Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Alkaline phosphatase (ALPL) inhibits the migration and invasion of LUAD cells by downregulating RhoA protein expression. a Western blot analysis of cell lysates from the indicated cells. b , e Western blot analysis of GFP-RhoA and RhoA expression in A549 (ALPL) and HCC827 (ALPL) cells stably transfected with GFP-RhoA or a vector control plasmid. GAPDH was used as a loading control. Transwell assays were performed to determine the effect of RhoA overexpression on A549 (ALPL) ( c ) and HCC827 (ALPL) ( f ) cells, Scale bars: 100 µm. d , g The migration and invasion rate of A549 (ALPL) and HCC827 (ALPL) cells. Data were presented as the mean ± SD and analyzed by Student′s t -test, n = 3. Asterisks (*) indicate a significant increase relative to control vector cells ( P < 0.05)

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Migration, Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Over Expression

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Alkaline phosphatase (ALPL) inhibits RhoA mRNA transcription by downregulating c-Myc. a Semi-quantitative PCR was used to evaluate RhoA mRNA expression after ALPL overexpression in A549 and HCC827 cells. b RhoA mRNA expression was evaluated by qPCR after ALPL overexpression in A549 and HCC827 cells. c Dual-luciferase reporter assays were used to detect RhoA promoter activity after ALPL overexpression in A549 and HCC827 cells. d TFANSFAC Transcription Factor Binding Sites Software (Biological Database, Wolfenbuttel, Germany) was used for bioinformatics analysis of the RhoA promoter region. Transcription factors for which potentially conserved binding sites were identified in the RhoA promoter region included c-Myc, c-Jun, Foxp3, and Sp1. e Western blot analysis of cell lysates from the indicated cells. GAPDH was used as an internal control. f Western blot analysis of c-Myc and RhoA expression in A549 (ALPL) cells stably transfected with c-Myc or a control plasmid. GAPDH was used as a loading control. g The RhoA mRNA expression level was evaluated by qPCR after overexpression of c-Myc in A549 (ALPL) cells. h Dual-luciferase reporter assays of the RhoA promoter activity after overexpression of c-Myc in A549 (ALPL) cells. i Transwell assays to determine the effect of overexpression of c-Myc on A549 (ALPL) cells, Scale bars: 100 µm. j The migration and invasion rate of A549 (ALPL) cells. All results were presented as the mean ± SD and analyzed by Student’s t -test, n = 3. Asterisks (*) represent statistical significance ( P < 0.05)

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Luciferase, Activity Assay, Binding Assay, Software, Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation, Migration

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Alkaline phosphatase (ALPL) promotes the degradation of c-Myc by activating ERK. a qPCR analysis of the levels of c-Myc mRNA in A549 and HCC827 cells overexpressing ALPL. ns, not significant. b The rate of c-Myc protein degradation in A549 (Vector) and A549 (ALPL) cells. GAPDH was used as an internal control. c Western blot analysis of the levels of c-Myc and p-c-Myc (Thr58/Ser62) in A549 (Vector) and A549 (ALPL) cells after treatment with MG132. d Western blot analysis of the indicated cells for levels of the ubiquitin-related molecules ERK1/2, GSK-3β, p-ERK1/2, and p-GSK-3α/β. GAPDH was used as an internal control. e Western blot analysis of the levels of p-ERK1/2, ERK1/2, c-Myc, and p-c-Myc (Thr58/Ser62) in A549 (ALPL) cells after treatment with PD98059 (100 μM)

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Plasmid Preparation, Control, Western Blot, Ubiquitin Proteomics

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: The expression of ALPL, p-ERK, c-Myc and RhoA were determined by IHC staining in the metastatic lung tumors tissues. a Representative IHC images showing the expression of ALPL, p-ERK, c-Myc and RhoA in metastatic lung tumors tissues, n = 9, Scale bars: 400 µm or 100 µm. Protein expression levels of p-ERK ( b ), c-Myc ( c ), and RhoA ( d ) were analyzed by calculating the integrated optical density per stained area (IOD/area) using Image-Pro Plus version 6.0, and the correlation between p-ERK/c-Myc/RhoA and ALPL was analyzed by Pearson’s Chi-square test

