ankrd49  (Proteintech)

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 expression correlates with EMT markers in LUAD ( A ) Representative immunohistochemical staining of ANKRD49, E-cadherin, α-SMA, TGF-β1, and PKNOX1 in LUAD tissues (scale bar: 200 μm), with corresponding magnified views (scale bar: 50 μm). ( B ) Correlation analysis between ANKRD49 expression and E-cadherin, α-SMA, TGF-β1, or PKNOX1 in 89 LUAD specimens.

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 induces EMT in NSCLC cells. ( A ) Morphological changes in ANKRD49-OE versus control A549 and H1299 cells, exhibiting characteristic EMT features (scale bar: 100 μm). ( B , C ) Western blot analysis of EMT markers (E-cadherin, EpCAM, Vimentin, α-SMA, N-cadherin, Slug, Snail, Twist and ZEB1) in ( B ) ANKRD49-OE and ( C ) ANKRD49-KD A549 or H1299 cells. β-tubulin was used as a loading control.

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Control, Western Blot

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 promotes EMT and activates SMAD signaling in lung xenografts. Representative IHC staining of ANKRD49, E-cadherin, α-SMA, N-cadherin, EpCAM, Slug, Snail, ZEB1, p-SMAD2, and p-SMAD3 in lung xenografts derived from nude mice injected with ( A ) ANKRD49-OE A549 or ( B ) ANKRD49-OE H1299 cells via the tail vein. Scale bars: 100 μm.

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Immunohistochemistry, Derivative Assay, Injection

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 promotes EMT and activates SMAD signaling in subcutaneous tumor tissues Representative immunohistochemical staining of ANKRD49, E-cadherin, α-SMA, N-cadherin, EpCAM, Slug, Snail, ZEB1, P-SMAD2 and P-SMAD3 in subcutaneous tumor tissues from nude mice subcutaneously injected with ANKRD49-OE A549 cells ( A ) or ANKRD49-OE H1299 cells ( B ). Scale bars: 100 μm.

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Immunohistochemical staining, Staining, Injection

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 regulates TGF-β1 expression and secretion. (A, B) Western blot analysis of intracellular TGF-β1 protein levels in ( A ) ANKRD49-OE and ( B ) ANKRD49-KD A549 or H1299 cells. GAPDH was used as a loading control. ( C , D ) ELISA quantification of secreted TGF-β1 in conditioned media from ( C ) ANKRD49-OE ( n = 3) and ( D ) ANKRD49-KD cells ( n = 3). ( E , G ) Representative IHC staining for TGF-β1 in ( E ) lung xenografts and ( G ) subcutaneous tumors from mice injected with ANKRD49-OE A549 or H1299 cells (scale bars: 100 μm). (F, H) Quantitative analysis of TGF-β1 staining intensity (average optical density) in the corresponding ( F ) lung and ( H ) subcutaneous tumor tissues ( n = 5 mice/group). Quantitative data ( C , D , F , H ) were presented as mean ± SEM and statistical analysis was performed using unpaired Student’s t -test ( C , F , H ) or one-way ANOVA with Tukey’s post hoc test ( D ).

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Injection, Staining

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 activates the TGF-β/SMAD signaling pathway. ( A ) Western blot analysis of p-SMAD2/3 levels in ANKRD49-OE A549 or H1299 cells. GAPDH served as the loading control ( B ) ANKRD49 enhances SMAD-dependent transcriptional activity. Luciferase reporter assays in 293 T cells co-transfected with (SBE)4(BV)-Luc reporter and Flag- ANKRD49 plasmids. Data represented mean ± SEM ( n = 3) and were analyzed by Unpaired Student’s t test. ( C ) Western blot showing p-SMAD2/3 levels in ANKRD49 -KD A549 or H1299 cells. GAPDH was used as the loading control. ( D , E ) Western blot analysis of EMT markers (E-cadherin, N-cadherin, α-SMA) and p-SMAD2/3 in ANKRD49-OE A549 ( D ) or H1299 cells ( E ) treated with or without 5 µM Galunisertib. GAPDH served as the loading control.

