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: