Journal: Scientific Reports

Article Title: Smurf1 promotes gastric cancer progression by regulating Axin2-dependent Wnt signaling pathway

doi: 10.1038/s41598-025-23707-3

Figure Lengend Snippet: Smurf1 promoted Axin2 ubiquitination and ubiquitin-dependent degradation. ( A ) qRT-PCR analysis of Axin1 and Axin2 mRNA levels in AGS cells after Smurf1 overexpression. ( B ) Western blot analysis of Axin1 and Axin2 protein levels in AGS cells after Smurf1 overexpression. ( C ) Co-IP assay of the direct interaction between Smurf1 and Axin2 in AGS cells using the Smurf1 antibody, followed by western blot analysis using the Axin2 antibody. ( D and E ) After Smurf1 overexpression, de novo protein synthesis was inhibited in AGS cells using 40 µM of Chx, and then Axin2 protein levels were measured using western blot assays at different time points. ( F ) After Smurf1 overexpression in AGS cells, a Co-IP assay was carried out using the Axin2 antibody, followed by western blot analysis using the ubiquitin antibody (Left: Smurf1 OE + Axin2 IP + Ub WB; Middle: no Smurf1 OE + Axin2 IP + Ub WB; Right: Smurf1 OE + IgG IP + Ub WB. OE: overexpression, Ub: ubiquitin, WB: western blot). ( G ) After Smurf1 overexpression and treatment with 20 µM of MG132 in AGS cells, Axin2 protein levels were measured using the western blot assay. The results were shown as the median (1st quartile and 3rd quartile). * p < 0.05.

Article Snippet: For the ubiquitination analysis, western blot was carried out to assess Axin2 protein in AGS cells following Smurf1 overexpression and MG132 (20 μM, MCE) treatment for 24 h.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Over Expression, Western Blot, Co-Immunoprecipitation Assay

Journal: International journal of oncology

Article Title: USP22 promotes the G1/S phase transition by upregulating FoxM1 expression via β-catenin nuclear localization and is associated with poor prognosis in stage II pancreatic ductal adenocarcinoma.

doi: 10.3892/ijo.2014.2531

Figure Lengend Snippet: Figure 6. USP22 expression affects FoxM1 expression and Wnt/β-catenin pathway activation. (A) Triple IF staining for FoxM1 (red), β-catenin (green) and nuclei (DAPI, blue) was performed on PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM). (B) Triple IF staining for FoxM1 (red), β-catenin (green) and nuclei (DAPI, blue) was performed on CFPAC-1 cells that were transfected by vector USP22-1 (2 µg) or USP22-2 (10 µg). (C and D) RT-PCR and western blot analysis of FoxM1 and β-catenin in PANC-1 cells that were transfected by control- siRNA, USP22‑siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM) (left panel) and CFPAC-1 cells that were transfected by vector, USP22-1 (2 µg) or USP22-2 (10 µg) (right panel). (E) Cellular levels of Axin2, c-Myc and LEF-1 in PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22‑siRNA-2 (150 nM) (left panel) and CFPAC-1 cells that were transfected by vector, USP22-1 (2 µg) or USP22-2 (10 µg) (right panel). (F) Activities of TOP-Flash and FOP-Flash in PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM) (left panel) and CFPAC-1 cells that were transfected by vector, USP22-1 (2 µg) or USP22-2 (10 µg) (right panel). (G) Cytoplasmic and nuclear levels of FoxM1 and β-catenin in PANC-1 cells that were transfected by control-siRNA, USP22-siRNA-1 (50 nM) or USP22-siRNA-2 (150 nM). (H) Cytoplasmic and nuclear levels of FoxM1 and β-catenin in CFPAC-1 cells that were transfected by vector USP22-1 (2 µg) or USP22-2 (10 µg).

Article Snippet: Membranes were blocked in a buffer (TBS: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5% bovine serum albumin and 0.1% Tween-20, followed by incubation with the primary antibodys USP22 (ab4812, 1:2,000, Abcam), FoxM1 (SC-502, 1:200, Santa Cruz Biotechnology), cyclin D1 (60186-1-lg, 1:100), p21 (10355-1-AP, 1:100), p27 (10567-1-AP, 1:100), cdk4 (11026-1-AP, 1:500), cdk6 (14052-1-AP, 1:500, all from Proteintech, Chicago, IL, USA), β-catenin (SC-7963, 1:500, Santa Cruz Biotechnology), Axin2 (EPR2005-2, 1:2,000), LEF (EP2030Y, 1:2,000, both from Abcam), c-Myc (10057-1-AP, 1:200) and LMNB1 (12987-1-AP, 1:1,000) or β-actin (20536-1-AP, 1:2,000, all from Proteintech) diluted in the same buffer.