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Western Blot, Control, Activity Assay, Luciferase, Transfection

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: PKNOX1 binds the TGF-β1 promoter and enhances its transcriptional activity. ( A ) Schematic of the TGF-β1 promoter region shows two predicted PKNOX1 binding sites (positions − 1455 ~ −1441 and − 1511 ~ −1500 relative to TSS). TSS, transcription start site. ( B ) Luciferase reporter assays in 293 T cells demonstrated that TGF-β1 promoter activity was significantly activated by PKNOX1 overexpression and further enhanced with ANKRD49 co-expression. pGL3-Basic-Ctrl (empty promoter vector) or p3×Flag-Ctrl (empty expression vector) served as control ( n = 3). ( C ) Functional validation of PKNOX1 binding sites in the TGF-β1 promoter. Only Mut1 (disrupting − 1455 ~ −1442 site) abolished PKNOX1-mediated activation ( n = 3). ( D ) ChIP-qPCR validation of PKNOX1 binding to TGF-β1 promoter. Significant enrichment at −1458 to −1347 region (containing − 1455 ~ −1442 site). No enrichment at −1605 to −1470 region (containing − 1511 ~ −1500 site). Enhanced binding in ANKRD49-OE cells ( n = 3). Data were presented as mean ± SEM and statistical analysis was performed using Two-way ANOVA followed by Tukey’s HSD test (B and D) or Unpaired Student’s t test ( C ).

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Activity Assay, Binding Assay, Luciferase, Over Expression, Expressing, Plasmid Preparation, Control, Functional Assay, Biomarker Discovery, Activation Assay, ChIP-qPCR

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: ANKRD49 regulates TGF-β1 expression through PKNOX1 interaction. ( A , C ) RT-qPCR analysis of PKNOX1 in ( A ) ANKRD49-OE A549 or H1299 cells and ( C ) ANKRD49-KD A549 or H1299 cells. ( B , D ) Western blot analysis of PKNOX1 protein levels in ANKRD49-OE A549 or H1299 cells ( B ) and ANKRD49-KD A549 or H1299 cells ( D ). GAPDH served as loading control. ( E ) Subcellular localization of PKNOX1 in ANKRD49-OE cells. Cytoplasmic and nuclear fractions were analyzed by Western blot. GAPDH served as a cytoplasmic marker, Lamin B or Histone H3 served as a nuclear marker. ( F ) Rescue experiment showing TGF-β1 and PKNOX1 levels in ANKRD49-OE cells treated with PKNOX1 siRNA or control siRNA. GAPDH was a loading control. ( G ) Immunofluorescence microscopy demonstrating ANKRD49 (red) and PKNOX1 (green) co-localization in 293 T cells. Scale bar, 100 μm. ( H ) HEK 293 T cells were co-transfected with Flag-ANKRD49 and PKNOX1-GFP and co-immunoprecipitation (Co-IP) was performed using Flag antibody for pulldown. Data represented mean ± SEM ( n = 3). A and C, Unpaired Student’s t test. B and D, One-way ANOVA followed by Tukey’s HSD test

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Marker, Immunofluorescence, Microscopy, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cancer Cell International

Article Title: ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis

doi: 10.1186/s12935-026-04240-3

Figure Lengend Snippet: Mechanism pattern of the ANKRD49/PKNOX1/TGF-β1/SMAD regulatory and function network

Article Snippet: After blocking with 5% skimmed milk or 5% BSA, the membranes were incubated with indicated primary antibodies at 4 °C overnight, and then incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies at room temperature for 1 h. The primary antibodies used in this study are as following: ANKRD49 (Cat# 25034-1-AP, 1:1000), PKNOX1 (Cat# 10614-1-AP, 1:750), Twist (Cat# 25465-1-AP, 1:1000, Protein Tech, USA), E-cadherin (Cat# 3195 S, 1:1000), EpCAM (Cat# 93790, 1:1000), N-cadherin (Cat# 13116, 1:1000), ZEB1(Cat# 70512, 1:1000), Vimentin (Cat# 5741 S, 1:1000, Cell Signaling Technology, USA), Slug (Cat# sc-166476, 1:1000), Snail(Cat# sc-2719777, 1:1000), α-SMA (Cat# sc-53142, 1:1000, Santa Cruz Biotechnology, USA), TGF-β1 (Cat# BA0290, 1:1000, Boster Biological Technology, China), SMAD2 (Cat# D155233, 1:1000), SMAD3 (Cat# D161451, 1:1000, Sangon Biotech, China), P-SMAD2 (Cat# AP0269, 1:1000, ABclonal, China) and P-SMAD3 (Cat# AF1759, 1:1000, Beyotime Biotechnology, China), Tubulin monoclonal antibody (Cat# CPA9126, 1:5000, Cohesion Biosciences, UK) or GAPDH monoclonal antibody (Cat# bsm-33033 M, 1:5000, Bioss Biotechnology, China).

