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: 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: Journal of Cellular and Molecular Medicine

Article Title: MiR‐17 family‐mediated regulation of Pknox1 influences hepatic steatosis and insulin signaling

doi: 10.1111/jcmm.13902

Figure Lengend Snippet: Pknox1 expression level is inversely correlated with miR‐17 family. A, Liver histology as determined by H&E. B, Blood GLU and serum TG , ALT , and AST contents were significantly elevated in the rat model of T2 DM complicated with NAFLD . C, The hepatic expression levels of miR‐17 family were performed by real time‐ PCR ( qPCR ). D, The protein levels of Pknox1, total IRS , and serine phosphorylation of IRS 1 ( pS ‐ IRS ) in liver of the rat model were determined by Western blot analysis. E, The relative expression levels of miR‐17 and miR‐20a were performed by qPCR in FFA ‐treated HepG2 and L02 cells. F, The protein levels of Pknox1 in FFA ‐treated HepG2 and L02 cells were determined by Western blot analysis. Data are presented as the mean ± SD. (* P < 0.05, ** P < 0.01, n = 3)

Article Snippet: SiRNA against Pknox1 (si‐Pknox1) and scrambled siRNA (si‐Ctrl) were purchased from RiboBio.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐17 family‐mediated regulation of Pknox1 influences hepatic steatosis and insulin signaling

doi: 10.1111/jcmm.13902

Figure Lengend Snippet: Validation of Pknox1 as the direct target of miR‐17 family. A, Wild‐type ( WT ) and mutant (Mut) 3′‐ UTR of Pknox1 were cloned into a pmiR‐ RBREPORT ™ vector. B, Dual‐luciferase activity of the WT and Mut Pknox1 3′‐ UTR reporter constructs in the presence of miR‐17or control mi RNA (miR‐ NC ). Pknox1‐Mut1, Pknox1‐Mut2, and Pknox1‐Mut1 + 2 were represented as mutation of miR‐17‐targeted region 1 and 2 individually or of both regions in the Pknox1 3′‐ UTR , respectively. (C and E) qPCR detection of miR‐17 and miR‐20a levels in HepG2 and L02 cells transfected with their mimics (C) or inhibitors (E) or the controls. (D and F) Western blot analysis of Pknox1 expression in HepG2 and L02 cells transfected with miR‐17 and miR‐20a mimics (D) or inhibitors (F) or the controls. Data are presented as the mean ± SD. (* P < 0.05, ** P < 0.01, n = 3)

Article Snippet: SiRNA against Pknox1 (si‐Pknox1) and scrambled siRNA (si‐Ctrl) were purchased from RiboBio.

Techniques: Mutagenesis, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Construct, Transfection, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐17 family‐mediated regulation of Pknox1 influences hepatic steatosis and insulin signaling

doi: 10.1111/jcmm.13902

Figure Lengend Snippet: MiR‐17‐mediated regulation of Pknox1 affects insulin signaling in hepatocytes. A, Western blot analysis of Pknox1 expression in HepG2 and L02 cells transfected with Pknox1 si RNA (si‐Pknox1) or control si RNA (si‐Ctrl). B, HepG2 cells were transfected with si‐Pknox1 or miR‐17 mimic or their corresponding control for 48 h, then stimulated with insulin (200 n mol L −1 ) for 10 min. The IR , pY ‐ IR , IRS , pY ‐ IRS , pS ‐ IRS , AKT and pS ‐ AKT levels in HepG2 cells were determined by Western blot analysis. C, Western blot analysis of Pknox1 expression in HCC cells transfected with Pknox1 overexpression plasmid (Pknox1) or control plasmid (Ctrl). D, Restoration of Pknox1 level in HepG2 cells by transfection with Pknox1 plasmid reversed the effects of miR‐17 on the expressions of insulin signal transduction‐related genes. E, MiR‐17 inhibition impaired the insulin receptor signaling as determined by Western blot analysis of these genes. IR , insulin receptor; pY ‐ IR , Tyrosine phosphorylation of IR ; IRS , insulin receptor substrate; pY ‐ IRS , Tyrosine phosphorylation of IRS ; pS ‐ IRS , Serine phosphorylation of IRS 1; pS ‐ AKT , Serine phosphorylation of AKT

Article Snippet: SiRNA against Pknox1 (si‐Pknox1) and scrambled siRNA (si‐Ctrl) were purchased from RiboBio.

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Transduction, Inhibition

Journal: Journal of Cellular and Molecular Medicine

Article Title: MiR‐17 family‐mediated regulation of Pknox1 influences hepatic steatosis and insulin signaling

doi: 10.1111/jcmm.13902

Figure Lengend Snippet: MiR‐17‐mediated regulation of Pknox1 affects hepatocyte steatosis. A, HepG2 and L02 cells were transfected with si‐Pknox1 or miR‐17 mimic or their corresponding control for 48 h, then exposed to FFA for 48 h. Intracellular TG contents in HepG2 and L02 cells were decreased by si‐Pknox1 or miR‐17 mimic transfection. B, Restoration of Pknox1 level in HepG2 and L02 cells by transfection with Pknox1 plasmid reversed the inhibitory effect of miR‐17 on TG content. C, MiR‐17 inhibition promoted lipogenesis in HepG2 and L02 cells. (D‐F) Representative image of Oil Red O staining of HepG2 cells corresponding to the above groups. G, Relative mRNA expression levels of genes involved in lipogenesis, TG synthesis, and lipolysis. Data are presented as the mean ± SD. (* P < 0.05, ** P < 0.01, n = 3). ACC , acetyl‐CoA carboxylase; FASN , fatty acid synthase; SREBP 1c, sterol regulatory element binding protein 1; SCD ‐1, Stearoyl‐CoA desaturase; GPAM , glycerol‐3‐phosphate acyltransferase; AGPAT 6, acylglycerol‐phosphate acyltransferase 6; LPIN 1, lipin1; DGAT 1, diacylglycerol acyltransferase 1; ATGL , adipose triglyceride lipase; HSL , hormone‐sensitive lipase; MGL , monoacylglycerol lipase

Article Snippet: SiRNA against Pknox1 (si‐Pknox1) and scrambled siRNA (si‐Ctrl) were purchased from RiboBio.

Techniques: Transfection, Plasmid Preparation, Inhibition, Staining, Expressing, Binding Assay