Techniques: Expressing, Activation Assay, Staining, Transfection, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors

doi: 10.1038/s41598-018-36447-4

Figure Lengend Snippet: MSC2504877 is a novel, drug-like, small molecule tankyrase inhibitor. ( A ) Structure of MSC2504877. ( B ) Dose-response curve illustrating the inhibition of recombinant tankyrase (TNKS) or PARP1 with MSC2504877A. Tankyrase activity was assayed using the PARP domain of recombinant human Tankyrase (TNKS) or PARP1 in an ELISA assay. Mean dose response data from three independent experiments is shown. Error bars represent standard error of the mean (SEM). ( C ) Table illustrating IC 50 concentrations obtained for MSC2504877A and two toolbox tankyrase inhibitors. TNKS, TNKS2 and PARP1 activity was determined as in ( B ). ( D ) Western blot illustrating tankyrase stabilisation, suppression of β-catenin and stabilisation of Axin 2 protein levels in COLO320DM (APC mutant) colorectal tumour cells exposed to MSC2504877 for 24 hours as shown. α-Tubulin was used as loading control ( E , F ). Luminex antibody-based detection of AXIN2 and TNKS in COLO320DM cells exposed MSC2504877 in vitro . Dose response data from three independent experiments are shown; error bars represent standard deviations ( G , H ) Luminex detection of AXIN2 and TNKS in COLO320DM xenografts. CB17 SCID mice received 30 mg/kg MSC2504877 via an oral route. At the time points indicated, mice were sacrificed and xenografts recovered. Each data point indicates data from one animal. ( I ) Plasma concentration time profile of MSC2504877 after one single oral dose of 30 mg/kg.

Article Snippet: A monoclonal AXIN2 antibody (R&D Systems #MAB6078) bound to fluorescent carboxybeads was used for isolation of AXIN2 from lysates.

Techniques: Inhibition, Recombinant, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Mutagenesis, Control, Luminex, In Vitro, Clinical Proteomics, Concentration Assay

Journal: Scientific Reports

Article Title: Midbrain tectal stem cells display diverse regenerative capacities in zebrafish

doi: 10.1038/s41598-019-40734-z

Figure Lengend Snippet: Wnt/β-catenin signalling in stem cell zone 1 (qRG-L) and zone 2 (TMZi) following tectal lesion. (a) Experimental design to study Wnt/β-catenin signalling at 3-dpl. (b–d) Physiological levels of β-catenin expression in the neural layer (Neu-L) and qRG layer (qRG-L; stem cell zone 1). A small number of β-catenin-positive neuronal cell bodies are seen in the Neu-L (yellow arrow). White box depicts higher magnification images of the qRG-L in ( c , d ). Split-channel images showing co-labelling of qRG ( c ) and β-catenin ( d ; white arrows). (e – g) Lesion-induced β-catenin staining in the neural layer (Neu-L) and qRG layer (qRG-L) showing upregulation in the proportion of neuronal cells expressing nuclear β-catenin. White box depicts higher magnification images of the qRG-L in ( f , g ). Split-channel images showing co-labelling of qRG ( f ) and β-catenin ( g ; white arrow). (h – k) Common expression pattern of Wnt activity observed in the qRG-L between control ( h , i ) and at 3-dpl ( j , k ) using the Wnt reporter lines Tg(TCFSiam:mCherry) and Tg(top:GFP ; white arrows). (l , m) β-catenin expression and EdU-labelling at the TMZi (stem cell zone 2; dashed white circles) under control conditions ( l ) and at 3-dpl ( m , orange asterisk), showing increased nuclear expression in putative neurons post-lesion. (n – p) Homeostatic levels of axin2 expression in the qRG layer ( o ) and TMZi ( p ; black dashed circle). Black boxes in ( n ) denote higher magnifications in ( o , p ). (q , s) axin2 expression 3-dpl in the qRG layer ( r ) and TMZi ( s ; black dashed circle). Black boxes in ( q) denote higher magnifications in ( r , s ). (t) Experimental design to study the requirement of Wnt/β-catenin signalling for the proliferative response of stem cells to tectal stab lesion using the heat-shock line Tg(hsp70l:dkk-1:gfp) . (u) EdU population size in the qRG layer and TMZi shows no change in proliferation post-injury in the absence of Wnt signalling (dkk+) compared with wildtype animals (dkk-) using the Tg(hsp70:dkk-1:gfp) transgenic line (unpaired t-test, two-tailed: qRG layer, p = 0.3081; NE-Ap zone, p = 0.4960). (v- w ”) Representative images of EdU + staining (pink) in the qRG layer (dashed lines) in dkk- (control; v- v”) and dkk + (w- w ” ) post-lesion. (x- y”) Representative images of EdU + staining (pink) in the TMZi (dashed lines) in dkk- (control; x- x ” ) and dkk + (y- y ” ) post-lesion. Orange asterisk denotes the lesioned hemisphere. Note GFP + expression observed in the dkk + (w”,y”) but not dkk- (v”,x”). Experimental replicates were combined for all statistical analyses. All data presented are mean ± S.E.M. Significance was accepted at * p < 0.05. In panels b,e, v–y, DAPI nuclear counterstaining (blue) was performed. In all cross-sectional images dorsal is oriented up. TMZi, internal tectal marginal zone; dpl, days post lesion.