Techniques:

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 1 ANKRD49 is upregulated in NSCLC. (A) The mRNA levels of ANKRD49 in nine fresh NSCLC tissues and corresponding adjacent normal tissues were analyzed by RT-qPCR (N: normal tissues; T: tumorous tissues). (B) The mRNA levels of ANKRD49 were assessed by RT-qPCR in the human bronchial epithelial cell line (HBEC) and seven NSCLC cell lines. Data are expressed as means ± standard deviation. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding adjacent normal lung tissues or HBEC cells

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Quantitative RT-PCR, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 2 ANKRD49 potentiates migration and invasion of H1299 cells. (A, B) Identification of ANKRD49-OE or ANKRD49-sh H1299 cells was validated by RT- qPCR and Western blot. (C, D) Wound healing assay was used to measure migration of ANKRD49-OE and ANKRD49-sh H1299 cells, representative images were taken at a magnification of 40×, at 0, 24 and 48 h. (E, F) Transwell migration and invasion assays were performed to detect migration and invasion of ANKRD49-OE and ANKRD49-sh H1299 cells. Representative images were taken at 200× magnification. Statistical analysis from five random fields was conducted. All experiments were repeated independently three times. Data are expressed as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV5 group, #P < 0.05, ##P < 0.01 vs. LV3 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Migration, Quantitative RT-PCR, Western Blot, Wound Healing Assay, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 3 ANKRD49 upregulates MMP-2/MMP-9 expression in H1299 cells. (A, B) The mRNA and protein levels of MMP-2 and MMP-9 in ANKRD49-OE and ANKRD49-sh H1299 cells were detected using RT-qPCR and Western blot assays. (C, D) The activity of MMP-2 and MMP-9 in ANKRD49-OE and ANKRD49- sh H1299 cells was detected by gelatin zymography. (E, F) A wound healing assay was conducted to assess the effect of MMPs inhibitor (ilomastat) on the migration of ANKRD49-OE H1299 cells; representative images were taken at 40× magnification at 0 and 24 h. Data are expressed as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV5 group, #P < 0.05, ##P < 0.01 vs. LV3 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Zymography, Wound Healing Assay, Migration, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 4 ANKRD49 activates JNK pathway to regulate the expression of MMP-2/MMP-9 in H1299 cells. (A) The MAPK protein levels in ANKRD49-OE and ANKRD49-sh H1299 cells were assessed by Western blot. (B, C) The effects of JNK inhibitor or p38 MAPK inhibitor on the levels of MMP-2/MMP-9 in ANKRD49-OE H1299 cells were tested by Western blot. β-Tubulin served as an internal control. (D, E) A wound healing assay was conducted to assess the effect of JNK inhibitor (SP600125) or p38 inhibitor (SB203580) on the migration of ANKRD49-OE H1299 cells; representative images were taken at 40× magnification at 0 and 24 h. (F) The levels of p-ATF2 and p-c-Jun in ANKRD49-OE and ANKRD49-sh H1299 cells were measured by Western blot. (G, H) The levels of p-ATF2 and p-c-Jun in ANKRD49-OE H1299 cells treated with SP600125, SB203580 or DMSO were detected by Western blot. Data are expressed as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV5 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Expressing, Western Blot, Control, Wound Healing Assay, Migration, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 5 ANKRD49 activates c-Jun/ATF2 transcription factor to regulate the expression of MMP-2/MMP-9 in H1299 cells. (A) The levels of ATF2 and c-Jun in the nuclear fraction of ANKRD49-OE or LV5 H1299 cells were measured by Western blot. (B) The levels of p-ATF2 and p-c-Jun in the nuclear fraction of ANKRD49-OE or LV5 H1299 cells were measured by Western blot. (C) Co-immunoprecipitation (Co-IP) analysis was performed to assess the interaction between p-ATF2 and p-c-Jun in the nucleus of ANKRD49-OE or vector H1299 cells. (D) Representative immunofluorescence images of ANKRD49 induced nuclear co-location of p-ATF2 and p-c-Jun. Scale bar:100 μm. (E-G) Chromatin immunoprecipitating (CHIP) assay was carried out to analyze the binding of p-ATF2 or p-c-Jun with the promoter of MMP-2 or MMP-9. GAPDH, β-actin, PARP and Histone-H3 served as the internal control for cytosol and nuclear fractions, respectively. Data are expressed as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV5 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunofluorescence, Binding Assay, Control, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 6 Knockdown of ANKRD49 inhibits migration, invasion of H1703 cells. (A, B) The stable ANKRD49-sh H1703 cells was validated by RT-qPCR and Western blot. β-actin served as an internal control. (C, D) Wound healing assay was performed to detect migration of ANKRD49-sh H1703 cells, represen tative images were taken at 40× magnification at 0, 24 and 48 h. (E, F) Transwell migration and invasion assays were conducted to evaluate the migration and invasion of ANKRD49-sh H1703 cells. Representative images were taken at 200× magnification. Data are expressed as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV3 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Knockdown, Migration, Quantitative RT-PCR, Western Blot, Control, Wound Healing Assay, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 7 Knockdown of ANKRD49 downregulates MMP-2/MMP-9 expression of H1703 cells. (A, B) The mRNA and protein levels of MMP-2 and MMP-9 in ANKRD49-sh H1703 cells were analyzed by RT-qPCR and Western blot. (C, D) The activities of MMP-2 and MMP-9 in ANKRD49-sh H1703 cells were de tected by gelatin zymography. (E) The MAPK proteins in ANKRD49-sh H1703 cells were detected by Western blot. GAPDH served as an internal control. (F) The levels of p-ATF2 and p-c-Jun in ANKRD49-sh H1703 cells were measured by Western blot. (G, H) The levels of JNK, p-JNK, p38, p-p38, MMP-2 and MMP-9 in ANKRD49-sh H1703 cells treated with SP600125, SB203580, Anisomycin or DMSO were measured by Western blot. β-Tubulin served as an inter nal control. Data are expressed as means ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LV3 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Zymography, Control, Standard Deviation