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Schematic diagram of the pathway by which ALPL downregulates RhoA via c-Myc. ① ALPL leads to an accumulation of phosphorylated (p)-ERK. ② p-ERK promotes c-Myc phosphorylation at Thr58 and Ser62 which is then degradated through ubiquitin–proteasome system. ③ The reduction of c-Myc leads to a decrease in RhoA transcription, which eventually suppresses LUAD cell metastasis. The dotted line indicates a multi-process, and the solid line indicates a single process

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Phospho-proteomics, Ubiquitin Proteomics

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Alkaline phosphatase (ALPL) inhibits the migration and invasion of LUAD cells by downregulating RhoA protein expression. a Western blot analysis of cell lysates from the indicated cells. b , e Western blot analysis of GFP-RhoA and RhoA expression in A549 (ALPL) and HCC827 (ALPL) cells stably transfected with GFP-RhoA or a vector control plasmid. GAPDH was used as a loading control. Transwell assays were performed to determine the effect of RhoA overexpression on A549 (ALPL) ( c ) and HCC827 (ALPL) ( f ) cells, Scale bars: 100 µm. d , g The migration and invasion rate of A549 (ALPL) and HCC827 (ALPL) cells. Data were presented as the mean ± SD and analyzed by Student′s t -test, n = 3. Asterisks (*) indicate a significant increase relative to control vector cells ( P < 0.05)

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Migration, Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Over Expression

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Alkaline phosphatase (ALPL) inhibits RhoA mRNA transcription by downregulating c-Myc. a Semi-quantitative PCR was used to evaluate RhoA mRNA expression after ALPL overexpression in A549 and HCC827 cells. b RhoA mRNA expression was evaluated by qPCR after ALPL overexpression in A549 and HCC827 cells. c Dual-luciferase reporter assays were used to detect RhoA promoter activity after ALPL overexpression in A549 and HCC827 cells. d TFANSFAC Transcription Factor Binding Sites Software (Biological Database, Wolfenbuttel, Germany) was used for bioinformatics analysis of the RhoA promoter region. Transcription factors for which potentially conserved binding sites were identified in the RhoA promoter region included c-Myc, c-Jun, Foxp3, and Sp1. e Western blot analysis of cell lysates from the indicated cells. GAPDH was used as an internal control. f Western blot analysis of c-Myc and RhoA expression in A549 (ALPL) cells stably transfected with c-Myc or a control plasmid. GAPDH was used as a loading control. g The RhoA mRNA expression level was evaluated by qPCR after overexpression of c-Myc in A549 (ALPL) cells. h Dual-luciferase reporter assays of the RhoA promoter activity after overexpression of c-Myc in A549 (ALPL) cells. i Transwell assays to determine the effect of overexpression of c-Myc on A549 (ALPL) cells, Scale bars: 100 µm. j The migration and invasion rate of A549 (ALPL) cells. All results were presented as the mean ± SD and analyzed by Student’s t -test, n = 3. Asterisks (*) represent statistical significance ( P < 0.05)

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Luciferase, Activity Assay, Binding Assay, Software, Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation, Migration

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: The expression of ALPL, p-ERK, c-Myc and RhoA were determined by IHC staining in the metastatic lung tumors tissues. a Representative IHC images showing the expression of ALPL, p-ERK, c-Myc and RhoA in metastatic lung tumors tissues, n = 9, Scale bars: 400 µm or 100 µm. Protein expression levels of p-ERK ( b ), c-Myc ( c ), and RhoA ( d ) were analyzed by calculating the integrated optical density per stained area (IOD/area) using Image-Pro Plus version 6.0, and the correlation between p-ERK/c-Myc/RhoA and ALPL was analyzed by Pearson’s Chi-square test

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cancer Cell International

Article Title: Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

doi: 10.1186/s12935-021-01919-7

Figure Lengend Snippet: Schematic diagram of the pathway by which ALPL downregulates RhoA via c-Myc. ① ALPL leads to an accumulation of phosphorylated (p)-ERK. ② p-ERK promotes c-Myc phosphorylation at Thr58 and Ser62 which is then degradated through ubiquitin–proteasome system. ③ The reduction of c-Myc leads to a decrease in RhoA transcription, which eventually suppresses LUAD cell metastasis. The dotted line indicates a multi-process, and the solid line indicates a single process

Article Snippet: Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China).

Techniques: Phospho-proteomics, Ubiquitin Proteomics