Article Snippet: The axin2 in situ probe was generated in our lab from the ZP60 Axin2 plasmid (Addgene #16882) in a pSPORT1 vector, digested with asp718 and transcribed with SP6 RNA polymerase.

Techniques: Expressing, Staining, Activity Assay, Control, Transgenic Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Midbrain tectal stem cells display diverse regenerative capacities in zebrafish

doi: 10.1038/s41598-019-40734-z

Figure Lengend Snippet: Wnt/β-catenin signalling in stem cell zone 1 (qRG-L) and zone 2 (TMZi) following tectal lesion. (a) Experimental design to study Wnt/β-catenin signalling at 3-dpl. (b–d) Physiological levels of β-catenin expression in the neural layer (Neu-L) and qRG layer (qRG-L; stem cell zone 1). A small number of β-catenin-positive neuronal cell bodies are seen in the Neu-L (yellow arrow). White box depicts higher magnification images of the qRG-L in ( c , d ). Split-channel images showing co-labelling of qRG ( c ) and β-catenin ( d ; white arrows). (e – g) Lesion-induced β-catenin staining in the neural layer (Neu-L) and qRG layer (qRG-L) showing upregulation in the proportion of neuronal cells expressing nuclear β-catenin. White box depicts higher magnification images of the qRG-L in ( f , g ). Split-channel images showing co-labelling of qRG ( f ) and β-catenin ( g ; white arrow). (h – k) Common expression pattern of Wnt activity observed in the qRG-L between control ( h , i ) and at 3-dpl ( j , k ) using the Wnt reporter lines Tg(TCFSiam:mCherry) and Tg(top:GFP ; white arrows). (l , m) β-catenin expression and EdU-labelling at the TMZi (stem cell zone 2; dashed white circles) under control conditions ( l ) and at 3-dpl ( m , orange asterisk), showing increased nuclear expression in putative neurons post-lesion. (n – p) Homeostatic levels of axin2 expression in the qRG layer ( o ) and TMZi ( p ; black dashed circle). Black boxes in ( n ) denote higher magnifications in ( o , p ). (q , s) axin2 expression 3-dpl in the qRG layer ( r ) and TMZi ( s ; black dashed circle). Black boxes in ( q) denote higher magnifications in ( r , s ). (t) Experimental design to study the requirement of Wnt/β-catenin signalling for the proliferative response of stem cells to tectal stab lesion using the heat-shock line Tg(hsp70l:dkk-1:gfp) . (u) EdU population size in the qRG layer and TMZi shows no change in proliferation post-injury in the absence of Wnt signalling (dkk+) compared with wildtype animals (dkk-) using the Tg(hsp70:dkk-1:gfp) transgenic line (unpaired t-test, two-tailed: qRG layer, p = 0.3081; NE-Ap zone, p = 0.4960). (v- w ”) Representative images of EdU + staining (pink) in the qRG layer (dashed lines) in dkk- (control; v- v”) and dkk + (w- w ” ) post-lesion. (x- y”) Representative images of EdU + staining (pink) in the TMZi (dashed lines) in dkk- (control; x- x ” ) and dkk + (y- y ” ) post-lesion. Orange asterisk denotes the lesioned hemisphere. Note GFP + expression observed in the dkk + (w”,y”) but not dkk- (v”,x”). Experimental replicates were combined for all statistical analyses. All data presented are mean ± S.E.M. Significance was accepted at * p < 0.05. In panels b,e, v–y, DAPI nuclear counterstaining (blue) was performed. In all cross-sectional images dorsal is oriented up. TMZi, internal tectal marginal zone; dpl, days post lesion.

Article Snippet: The axin2 in situ probe was generated in our lab from the ZP60 Axin2 plasmid (Addgene #16882) in a pSPORT1 vector, digested with asp718 and transcribed with SP6 RNA polymerase.

Techniques: Expressing, Staining, Activity Assay, Control, Transgenic Assay, Two Tailed Test

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effect of Pulsed Electromagnetic Field on Bone Formation and Lipid Metabolism of Glucocorticoid-Induced Osteoporosis Rats through Canonical Wnt Signaling Pathway

doi: 10.1155/2016/4927035

Figure Lengend Snippet: Primer sequences for real-time PCR analysis.

Article Snippet: The PVDF membrane was blocked for 2 h at room temperature in TBS-Tween 20 (TBST) buffer containing 5% BSA, washed with TBST three times, and incubated overnight at 4°C with 1/500 dilution of Wnt10b antibodies, LRP5 antibodies, β -catenin antibodies, Axin2 antibodies, OPG antibodies, RANKL antibodies, Dkk-1 antibodies, SOST antibodies, Runx2 antibodies, PPAR- γ antibodies, C/EBP α antibodies, FABP4 antibodies (all purchased from Santa Cruz Biotech), and GAPDH antibody (1 : 1000, BOSTER, China), respectively.

Techniques: Real-time Polymerase Chain Reaction, Sequencing