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 8 Overexpression of ANKRD49 promotes the migration and invasion of H1299 cells in nude mice. (A) Representative lung images of mice injected with ANKRD49-OE-H1299 or LV5-H1299 cells. White arrows manifest the metastasis nodules on the lung. (B) Statistical analysis of the number of metas tasis nodules on the lung was illustrated. (C) Representative images of HE staining for lung metastases. Green arrows indicated metastatic H1299 cells. (D) Representative images of IHC staining for NAPSA and NKX2-1. Scale bars represent 100 μm. (E, F) Representative images of IHC staining for ANKRD49, MMP-2, MMP-9, p-JNK, p-ATF2 or p-c-Jun. Scale bars represent 100 μm. (G) Quantitative analysis of IHC staining. **P < 0.01, ***P < 0.001 vs. LV5 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Over Expression, Migration, Injection, Staining, Immunohistochemistry

Journal: BMC cancer

Article Title: ANKRD49 promotes the metastasis of NSCLC via activating JNK-ATF2/c-Jun-MMP-2/9 axis.

doi: 10.1186/s12885-023-11612-9

Figure Lengend Snippet: Fig. 9 Knockdown of ANKRD49 declines the migration and invasion of H1703 cells in nude mice. (A) Representative lung images of mice injected with ANKRD49-sh or LV3 H1703 cells. White arrows manifest the metastasis nodules on the lung. (B) Statistical analysis of the number of metastasis nodules on the lung was illustrated. (C) Representative images of HE staining for lung metastases. Green arrows indicated metastatic H1703 cells. (D) Representative images of IHC staining for p63. Scale bars represent 100 μm. (E, F) Representative images of IHC staining for ANKRD49, MMP-2, MMP-9, p-JNK, p-ATF2 or p-c-Jun. Scale bars represent 100 μm. (G) Quantitative analysis of IHC staining. **P < 0.01, ***P < 0.001 vs. LV3 group

Article Snippet: Rabbit anti-ANKRD49 primary antibody (Proteintech, Cat# 25034-1-AP, RRID:AB_2879860), rabbit anti-MMP-2 polyclonal antibody (Cat# bs-4599R, RRID:AB_11083963), rabbit anti-MMP-9 polyclonal antibody (Cat# bs-0397R, RRID:AB_10853038), rabbit anti-c-Jun polyclonal antibody (Cat# bs-0670R, RRID:AB_10857880), rabbit anti-p-ATF2 polyclonal molecular mechanisms of ANKRD49’s function is different from those found in A549 cells.

