing, In Vivo, RNA Extraction, Control, Software, Western Blot, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Rspo2 inhibits TCF3 phosphorylation to antagonize Wnt signaling during vertebrate anteroposterior axis specification

doi: 10.1038/s41598-021-92824-6

Figure Lengend Snippet: Rspo2 inhibits TCF3 phosphorylation. ( A ) Schematic of Rspo2 deletion constructs. SP, signal peptide; FU1, furin-like domain 1; FU2, furin-like domain 2; TSP, thrombospondin type 1 domain; BR, the basic amino acid-rich domain. ( B , C ) Effects of Rspo2 constructs on Wnt-dependent Dvl2 phosphorylation ( B ) and TCF3 phosphorylation and β-catenin levels ( C ). Four-cell stage embryos were injected animally with Wnt8 DNA (50 pg or 100 pg) or Wnt8, Wnt3a or Wnt5a RNAs (1 ng each) and Rspo2, Rspo∆F or Rspo∆T RNAs (0.5 ng each) as indicated. Ectoderm explants were dissected at stage 9 and cultured until stage 12 for immunoblotting with antibodies against Dvl2, TCF3, ABC (non-phosphorylated β-catenin). Arrowheads indicate the position of phosphorylated (upshifted) and non-phosphorylated Dvl2 or TCF3 proteins. Erk1 controls for loading. ( D ) Effects of Rspo2 constructs (0.5 ng each) on TCF3 phosphorylated by endogenous signals. Dorsal marginal zone (D) and ventral marginal zone (V) were dissected from the control and injected embryos at stage 10 and cultured until stage 12.5 for immunoblotting with anti-TCF3 antibodies as shown. Control D and V groups were run in the same gel but separately from the other groups (see Supplementary Fig. ). ( E ) Effects of Rspo2 depletion on TCF3 phosphorylation by endogenous signals. DMZ and VMZ explants of embryos injected with control MO (COMO, 20 ng) or RMO ATG (20 ng) were dissected and analyzed by immunoblotting as in ( B , C ).

Article Snippet: The following primary antibodies were used: mouse anti-FLAG (M2, Sigma), mouse anti-non-phosphorylated β-catenin (ABC; Upstate Biotechnology), rabbit anti-XTCF3N , rabbit anti-Dvl2 .

Techniques: Construct, Injection, Cell Culture, Western Blot