Techniques: Knockdown, Migration, Injection, Staining, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: ANKRD49 is highly expressed in LUAD and correlates with poor prognosis. (A) Immunohistochemical determination of ANKRD49 in lung adenocarcinoma and its adjacent non‐cancerous tissues. Representative images are shown for negative ANKRD49 staining in adjacent non‐tumorous tissues (a) and weak ANKRD49 staining (b), moderate ANKRD49 staining, (b) and strong ANKRD49 staining (d) in tumour tissues. Scale bar is 50 μm. (B) Quantitative analysis of immunostaining intensities of ANKRD49 in lung adenocarcinoma tissues and their adjacent non‐tumorous tissues using the H ‐score method. (C) Clinicopathological parameters associated with overall survival in lung adenocarcinoma based on Kaplan–Meier survival curves. These parameters include overall survival rate (a), TNM stage (b), lymph node metastasis (c), differentiation (d), distant metastasis (e), gender (f), age (g), histological grade (h), and tumour size (i). The horizontal axis represents overall survival time (months), and the vertical axis represents overall survival rate (%)

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Immunohistochemical staining, Staining, Immunostaining

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Correlation between ANKRD49 and clinicopathalogical characteristics in LUAD tissues

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Cox's proportional hazard model analysis of prosnostic factors in LUAD

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Significance Assay, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Effect of ANKRD49 overexpression and knockdown in A549 cells on cell migration and invasion. (A) Effect of ANKRD49 overexpression on A549 cell migration determined by scratch healing analysis after 24 h and 48 h; (B) Width of scratch wound after 24 h and 48 h. (C) Effect of ANKRD49 knockdown on A549 cell migration determined by scratch healing analysis after 48 h. (D) Width of scratch wound after 24 h and 48 h. (E) Matrigel invasion and migration assay of A549 cells following overexpression of ANKRD49 (ANKRD49 OE), compared to control cells (vector) in a 200× light by crystal violet staining. (F) Matrigel invasion analysis of ANKRD49 OE A549 cells or its vector control cells under a light scope in three randomly selected areas. (G) Transwell migration analysis of ANKRD49 OE A549 cells or its vector counterpart cells under a light scope in three randomly selected areas. (H) Matrigel invasion and migration assay of A549 cells after ANKRD49 knockdowns (ANKRD49 KD702 and ANKRD49 KD829), compared to their shcontrol cells by crystal violet staining. (I). Matrigel invasion analysis of A549 cells stably transfected with shcontrol, ANKRD49 KD702, or ANKRD49 KD829 under a light scope in three randomly selected areas. (J) Transwell migration analysis of A549 cells stably transfected with shcontrol, ANKRD49 KD702, or ANKRD49 KD829 vectors under a light scope in three randomly selected areas. Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01; ns, no significance

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Over Expression, Knockdown, Migration, Control, Plasmid Preparation, Staining, Stable Transfection, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Effect of ANKRD49 expression on the invasion and metastasis and P38 MAPK/ATF‐2/MMPs signalling in nude mice in vivo. (A) Visible metastatic nodules and hemorrhagic spots on the lungs of the mice injected with ANKRD49 OE A549 cells but not their vector‐A549 cells. (B) Body weights and the number of metastatic nodules and hemorrhagic spots of the mice were determined following the injections of ANKRD49 OE A549 cells or their control vector cells ( n = 5). (C) Immunoreactivities of ANKRD49, NKX2‐1, Napsin A, Fibronectin, MMP‐2, MMP‐9, p‐P38, and p‐ATF‐2 in lung tissues of the mice injected with ANKRD49 OE A549 cells or control vector‐A549 cells. Scale bar 50 μm. (D) HE staining of nude mouse lung tissue. Scale bar: 50 μm. (E) H ‐score analysis of ANKRD49, NKX2‐1, Napsin A, Fibronectin, MMP‐2, MMP‐9, p‐P38, and p‐ATF‐2 immunoreactivities in lung tissues of the mice injected with ANKRD49 OE A549 cells or control vector‐A549 cells. (F) IHC score relevance of ANKRD49 and MMP‐2, MMP‐9, p‐P38, and p‐ATF‐2. Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Expressing, In Vivo, Injection, Plasmid Preparation, Control, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Effect of ANKRD49 on MMP‐2 and MMP‐9 expression, their enzyme activities, and the expression and activity of ATF‐2 transcription factor in A549 cells. (A) Expression levels of MMP‐2 and MMP‐9 mRNAs measured by q‐RT‐PCR in ANKRD49 OE A549 cells and their vector control cells. Data were calculated using 2 −ΔΔC t relative quantitative analysis. (B) Protein levels of MMP‐2 and MMP‐9 in ANKRD49 OE A549 cells or their vector control cells as determined by Western blot analysis. β‐Actin was used as the loading control. (C) Expression levels of MMP‐2 and MMP‐9 mRNAs were measured by q‐RT‐PCR in A549 cells stably transfected with ANKRD49 KD702, ANKRD49 KD829, and their shcontrol. Data were calculated using 2 −ΔΔCt relative quantitative analysis. (D) Protein levels of MMP‐2 and MMP‐9 in A549 cells stably transfected with ANKRD49 KD702, ANKRD49 KD829, and their shcontrol as determined by Western blot analysis. The knockdown effect of ANKRD49 and β‐Actin was measured using the same PVDF membrane and presented in Figure A. (E) Enzyme activities of MMP‐2 and MMP‐9 were detected by gelatin zymography. (F) Grey value of MMP‐2 and MMP‐9 in ANKRD49 OE A549 cells and ANKRD49 KD A549 cells. (G) Scratch healing analysis of ANKRD49 overexpression on A549 cell migration in the presence or absence of the MMP inhibitor (ilomastat) in 72 h. (H) Protein levels of ERK, p‐ERK, JNK, p‐JNK, P38 and p‐P38 determined in ANKRD49 OE A549 cells and their vector control cells by Western blot analysis. β‐Actin was used as loading control. (I) Levels of protein expression for p‐ATF‐2, ATF‐2, p‐P38, P38, MMP‐2 and MMP‐9 as determined by Western blot in ANKRD49 OE A549 cells and their vector control cells in the presence of a P38/MAPK inhibitor (SB203580) or its absence (DMSO). β‐Actin was used as loading control. (J) Protein levels of p‐ATF‐2 and ATF‐2 determined in ANKRD49 OE A549 cells and their vector control cells by Western blot analysis. (K) Protein levels of p‐ATF‐2 and ATF‐2 in A549 cells stably transfected with ANKRD49 KD702, ANKRD49 KD829, and their shcontrol as determined by Western blot analysis. β‐Actin was used as loading control. (L) Effect of ATF‐2 downregulation by RNA interference on MMP‐2 and MMP‐9 expression as determined by Western blot analysis in ANKRD49 OE A549 cells infected with lentivirus shatf2b or its vector control shatfNC. GAPDH was used as loading control. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001, and **** p < 0.0001. (M) Protein levels of ATF‐2 in the cytoplasm and nucleus compartments of ANKRD49 OE A549 cells and their vector control cells as determined by Western blot analysis. GAPDH was used as cytoplasm loading control. LaminB was used as nucleus loading control

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Control, Western Blot, Stable Transfection, Transfection, Knockdown, Membrane, Zymography, Over Expression, Migration, Infection

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Immunofluorescence analysis of the nuclear distribution of ATF‐2 in vector‐ or ANKRD49 OE‐A549 cells. Representative immunofluorescence images (A) and percentage analysis of nuclear positive staining (B) are shown. Scar bar: 50 μm; Data are shown as mean ± SEM. ** p < 0.01

Article Snippet: After blocking with 5% skim‐milk, the membranes were immunoblotted with a rabbit polyclonal or monoclonal antibodies for ANKRD49 (1:500, 25,034‐1‐AP, Protein Tech, USA), MMP‐2 (bs‐4599R), MMP‐9 (bs‐4599R), and p‐ATF‐2 (1:1000, bsm‐52134R, Bioss Biotechnology, China); P38 monoclonal antibody (8690), p‐P38 (4511), SAPK/JNK monoclonal antibody (9258P), and p‐JNK (1:1000, 4668P, Cell Signalling Technology, USA); ATF‐2 (1:1000, BS1022, BioWorld, USA); β‐actin monoclonal antibody (AP0060), GAPDH monoclonal antibody (AP0066), or LaminB (1:5000, AP6001, BioWorld, USA).

Techniques: Immunofluorescence, Plasmid Preparation, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: ANKRD49 is highly expressed in LUAD and correlates with poor prognosis. (A) Immunohistochemical determination of ANKRD49 in lung adenocarcinoma and its adjacent non‐cancerous tissues. Representative images are shown for negative ANKRD49 staining in adjacent non‐tumorous tissues (a) and weak ANKRD49 staining (b), moderate ANKRD49 staining, (b) and strong ANKRD49 staining (d) in tumour tissues. Scale bar is 50 μm. (B) Quantitative analysis of immunostaining intensities of ANKRD49 in lung adenocarcinoma tissues and their adjacent non‐tumorous tissues using the H ‐score method. (C) Clinicopathological parameters associated with overall survival in lung adenocarcinoma based on Kaplan–Meier survival curves. These parameters include overall survival rate (a), TNM stage (b), lymph node metastasis (c), differentiation (d), distant metastasis (e), gender (f), age (g), histological grade (h), and tumour size (i). The horizontal axis represents overall survival time (months), and the vertical axis represents overall survival rate (%)

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Immunohistochemical staining, Staining, Immunostaining

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Correlation between ANKRD49 and clinicopathalogical characteristics in LUAD tissues

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Cox's proportional hazard model analysis of prosnostic factors in LUAD

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Significance Assay, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Effect of ANKRD49 overexpression and knockdown in A549 cells on cell migration and invasion. (A) Effect of ANKRD49 overexpression on A549 cell migration determined by scratch healing analysis after 24 h and 48 h; (B) Width of scratch wound after 24 h and 48 h. (C) Effect of ANKRD49 knockdown on A549 cell migration determined by scratch healing analysis after 48 h. (D) Width of scratch wound after 24 h and 48 h. (E) Matrigel invasion and migration assay of A549 cells following overexpression of ANKRD49 (ANKRD49 OE), compared to control cells (vector) in a 200× light by crystal violet staining. (F) Matrigel invasion analysis of ANKRD49 OE A549 cells or its vector control cells under a light scope in three randomly selected areas. (G) Transwell migration analysis of ANKRD49 OE A549 cells or its vector counterpart cells under a light scope in three randomly selected areas. (H) Matrigel invasion and migration assay of A549 cells after ANKRD49 knockdowns (ANKRD49 KD702 and ANKRD49 KD829), compared to their shcontrol cells by crystal violet staining. (I). Matrigel invasion analysis of A549 cells stably transfected with shcontrol, ANKRD49 KD702, or ANKRD49 KD829 under a light scope in three randomly selected areas. (J) Transwell migration analysis of A549 cells stably transfected with shcontrol, ANKRD49 KD702, or ANKRD49 KD829 vectors under a light scope in three randomly selected areas. Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01; ns, no significance

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Over Expression, Knockdown, Migration, Control, Plasmid Preparation, Staining, Stable Transfection, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Effect of ANKRD49 expression on the invasion and metastasis and P38 MAPK/ATF‐2/MMPs signalling in nude mice in vivo. (A) Visible metastatic nodules and hemorrhagic spots on the lungs of the mice injected with ANKRD49 OE A549 cells but not their vector‐A549 cells. (B) Body weights and the number of metastatic nodules and hemorrhagic spots of the mice were determined following the injections of ANKRD49 OE A549 cells or their control vector cells ( n = 5). (C) Immunoreactivities of ANKRD49, NKX2‐1, Napsin A, Fibronectin, MMP‐2, MMP‐9, p‐P38, and p‐ATF‐2 in lung tissues of the mice injected with ANKRD49 OE A549 cells or control vector‐A549 cells. Scale bar 50 μm. (D) HE staining of nude mouse lung tissue. Scale bar: 50 μm. (E) H ‐score analysis of ANKRD49, NKX2‐1, Napsin A, Fibronectin, MMP‐2, MMP‐9, p‐P38, and p‐ATF‐2 immunoreactivities in lung tissues of the mice injected with ANKRD49 OE A549 cells or control vector‐A549 cells. (F) IHC score relevance of ANKRD49 and MMP‐2, MMP‐9, p‐P38, and p‐ATF‐2. Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, no significance

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Expressing, In Vivo, Injection, Plasmid Preparation, Control, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Effect of ANKRD49 on MMP‐2 and MMP‐9 expression, their enzyme activities, and the expression and activity of ATF‐2 transcription factor in A549 cells. (A) Expression levels of MMP‐2 and MMP‐9 mRNAs measured by q‐RT‐PCR in ANKRD49 OE A549 cells and their vector control cells. Data were calculated using 2 −ΔΔC t relative quantitative analysis. (B) Protein levels of MMP‐2 and MMP‐9 in ANKRD49 OE A549 cells or their vector control cells as determined by Western blot analysis. β‐Actin was used as the loading control. (C) Expression levels of MMP‐2 and MMP‐9 mRNAs were measured by q‐RT‐PCR in A549 cells stably transfected with ANKRD49 KD702, ANKRD49 KD829, and their shcontrol. Data were calculated using 2 −ΔΔCt relative quantitative analysis. (D) Protein levels of MMP‐2 and MMP‐9 in A549 cells stably transfected with ANKRD49 KD702, ANKRD49 KD829, and their shcontrol as determined by Western blot analysis. The knockdown effect of ANKRD49 and β‐Actin was measured using the same PVDF membrane and presented in Figure A. (E) Enzyme activities of MMP‐2 and MMP‐9 were detected by gelatin zymography. (F) Grey value of MMP‐2 and MMP‐9 in ANKRD49 OE A549 cells and ANKRD49 KD A549 cells. (G) Scratch healing analysis of ANKRD49 overexpression on A549 cell migration in the presence or absence of the MMP inhibitor (ilomastat) in 72 h. (H) Protein levels of ERK, p‐ERK, JNK, p‐JNK, P38 and p‐P38 determined in ANKRD49 OE A549 cells and their vector control cells by Western blot analysis. β‐Actin was used as loading control. (I) Levels of protein expression for p‐ATF‐2, ATF‐2, p‐P38, P38, MMP‐2 and MMP‐9 as determined by Western blot in ANKRD49 OE A549 cells and their vector control cells in the presence of a P38/MAPK inhibitor (SB203580) or its absence (DMSO). β‐Actin was used as loading control. (J) Protein levels of p‐ATF‐2 and ATF‐2 determined in ANKRD49 OE A549 cells and their vector control cells by Western blot analysis. (K) Protein levels of p‐ATF‐2 and ATF‐2 in A549 cells stably transfected with ANKRD49 KD702, ANKRD49 KD829, and their shcontrol as determined by Western blot analysis. β‐Actin was used as loading control. (L) Effect of ATF‐2 downregulation by RNA interference on MMP‐2 and MMP‐9 expression as determined by Western blot analysis in ANKRD49 OE A549 cells infected with lentivirus shatf2b or its vector control shatfNC. GAPDH was used as loading control. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.0001, and **** p < 0.0001. (M) Protein levels of ATF‐2 in the cytoplasm and nucleus compartments of ANKRD49 OE A549 cells and their vector control cells as determined by Western blot analysis. GAPDH was used as cytoplasm loading control. LaminB was used as nucleus loading control

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Control, Western Blot, Stable Transfection, Transfection, Knockdown, Membrane, Zymography, Over Expression, Migration, Infection

Journal: Journal of Cellular and Molecular Medicine

Article Title: ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38 / ATF ‐2 signalling pathway

doi: 10.1111/jcmm.17464

Figure Lengend Snippet: Immunofluorescence analysis of the nuclear distribution of ATF‐2 in vector‐ or ANKRD49 OE‐A549 cells. Representative immunofluorescence images (A) and percentage analysis of nuclear positive staining (B) are shown. Scar bar: 50 μm; Data are shown as mean ± SEM. ** p < 0.01

Article Snippet: Briefly, paraffin‐embedded sections were sliced 4‐μm‐thick and immunohistochemically stained using a rabbit anti‐human ANKRD49 primary antibody (25034‐1‐AP, ProteinTech, USA) at 1:100 dilution in an antibody dilution buffer.

Techniques: Immunofluorescence, Plasmid Preparation, Staining